Oxidative stress (OS) plays a major role on tissue function. Several catabolic or stress conditions exacerbate OS, inducing organ deterioration. Haptoglobin (Hp) is a circulating acute phase protein, produced by liver and adipose tissue, and has an important anti-oxidant function. Hp is induced in pro-oxidative conditions such as systemic inflammation or obesity. The role of systemic factors that modulate oxidative stress inside muscle cells is still poorly investigated.-/-) to determine the role of this protein and therefore, of systemic OS in maintenance of muscle mass and function. Absence of Hp caused muscle atrophy and weakness due to activation of an atrophy program. When animals were stressed by acute exercise or by high fat diet (HFD), OS, muscle atrophy and force drop were exacerbated in Hp-/-. Depending from the stress condition, autophagy-lysosome and ubiquitin-proteasome systems were differently induced.We used Hp knockout mice (HpHp is required to prevent OS and the activation of pathways leading to muscle atrophy and weakness in normal condition and upon metabolic challenges. Haptoglobin (Hp) is an acute-phase plasma glycoprotein produced mainly by hepatocytes Hp is part of the acute phase response to inflammation for which is considered a marker in the clinical practice If on one side Hp induction in conditions of enhanced OS, as in the adipose tissue of obese subjects, is protective, on the other its proven chemotactic potential for macrophages Oxidation can alter the structure and function of lipids, proteins and nucleic acids, leading to cellular injury and even cell death. Several lines of evidence suggest that OS leads to muscle atrophy, a feature often associated to conditions characterized by an impaired mitochondrial function The muscle mass is affected by the balance between synthesis and degradation of intracellular components. Muscle atrophy occurs when the degradation rate is higher than the synthesis rate.The Phosphatidylinositide 3-kinase (PI3K)/Akt signaling cascade plays a key role in the regulation of muscle mass The ubiquitin-proteasome system (UPS) consists of concerted actions of enzymes that link chains of the polypeptide ubiquitin onto proteins to mark them for degradation in a large multicatalytic protease complex, the proteasome in vivoThe other proteolytic system that is activated in catabolic conditions and that is under FOXO regulation is macroautophagy, hereafter named autophagy In the present study we explored how lack of a factor that regulates systemic OS, like Hp, may impact on skeletal muscle size and function in both normal situation, and conditions that are known to increase ROS production and OS, either acutely, such as physical exercise, or chronically herein induced by High Fat Diet (HFD). The data revealed a critical role of Hp in preventing protein oxidation and weakness in skeletal muscle.-/- and Wild Type (WT) mice in terms of mass and expression of inflammatory and mitochondrial biogenesis markers Extensor Digitorum Longus, EDL) and slow-twitch (soleus) muscle. In standard conditions Hp deficiency did not show any significant increase of oxidized proteins in any of the two muscles , catalaseSulfiredoxin 1 (Srx 1), we did not find any significant induction. A trend of induction was observed in Glutaredoxin (Glrx), a key modulator of the intracellular redox state Previous studies from our lab showed that Hp deficiency does not result in any relevant metabolic abnormality under normal diet -/- mice . When we-/- mice. No significant difference was found for IL-6, IL-1b, Tumor Necrosis Factor α (TNFα), Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor FGF inducible 14 protein (Fn14), suggesting that inflammation is not present in muscle of Hp knockout mice  and large mitochondrial-poor glycolytic fibers (50.7% and 51.3% for WT and Hp-/- respectively) was identical between WT and Hp-/- mice  and Bnip3 . The final body weight was of 42.29±0.98 gr in HFD WT and 41.96±0.54 gr in HFD Hp-/-We then studied the role of Hp as scavenger factor of OS when ROS are chronically higher, such as during systemic pro-inflammatory conditions like obesity. Mice were fed with HFD for 12 weeks. This resulted in elevation of plasma Hp by 3 fold in WT mice -/- mice doubled muscle loss compared to WT. Indeed Hp-deficient fibers were already 10% smaller and obesity caused a further 20% decrease: this effect can be clearly observed both in the comparison between the means  is deleterious for muscle mass, because activates signaling pathways that promote protein breakdown and suppress protein synthesis, causing atrophy of muscle cells. During chronic inflammation two major mechanisms are believed to cause muscle atrophy, namely the increase of oxidative stress (OS) and insulin resistance. Both these conditions are described to activate an atrophy program and therefore muscle loss -/-) mice. Moreover, Hp-/- mice were also studied in conditions that are known to induce OS such as exercise and obesity. Interestingly, the signaling pathways activated by these two stresses are different.Haptoglobin (Hp) is among the different factors that are secreted by the liver in response of TNFα and IL-6 stimulation -/- young mice do not exhibit any ouvert pathological feature, they are normally viable and fertile and their metabolic profile is not altered Bnip3, is also significantly induced in knockout mice. Among the mTOR downstream targets only 4EBP is altered, suggesting that it might contribute to the phenotype of Hp-/- mice through a slight reduction of protein synthesis. These findings well fit with the concept that muscle loss is mainly related to enhancement of protein breakdown instead of inhibition of protein synthesis In basal condition the HpNrf2. However, at this age the ROS scavenger genes that we analyzed were not significantly induced. We could only detect a trend of induction for Glrx. One potential interpretation is that in basal condition, at 5 months of age, the redox state starts to be moderately altered and activates the antioxidant response, which however is impaired or is just at the beginning of the induction. When animals were exposed to challenging, such as by exercise, the knockout became more prone to oxidative damage. Indeed both glycolytic and oxidative muscles showed an increase of carbonylation. This does not seem to be related to inflammation since exercise elicits an anti-inflammatory action in both genotypes. The susceptibility to oxidative stress in Hp deficient mice seems to be related to the inefficient activation of an anti-oxidant response. In fact, SOD1 and Srx 1 are induced in wild type but not in knockout while Glrx is suppressed in Hp-/-.Notably the adult mice, in which the level of carbonylated proteins is not changed, show some signs of activation of an antioxidant response as revealed by a transcriptional upregulation of PGC1alpha is not induced by exercise when Hp is absent. Therefore, it is possible that mitochondria of knockout mice start to generate more ROS due to the impaired PGC1alpha response and consequent alteration of mitochondrial fusion or biogenesis.It is of interest to underline that -/-. This finding implies that exercise affects mitochondrial network and ROS production, that need to be regulated in order to prevent alteration of sarcomeric proteins, that ultimately impact on force generation.The signaling pathways related to degradation and anabolic signaling show minor changes that can not explain the exercise intolerance of HpSOD 1 and Catalase are induced in Hp-/-mice. However mitochondria are greatly compromised in knockout mice and therefore, it is possible that the antioxidant response may not be sufficient to counteract mitochondrial-dependent ROS production. Differently from exercise, obesity induces autophagy and expression of MuRF1, suggesting that the degradation systems contribute to muscle atrophy in Hp KO mice.Obesity poses a different scenario from exercise. Similar to exercise, high fat diet leads to oxidative stress, but in this case Bnip3, an important gene for the selective removal of damaged mitochondria via autophagy (mitophagy) implies that this system may play an additional role in buffering mitochondrial dysfunction to limit ROS production and in inducing muscle atrophy The obesity-related activation of autophagy-lysosome system, together with the upregulation of These findings open a new view on the effects that pro-inflammatory cytokines elicit on muscle mass and ROS production. Here we provide evidence that inflammatory cytokines are also able to activate a response pathway (i.e. upregulation of Hp) that is important to reduce the detrimental effects of systemic inflammation and oxidative stress during catabolic or stress conditions.The experimental protocols followed the Principles of Laboratory Animal Care and a specific authorization was issued by Italian Ministry of Education (114/2003-A of 16/09/2003) to CNR Animal Facilities, where mice were housed. Mice were sacrificed by cervical dislocation following isoflurane anaesthesia , and all efforts were made to minimize suffering.-/- mice was described elsewhere Tibialis anterior, gastrocnemius and soleus muscles were dissected from fed animals and frozen in liquid nitrogen for molecular assessment, and in 2-methylbutane for histological analysis. The number of animals studied in each experiment is indicated in the figure legends.The strategy used to target the Hp locus and generate HpHFD and RUN underwent treatments that are described below.Animals referred to as ad libitum access to food and water. Animals that did not reach 40 g of weight at the end of the diet, similarly represented in the two genotypes, were excluded from further investigations.Animals were fed with HFD  for 12 weeks. Animals had Mice were trained for 2 days (day 1 and 2) on the rotarod RS  for acclimation purposes prior to the actual fatigue task. At day 1 mice were placed on the rotarod (speed 13 rpm) for 15 min. They were replaced on the rotarod if they fell off. After 30 min of rest, mice were placed again on the rotarod for 15 min with a speed from 13 to 20 rpm. At day 2 training regimen was similar to the day 1 protocol, but the speed was gradually adjusted from 13 to 20 rpm in a ramp of 15 min. The second run had a similar speed but the ramp was contracted to 180 s. At day 3 the fatigue task consisted of three rounds of 1 h each  with a 15 min of rest between the first and the second task and 10 min of rest between the second and the third task. Mice were placed on the rotarod and ran at a speed ramping, in 180 s, from 13 to 20 rpm. They maintained a constant speed afterwards. The second task was similar to the first one. During the third task the speed was higher (13 to 21 rpm in a ramp of 180 s). If a mouse fell off four times within 60 s, the mouse was termed fatigued and the task was stopped. The number of falls and the duration time of consecutive exercising was recorded for each mouse. This experiment was performed in a blind fashion, with the operator unaware of the mouse genotype.Grip strength was measured each day at baseline and the end of the task. The grip-strength test apparatus consisted of a grasping trapeze connected to a force transducer . Each mouse was held by the base of the tail and placed in front of the grasping trapeze. Once the mouse grasped the trapeze, it was slowly pulled back until the pulling force overcame the mouse's grip strength. The grip-strength meter expresses the grip force in grams. Each mouse was submitted to five trials that were separated by an inter-trial interval of 15 min. Final score represents the average of the five trials for each mouse. The grip force was measured before and after the fatigue task both during the acclimation training and the actual fatigue task.Tibialis anterior muscle was frozen in melting isopentane cooled in liquid nitrogen; 10 µm transverse sections were cut on a cryostat. Skeletal muscle frozen sections were subjected to Hematoxylin and Eosin (H&E) staining Fiber Cross-Sectional Areas (CSA) were measured using IMAGEJ software  by an observer unaware of the group assignments. A minimum of 1500 fibers evaluated in at least 3 different animals were analyzed for each group. The fields were randomly selected to measure the fiber area, and all the fibers encompassed in those fields were evaluated. This analysis was carried out in a blind fashion, with the operator unaware of the mice genotype.Muscles and cells were lysed and immunoblotted as described earlier Protein lysates were prepared with the same procedure as for immunoblotting, but DTT was added to the lysis buffer to a final 50 mM concentration, as suggested in the manufacturer's protocol. OxyBlot Protein Oxidation Detection Kit (Millipore) was used for the procedure: 10 µg of protein were used per reaction.flexor digitorum brevis muscles. Mitochondrial membrane potential was measured by epifluorescence microscopy based on the accumulation of TMRM fluorescence, as previously described Muscle fibers were isolated from Nrf2 (Mm00477784_m1), SOD1 (Mm01344266_m1), CAT (Mm00437992_m1), Srx 1 (Mm00769566_m1), Glrx (Mm00728386_s1), TNFα (Mm 00443258_m1), IL-1b (Mm 00434228_m1), IL-6 (Mm 00446191_m1), TWEAK (Mm02583406_s1), Fn14 (Mm01302476_g1), PGC1α (Mm01208835_m1), Nrf1 (Mm00447996_m1) and Tfam (Mm00447485_m1) inventoried gene expression Taqman assays were purchased from Life Technologies-Applied Biosystems . A taqman assay for GAPDH (Mm99999915_g1) was employed for normalization.Total RNA was prepared from skeletal muscle using the Promega SV Total RNA Isolation kit. Complementary DNA generated from 400 ng of RNA with SuperScript III reverse transcriptase (Life Technologies), was analyzed by quantitative real-time RT-PCR using the QuantiTect SYBR Green PCR kit . Signals were normalized to GAPDH expression. Sequences of oligonucleotide primers used for SYBR green are shown in post-hoc test were used to compare more than 2 groups. When only two groups were compared, the Student's t-test was applied assuming equal variances. Statistical evaluation was performed using GraphPad Prism 3 . The difference among groups was considered significant at P<0.05.All values are expressed as mean ± Standard Error of the Mean (SEM). 2-tailed Student's t-test was used for pairwise comparisons. 1-way and 2-way or matched 2-way ANOVA followed by (when appropriate) Bonferroni


Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In  Arabidopsis thaliana, maintenance of central stem cells is organized by a feedback loop between WUSCHEL, a homeobox transcription factor, and CLAVATA receptor-like kinases and ligand proteins . Therefore, we aimed to establish an early blindness induction system, useful for a spectrum of B. oleracea crop types and to apply this method to study the morphological, physiological and genetic mechanisms underlying SAM arrest in B. oleracea plants.To study and elucidate the basis of SAM arrest in B. oleracea seed lots from our laboratory collection of seed samples. These seed lots, left from previous seed research projects, represented several crop types. Seed lot numbers (in our collection), crop type and sensitivity for blindness are given in Supplementary Table B. oleracea var. alboglabra (A12DHd), and a Calabrese broccoli line, B. oleracea convar. botrytis var. italica (GDDH33), through microspore culture of the F1. Seeds from about 100 double haploid lines were multiplied by seed companies in The Netherlands at three different locations under protected cultivation with natural light conditions.To develop a protocol for blindness induction, we used various Subsequent experiments, to study morphological effects and gene expression, were performed with seeds from the sensitive seed lot 1645 from the green cabbage cultivar Stanton F1 and specific lines from the AGDH population.To develop an assay for detecting sensitivity to the induction of blindness, the seeds were imbibed at low temperatures for a period between one and 14 days in darkness. Dry seeds were sown in Petri dishes on a layer of three filter papers moistened with cold demineralized water and incubated at the tested induction temperature in the dark. Temperature was measured with data loggers  next to the Petri dishes. In the first experiments temperatures between 0 and 10.5°C were tested. As the lowest temperatures gave the strongest induction, a temperature between 0 and 1°C was used in most of the subsequent experiments. This was achieved by placing the Petri dishes with seeds in a Styrofoam box filled with melting ice. The box with seeds and ice was placed in an incubator at 4°C. Cold induction for the QTL analysis with a large number of lines required mores space and a cold room at 1.5°C was used. After the cold incubation the seeds were transferred to individual pots of a 240 pot tray  filled with washed coco-peat and placed in a growth chamber at 20°C with 18 h incandescent light (150 μmol). Moisture was provided to the seedlings with tap water during the first 2 weeks and a standard nutrient solution was applied in the 3rd week. Plants were scored by eye for the absence of a growing shoot 3 weeks after sowing, unless stated otherwise. Seedling behavior under non-inducing (control) conditions was studied by imbibing dry seeds directly at 20°C.For histological analysis the material was fixed in 4% paraformaldehyde for a maximum of 16 h. Afterward, the samples were transferred to 30% ethanol overnight, than dehydrated in steps of 30 min until incubation in 96% of ethanol. Subsequently, the Technovit protocol was followed according to the manufacturer . The Technovit blocks were cut with a microtome in sections of 3 μm and toluidine blue was used for staining. Digital pictures were made with the Zeiss Axioskop microscope. Images were taken with a Leica DFC320 digital camera attached to the microscope and processed using Adobe Photoshop for white balance and contrast correction. Electron microscopy pictures were made at the Electron Microscopy Centre using the FEI Magellan FESEM high resolution scanning electron microscope.For imaging nuclei with DNA replication activity, seeds were taken from the cold induction assay and directly placed on sterile growth medium  with Murashige and Skoog medium, Gamborg B5 Vitamins (Duchefa Biochemie) containing 10 μM EdU (Invitrogen) and germinated in a growth cabinet under 24 h light and at 17°C. After 48 h treatment time, shoot apices were dissected, dehydrated, and stained as described in For QTL mapping, double haploid lines of the AGDH population  were stuin silico PCR1. Then finally, the QTL was chosen as the smallest genomic region encapsulated by SSR amplicons which included all markers with LOD score ≥ 2.5. The QTL on chromosome C03 was determined to start at position 18434598 and end at 34773929 as defined by the amplified regions corresponding to the SSR markers: BoSF2985 and boa011. A total of 1354 genes are predicted to be encoded in this QTL-region.The physical contig corresponding to the C03 QTL region was identified by first determining the genomic region (on chromosomes C03) that could be amplified with the SSR primers flanking the QTL region using ® . RNA samples were treated with DNAse I. Sequencing libraries were prepared with the TruSeq RNA Sample Preparation kit  following the manufacturer manual. Paired-end sequencing was done on an Illiumina Hi-Seq 2000 machine with 100 bp read length.Total RNA was isolated at different time points, in triplicate after the induction treatment, from meristem enriched plant material  using the Spin Plant RNA Mini Kit from InvitrapB. oleracea2 using TopHat version 2.0.12 . The GO term annotation was formatted such that it could be used for enrichment analysis using BINGO (We assigned Gene ontology (GO) terms to g BlastP . The GO ng BINGO . The bacThe raw RNA-seq sequence reads were deposited at the NCBI Sequence Read Archive (SRA) under the BioProject number: PRJNA319618.A. thaliana genes active in the SAM were identified in B. oleracea by Blast (blastp) searches. Alignments of the best hits from the blast search were made using ClustalW . Gene expression was measured by quantitative RT-PCR (qRT-PCR) with four biological replicates, using SYBR green mix from Bio-Rad, and making use of the primer combinations indicated in Supplementary Table BoYLS8 gene was used as reference gene for normalization and data were analyzed using the ΔΔCT method . After 10 days of exposing seeds to cold incubation below 11°C, none had reached the phase of radicle protrusion, which occurred for most seeds within 24 h after transfer of the seeds from the cold to 20°C. After 14 days of cold incubation at 10.5°C some seeds showed radicle protrusion. The blind seedlings obtained from these cold treated seed batches exhibited a gradual variation in the severity of blindness, with either no leaves at all or a few aberrant leaves  temperatures on B. oleracea varieties, we tested four different genotypes (Table 1). The assay conditions resulted in general in a reduction of 10–20% in seed germination, but with a clear induction of blind plants in all four seed lots tested, showing that our induction method is applicable for several B. oleracea crop types . Subsequently, this result was confirmed with a larger number of seeds lots  and blind plant is depicted in Figure 2. The non-blind plant showed three expanded leaves, besides two small developing leaves emerging from the apex . In contrast, the blind plant had developed three expanded leaves only and stopped the production of subsequent leaves . Often the morphology of the non-blind and blind plants was very similar at this particular stage, but further leaf development was arrested in blind plants. At the position where new leaves usually emerge the arrested plant showed a dent and exhibited absence of a growing tip and further leaf initiation . We observed that the stage of development at which the arrest of the meristem occurs is variable since blind plants differed in their appearance from no leaves to plants with a few leaves, although these leaves can often be aberrantly shaped . The central zone displayed a group of small cells comprising the stem cells. The rib-zone showed the typical parallel file structure of cells . In general, the cell pattern of the blind seedlings was less organized and the small meristematic cells were replaced by less and large parenchyma-like cells. No clear organized L1 layer was visible in the blind seedlings at the position of the SAM in non-blind seedlings.To examine more closely the SAM area of blind plants, we performed histological analysis of normal and blind seedlings without visible leaves. Sections of 6-day old non-blind seedlings showed all three zones of a typical SAM: the central zone, the periphery zone and the rib zone . We observed a dome shaped meristem in non-blind seedlings with emerging leaf primordia at the flanks of the dome , while blind seedlings showed a flattened area at the position of the SAM with one leaf primordium in this case .To analyze the phenotype at an earlier stage, scanning electron microscopy pictures were taken . The sensitivity of some of the offspring DH lines in the AGDH population appeared to be even higher than that of the parents, indicating transgressive segregation for blindness under inducing conditions. In the ANOVA analysis no block effect was observed (P = 0.65), but an effect of genetic variation  and an effect of seed production location  were observed (Table 2). The latter indicates a significant effect of seed production on the sensitivity of the seeds. One QTL was identified on linkage group C03 . The QTL analysis further showed that the allele responsible for increased sensitivity to blindness induction is from the rapid-cycling Chinese kale parental line (A12DHd).Using the blindness assay described above, we determined the sensitivity for blindness in the AGDH Figure 5). Seedlings from those lines were used to identify genome-wide differentially expressed genes associated to blindness. To get an indication at what time-point after the cold induction the SAM loses its meristematic cells, we first performed expression measurements of the gene SHOOT MERISTEMLESS (STM), because it encodes a class-I KNOTTED-like homeobox transcription factor that is expressed throughout the SAM . Additionally, we measured the expression of BoSTM in 10 days old seedlings after the cold induction . Based on these results we selected day 2 after the cold induction as first time point for RNA-seq analysis. Unfortunately, plants are too small at this time point to determine their phenotype and hence, RNA samples are a mix of ‘to become’ non-blind and ‘to become’ blind seedlings (approximately 50%). In order to have also pure samples of blind and non-blind seedlings, we collected also material at day 7 after the cold induction, which is the earliest moment to distinguish flawlessly between blind and non-blind plants. RNA-seq was done on this material and differential gene expression was determined . To eliminate genes that are solely cold-responsive at day 2, we eliminated those genes that were significantly up- or down-regulated in both the sensitive and resistant genotypes upon the cold treatment. For time point 7 days after induction the comparison was made between sensitive induced blind seedlings and non-blind seedlings. These filter criteria resulted in the identification of 2962 and 5929 statistically significant differentially expressed genes at day 2  in these selected gene sets at 2 and 7 days after the cold induction. This analysis revealed that at day 2, especially genes related to photosynthesis and cell proliferation were differentially expressed  and WUSCHEL (WUS) expression, which represent meristematic cells, and cells marked by FILAMENTOUS FLOWER (FIL), representing differentiating cells at the flanks of the SAM. Potential functional homologs of these genes were identified in our RNA-seq dataset and their behavior was analyzed. As can be seen in Supplementary Table To get a further indication of genome-wide differentially expressed genes that might be related to meristem development and hence, the occurrence of blindness, we made use of a published  regions . In thisTables 3 and 4) are expressed in the Arabidopsis vegetative meristem and/or in the seed (during imbibition) according to previously published micro-array data , Minichromosome maintenance family protein PROLIFERA (PRL), A. THALIANA MutS HOMOLOG 2 and 6  and a HOMOLOG OF NUCLEOLAR PROTEIN 56 (NOP56) are differentially expressed when comparing samples from sensitive blind and sensitive non-blind seedlings at both time points.In total, 90 and 160 of the genes within the C03 QTL were differentially expressed at days 2 and 7, respectively. Most homologs of the genes present in the top 20 most significantly differentially expressed genes in the CO3 QTL region . However, the same set of genes showed down-regulation in the sensitive line 7 days after cold induction, except for three P-Type cyclins, which remained their higher expression levels also at day 7 .Since we identified GO-terms related to cell proliferation as being over-represented both at days 2 and 7 after cold treatment in seedlings from a blindness-sensitive line  and in all visible tissues: the SAM, hypocotyl and cotyledons. In the seedling from the sensitive line, DNA replication was observed in the hypocotyl and cotyledon tissues, but the nuclei in the SAM area did not incorporate EdU, demonstrating that blindness is related to an absence of DNA replication . DNA replication was also abolished in the leaf primordium of the sensitive line compared to the resistant one .To analyze the cell division activity Figure 8), which are not located in the C03 QTL region and are most likely indirectly affected by the blindness phenomenon, a number of other cell cycle related genes are located in the QTL region. RETINOBLASTOMA RELATED (RBR) gBol035455, a plant homolog of the tumor suppressor gene identified in animals  and related to the cell cycle are members of the Mini Chromosome Maintenance (MCM) 2-3-5 gene family. Genes of this family play important roles in both the initiation and the elongation phase of eukaryotic DNA replication  a member of the MCM genes from Arabidopsis , all located in QTL region, are the most likely candidates for being involved in the development of blindness in sensitive genotypes.Our study revealed that blindness in JJ, JK, RI, and SG contributed to the design of the research; performance of the research; data analysis, collection, or interpretation; and writing the manuscript. ES contributed to the bioinformatics analysis. GB and GA contributed to the design of the experiments, data interpretation and writing the manuscript. All authors have read and approved the 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.The authors declare that the research was 50% financed by the Dutch government and a cash (25%) and in-kind (25%) contribution by a consortium of companies .


Correct proportioning and mixing are essential to ensure cements attain their optimum physical properties. The aim of this experimental study was to evaluate the influence of various mixing techniques including manual, mechanical mixing, and ultrasonic vibration on push-out bond strength of calcium enriched mixture (CEM).p< 0.05) Ninety 2-mm-thick dentin disks were prepared from single-rooted human teeth and filled with CEM mixed with manual, trituration, or ultrasonic methods. Push-out bond strength values of the specimens were measured by a universal testing machine after 3 and 21 days. The samples were then examined under a stereomicroscope at 40× magnification to determine the nature of bond failure. Data were analyzed by Kruskal-Wallis and Mann-Whitney test. (The highest (7.59 MPa) and lowest (4.01 MPa) bond strength values were recorded in conventional method (after 21 days) and trituration method (after 3 days), respectively. There was no statistically significant difference between the three techniques in 3 and 21 days. According to the results, various mixing techniques had no effect on the push-out bond strength of CEM cement. Many types of dental cements are available as powder and liquid that should be mixed before application. Correct proportioning and mixing are essential to ensure that the cements attain their optimum physical properties. EncapsulWhilst trituration uses conventional mechanical energy, there might be a potential for ultrasonic energy to be more effective. Ultrasonic vibration has a dispersing effect on the particles of materials, which frequently cluster together. Ultrasonic treatment has been reported to be effective in increasing the compressive strength,-4 tensilet al. compared ultrasonication, trituration, and manual mixing and concluded that the application of ultrasonic energy to MTA produced a significantly higher surface microhardness value.[et al. reported that different mixing methods had no significant effect on the push-out bond strength of white MTA.[et al., however showed that mechanical mixing of encapsulated MTA resulted in higher compressive strength values than those mixed manually.[Little information is available on the effect of various mixing techniques on the physical properties of mineral trioxide aggregate-like (MTA) materials. Nekoofar ss value. On the ohite MTA. Basturk manually.Calcium enriched mixture (CEM) cement was introduced in 2008 with similar clinical application to MTA but different chemical compositions.-12 CEM iThere is no information about the effect of mixing techniques on the push-out bond strength of CEM cement. Therefore, the purpose of this study was to evaluate the effect of various mixing techniques including ultrasonic vibration, trituration, and manual method on push-out bond strength of CEM cement. Sixty freshly extracted human teeth including single rooted mandibular premolars or maxillary incisors that were either intact or contained only small carious lesion were used in this study. Teeth with cracks or internal resorption were excluded from the study. After removing the crowns by using a diamond disk, the middle thirds of the teeth were sectioned perpendicular to the root long axis to obtain 90 dentin disks with the thickness of 2±0.2 mm. A diamond saw microtome  was used to obtain root dentin slices. The internal disk canals space was enlarged with Gates Glidden burs  sizes 2 to 5 to achieve a standard diameter of 1.3 mm.[In groups 1 and 4 the CEM cement was mixed with conventional method; in groups 2 and 5, the CEM cement was mixed with trituration in an amalgamator  at the speed of 4500 revolutions/min for 30 second (customized encapsulated CEM); in groups 3 and 6, the CEM cement was mixed with an ultrasonic tip . The CEM cements in all instances were mixed according to the manufacturer’s recommendations. The samples were wrapped in wet pieces of gauze and kept in an incubator  at 37 ˚C and 95% humidity for 3 days  or 21 days . Push-out testThe push-out test was performed on the samples by using a universal testing machine  .The cured specimens were placed on a metal slab with a central hole and loaded with a 0.7-mm diameter cylindrical stainless steel plunger at a speed of 1mm/ min. The maximum load applied to the CEM cement at the time of dislodgement was registered in newton.2 calculated according to the following formula:To express the bond strength in megapascals (MPa), the recorded values were divided by the adhesion surface area of CEM in mm2πr × h, where π is the constant 3.14, r is the root canal radius, and h is the thickness of the root slice in millimetres.p<0.05)The modes of bond failure were evaluated under the light microscope  at 40× magnification. Each sample was categorized into one of the three failure modes as adhesive failure at the CEM and dentin interface, cohesive failure within the CEM, and mixed failure mode. The data were analyzed by using Kruskal-Wallis and Mann-Whitney test as post-hoc test. (The means and standard deviations (SD) of the push-out bond strength of the groups are shown in Although the groups that CEM was mixed with trituration (group 2 and 5) showed the lowest value of push-out bond strength, no statistically significant difference was found between the three techniques in 3 and 21 days .p<0.05)Inspection of the samples revealed the bond strength to be predominantly cohesive for conventional technique but mixed for trituration and ultrasonic techniques.  (p<0.05)In this study, push-out test was used to assess the bond strength between CEM cement and dentinal walls. Various methods have been described to evaluate the bonding quality of dental materials to dentin such as shear,-20 comprA scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA) study demonstrated that in the presence of normal saline as a storage solution, hydroxyapatite crystals are formed and precipitated over the surface of CEM cement. The composition and structure of precipitated crystals were comparable with that of standard hydroxyapatite. Thereforet al. evaluated the push-out bond strength of two root-end filling materials in root-end cavities prepared by Er,Cr:YSGG laser or ultrasonic technique and concluded that the bond strength of MTA and CEM to root-end cavities were comparable and higher in ultrasonically prepared cavities.[To achieve optimal properties, the particles of hydraulic cements should be thoroughly mixed with water. The mixing technique of cements is fundamental for producing effective contact between the powder particles and liquid and a final set material with optimal physical, chemical, and biological properties. It has bcavities.In the present study, the influence of various mixing techniques on push–out strength of CEM cement was evaluated for the first time. The results showed that trituration method compared with manual and ultrasonic techniques results in lower push-out strength; however, no statistically significant difference was found between the three mixing techniques. Another study on the effect of different mixing techniques on the compressive strength showed that mechanical mixing with amalgamator increased the compressive strength of CEM cement. The confet al.[et al.[Interestingly, the studies on MTA showed similar results. While Shahi et al. showed tl.[et al. reportedTherefore, one may assume that different mixing methods have impact on the compressive strength, not on the push-out strength of hydraulic cements like MTA and CEM.et al.[The results of the current study showed that there was no significant difference between the push out bond strength of similar groups in 3 and 21 days. This finding is in contrast with that of Rahimi et al. who repoet al. showed tIn this study inspection of the samples revealed the bond strength to be predominantly cohesive for manual technique, but mixed for trituration and ultrasonic techniques. Therefore, it can be concluded that mixing method can affect the pattern of bond failure. Two separate studies with manual mixing technique reported cohesive bond failure as the predominant bond failure for CEM, which is in agreement with the results of the current study.-32It should mention that the mechanical tests are unable to reflect the clinical situation; hence, future studies are required to determine the effect of these techniques on the bond strength of material in clinical applications. in vitro study, it can be concluded that different mixing techniques evaluated in this study have no effect on the push-out bond strength of the CEM cementWithin the limitation of this


Smyd gene family code for proteins containing a conserved core consisting of a SET domain interrupted by a MYND zinc finger. Smyd proteins are important in epigenetic control of development and carcinogenesis, through posttranslational modifications in histones and other proteins. Previous reports indicated that the Smyd family is quite variable in metazoans, so a rigorous phylogenetic reconstruction of this complex gene family is of central importance to understand its evolutionary history and functional diversification or conservation. We have performed a phylogenetic analysis of Smyd protein sequences, and our results show that the extant metazoan Smyd genes can be classified in three main classes, Smyd3 (which includes chordate-specific Smyd1 and Smyd2 genes), Smyd4 and Smyd5. In addition, there is an arthropod-specific class, SmydA. While the evolutionary history of the Smyd3 and Smyd5 classes is relatively simple, the Smyd4 class has suffered several events of gene loss, gene duplication and lineage-specific expansions in the animal phyla included in our analysis. A more specific study of the four Smyd4 genes in Drosophila melanogaster shows that they are not redundant, since their patterns of expression are different and knock-down of individual genes can have dramatic phenotypes despite the presence of the other family members.The  Smyd gene family is widespread in eukaryotes, and in mammals it has been related to epigenetic transcriptional control, development and cell proliferation SMYD1-3. activity –13. SMYDactivity , 15 and activity . Althougc muscle ; and SMYpression , 19.In comparison with SMYD1-3, little is known of SMYD4 and SMYD5. SMYD4 has been related to breast cancer , and worDrosophila has been used to understand the role of a Smyd gene illustrate the need to establish a rigorous phylogeny of gene families before we determine orthology relationships. CG14122 was singled out among all the possible the SMYD4 homologs and renamed Smyd4, despite the fact that there were several other candidates [Smyd family has suffered extensive lineage-specific expansion (LSE) in insects [Smyd family beyond the examples we have mentioned in vertebrates and insects, so we do not know the family members present in the different animal phyla, and their evolutionary history.But these two examples in which ndidates . Subsequ insects . Still, Drosophila melanogaster. But the use of model organisms has to be based in the careful establishment of molecular and functional homologies. The determination of the whole collection of homologous sequences and the establishment of their phylogenetic relationships is fundamental to understand their molecular evolution, to identify true orthologs and to classify them in sub-groups that probably reflect functional differences [Drosophila as an example, this species has single orthologs for Parkinson disease-related genes PINK1 and PARK2, and has been used to study the mitochondria-related pathomechanisms in Parkinsonism [The number of known rare diseases is in constant increase, but the resources that can be allocated to each one of them do not grow at the same rate. Therefore, one of the strategies that can be used in research on rare diseases is to use non-murine model organisms that are inexpensive and technically approachable, such as ferences . In manyinsonism –27.But the presence of clear one-to-one orthologs in the different species is not always the case. During evolution there are dramatic changes in gene number and structure, such as changes in ploidy, domain rearrangements and variations in gene copy number, both expansions and losses –30. CopyMesobuthus martensii scorpion, containing around 30,000 genes, more than any arthropod sequenced to date despite the fact that scorpions are considered living fossils among chelicerates [Quite often, large scale LSE are related with the acquisition of new functions required for adaptation of the species to their environment . LSE ofticerates . Among tDrosophila could also be a good model for SMYD4 gene function, we have reconstructed the phylogeny of Smyd genes in representative animal phyla. Our phylogeny will also be useful to understand the evolution of the Smyd family, to identify animal model organisms for other human SMYD genes and, in a wider scenario, to contribute to the understanding of gene expansion in evolution.In this scenario of gene gain, loss and shuffling, it is evident that identifying the two orthologs of a particular gene in different species can be sometimes difficult, if they exist at all. In order to see if Trichoplax adhaerens) and two cnidarians (Hydra magnipapillata and Nematostella vectensis). Among protostomes, we included sequences of the two main branches, Lophotrochozoa (the mollusk Lottia gigantea), and Ecdysozoa . As representatives of the deuterostomes we included a hemichordate , a tunicate , a cepalochordate (Branchiostoma floridae) and four vertebrate species .To construct a phylogeny of the Smyd family in metazoans, we chose species which represent a wide set of animal phyla, and for which there is a genome project with acceptable genome coverage and sequence quality . The tree topology regarding this branch with respect to the others is variable . In addition to the divergence in the SET/MYND core, these proteins have one or two TPR domains in their N terminus, and an additional MYND zinc finger in their C terminus.Two Smyd sequences from amphioxus and mollusk are so diverged that it is difficult to ascertain their origin. If we add Smyd protein sequences from the mollusks Smyd3 and Smyd5 are closest to each other and more distantly related to Smyd4, but the origin of SmydA and the mollusk/amphioxus group is not clear. In order to shed light on these questions, and to find out more about how the different classes originated, we extended the phylogeny by introducing Smyd sequences from other species. First, using the same similarity and co-linearity criteria employed in the metazoan dataset, we searched for sequences in two species basal to the metazoans, the choanoflagellate Monosiga brevicollis and the filose ameboid Capsaspora owczarzaki, where we found 3 and 4 sequences respectively. Smyd genes have also been found in plants [Arabidopsis thaliana five candidate Smyd proteins were defined: ATXR1, ATXR2, ATXR4, ASHR1 and ASHR2. We searched the A. thaliana protein sequence collection using the same criteria used to construct the original data set and identified only the last four, which is consistent with the fact that ATXR1 does not contain a Zn finger [Smyd genes originated from ancestral SET genes that also gave rise to yeast SET5 and SET6, in which the only recognizable domain was the SET domain [Saccharomyces cerevisiae SET5 and SET6 as candidate outgroups to root our trees.Our trees suggest that classes n plants . In Arabn finger . In addiT domain . TherefoSmyd3 and Smyd4 pre-date the appearance of metazoans. One protein sequence from each A. thaliana and both unicellular relatives of the metazoans now groups within the Smyd3 sequences; and sequences from A. thaliana and M. brevicollis with the Smyd4 class. These groupings are also supported by the presence and position of other domains not included in the alignment: C-terminal domain in both types of sequences, and an additional N-terminal TPR domain in Smyd4. There are no plant representatives of Smyd5, but this class contains one sequence from each M. brevicollis and C. owczarzaki, indicating that this class originated after the split from plants and fungi, but before the appearance of metazoans. These two sequences also have Smyd5-characteristic C-terminal domains rich in glutamic acid, aspartic acid and serine. One sequence from C. owczarzaki also groups with the atypical sequences from L. gigantea and B. floridae. Similar to them, it also has additional TPR repeats N-terminal to the SET-MYND core, but not an extra C-terminal MYND finger. The rest of sequences from A. thaliana and S. cerevisiae, and one sequence from C. owczarzaki, do not have further domains or noticeable features outside the SET-MYND core. It is surprising that none of these, especially the S. cerevisiae SET protein sequences, appear as an outgroup. A closer inspection of yeast SET5 and SET6 revealed that these proteins also contain putative Zn fingers similar to the MYND type ones (see below). It is thus possible that these are Smyd proteins rather than SET-only. In fact, it had been proposed that at least SET5 was actually a Smyd protein [Surprisingly, ML and NJ trees obtained with the alignment of the extended set of sequences , Smyd4 (4 members) and Smyd5 (1 member) match the typical domain structure of their counterparts in other animal species. Over half of the Smyd genes (9 members) belong to the SmydA class. As already explained above, SmydA proteins do not share domains with the other three classes, outside the SET/MYND core. A closer examination of the putative MYND domains revealed that the Zn finger in the interrupted SET domain is less similar to the MYND signature than the members of the other three groups (CG8503 and CG14590) have a second, perfectly canonical MYND finger just N-terminal to the SET domain  the serial name (Smyd3) could be used as a synonym. An exception would be CG14122, currently known as Smyd4, since this name could drive to confusion. In this case we would ask from the Drosophila community and the genomic annotation consortia that the current name is left as a synonym and the Smyd4 genes are renamed Smyd4-1 to -4, with Smyd4-1 assigned to CG1868 since it is closest to human SMYD4.Some of the D. melanogaster has four Smyd4 genes, one in the Smyd4 group, two in the Smyd4L group and one in the Smyd4I group. We ignore whether the multiple copies compared to other species are redundant or functionally relevant, for example to achieve high levels of expression, anatomical subdivision of their function, or to sub-specialization of their roles. To explore this, we studied their patterns of expression and the effects of down-regulation. The objective is not a detailed study of their function, but to define the biological relevance of gene expansion within the Smyd4 group.Smyd4 genes we used two complementary approaches. First, there are high-throughput analyses of gene expression by public consortia. We downloaded the anatomical gene expression data by FlyAtlas (http://flyatlas.org/), comprising the expression levels of each gene in a collection of 25 larval and adult tissues. In addition, we also used the data generated by modENCODE (http://www.modencode.org/) corresponding to expression levels at 30 time points along development, from fertilization to adult. These data provide a regulated expression fingerprint that can be used to compare expression profiles between genes. Second, we performed in situ hybridization with specific probes corresponding to all four genes in developing embryos. The results of these studies are shown in CG1868 with CG8378, and CG14122 with CG7759. Although within each group the absolute levels of expression for each data point are different, the relative levels between data points are remarkably similar. Therefore, each group has a characteristic temporal and spatial modulation. In situ hybridization reveals the same groups. In extended germ band embryos CG1868 and CG8378 are expressed more intensely in the presumptive primordia of the cephalic central nervous system and of the posterior gut  for two of the genes, CG1868  and CG14122, . For the other two genes there was only reduced viability 67.7% and 82% for CG7759 and CG8378 respectively. The retina is part of the peripheral nervous system, and it is a widely used model to study neurodegeneration. Loss of retinal cells affects the external appearance of the eye, producing the so-called rough eye phenotype, in which the units of the compound eye lose their stereotypical shape and organization. In the control eyes . According to our results, vertebrate Smyd4 and insect Smyd4 and Smyd4I genes are expressed in nervous system, mesodermal derivatives and digestive tract, while Smyd4L is expressed only in mesoderm. This points out to a duplication of Smyd4 as the origin of Smyd4I. Finally, Smyd4L was lost in the chordate ancestor, leaving the Smyd4 group as the only representative of this class.Within the metazoans, the ed first . Our resSmyd4 class in insects, arthropod genomes contain a further expanded class of Smyd genes, which we have called SmydA. These genes do not share domains with other Smyd genes outside the SET-MYND core, and also show a high divergence within this core. More research is required to understand why arthropods have suffered such an expansion in the number of Smyd genes and what their origin is.In addition to the expansion of the B. floridae is a species that shows several differences regarding Smyd gene complement compared to the related phyla of hemichordata and vertebrata. In addition to the expected Smyd genes, it has two more genes within the Smyd3 class, probably coming from a duplication which is different from the one that gave rise to vertebrate Smyd1 and Smyd2 genes. These could be further examples of amphioxus-specific gene duplications already described [Smyd gene, with N-terminal TPR domain and an additional MYND domain in the C-terminal position, which has putative orthologs in mollusk species but not in the rest of metazoan species. Our results present three types of evidence in favor of shared ancestry rather than convergence or independent origin: phylogeny of the SET-MYND core, BLASTP hits in searches against all metazoan sequences, and domain organization. Still, at this point it is impossible to be sure whether these genes represent a case of horizontal gene transfer or differential loss. Horizontal gene transfer is extremely rare in animals, probably due to the fact that the germ line is isolated from the somatic cells, leaving differential gene loss as the most likely explanation. In this respect, we also show that some Smyd-related sequences in B. floridae and L. gigantea are not correctly annotated. Obtaining genome sequences of more animal species, and a more rigorous annotation, will facilitate the reconstruction of the evolution of complex gene families.escribed , which aCamponotus floridanus and Harpegnathos saltator, the authors constructed two phylogenies of selected insect and vertebrate species corresponding to what they call SMYD1-3 and SMYD4 families. Although this study is not as exhaustive as ours, since their species represent only two phyla, and do not analyze any Smyd5 sequences it confirms some of our conclusions. For example, their tree shows the same three groups in their SMYD4 family (our Smyd4 class), with CG1868 as the closest homolog of human SMYD4. Their SMYD1-3 family tree includes two groups, one that would correspond to our Smyd3 class and another one corresponding to the SmydA group, which contains 3 genes from each ant species, and several of the insect sequences we have identified.Some of our findings are supported by an independent study . In a coSmyd genes, it becomes evident that previous assignments of gene names cannot be supported. The work in ant genomics we mention in the previous section is a first example [SMYD3 , due to its presence in what they incorrectly identify as a SMYD1-3 family. In fact, those genes belong to the SmydA group, and the true SMYD3 orthologs would be Hsal_08938 and Cflo_10149, the genes that group with human SMYD3 and fly buzidau. We have also mentioned that a D. melanogaster gene has been identified as the SMYD4 ortholog [CG14122 belongs in the Smyd4L group. The real ortholog candidate is CG1868 on the basis of phylogeny and pattern of expression.As a result of our phylogeny and classification of  example . The autortholog , but ourThese two examples highlight the pitfalls of attempting to classify genes in the absence of a rigorous phylogeny. Quite often, gene families and subfamilies are defined in function of the representatives found in vertebrate species. It is widely assumed that vertebrates represent the most complex genomes among metazoans, in other words, biomedicine quite often ignores evolution. If we want to take full advantage of the wide range of experimental models that can contribute to research in life and health sciences, we cannot afford to make such mistakes.Smyd3 and Smyd5, the Smyd4 class has suffered extensive expansion in the arthropod species. Our results seem to reflect that there has been expansion and a faster pace of divergence of arthropod genes within the Smyd4L group, so that they form separate subgroup with more than one representative; and a more dramatic event of the same type in the origin of the Smyd4I group. If we take into account the expression profiles of these genes in D. melanogaster, it is more likely that Smyd4I originates from Smyd4, since they share anatomical, developmental and temporal expression profiles.In contrast to classes Smyd4 genes begs the question of why so many of them. The patterns of expression of the four genes we investigated are highly regulated and not completely overlapping, which in principle rules out that the high copy number is just to ensure a high level of expression. Our knock-down experiments indicate that there is not functional redundancy among all of them, as down-regulation of individual genes always results in some kind of phenotype. Therefore, the most likely explanation is that LSE of the class has adaptive value in the arthropod lineage, and the different copies of Smyd4 have anatomical or molecular specialization. In a genome-wide analysis in Caenorhabditis elegans [Smyd4 genes has helped the different arthropod species to adapt their physiology to their environmental, ecological and/or ethological features. This makes sense if, as evidence from other Smyd family members seems to indicate, the major role of this type of proteins is epigenetic control of gene expression. Bonasio et al. (2010) found that among the Smyd genes in their ant species there were differences in gene expression depending on the caste and developmental stage, pointing to an important role of Smyd genes in transcriptional control. All insect species have a similar body plan, so it is to be expected that the genes controlling basic development are conserved in sequence and number. In contrast, they have a wide diversity regarding other biological features: body size, social structure, behavior, life cycle, feeding, ecology, etc. It is possible that Smyd4 genes are part of the genetic and epigenetic toolkit responsible for this variability.But this multiplicity of  elegans , phenotySMYD4, the use of Drosophila to study gene function is of relatively limited interest. The main advantage of this model organism is the range of techniques in advanced genetics, including transgenics, RNAi, somatic clones and gene editing. The genes of the Smyd4 class are not redundant on the basis of their patterns of expression and mutant phenotypes. We show that some experimental approaches would be still possible and informative; such as in vivo localization of proteins in transgenics. Human SMYD4 protein fused to GFP has a sub-cellular localization pattern in Drosophila adult muscle, in sarcomeres and nucleus, that is almost identical to murine Smyd1 [Smyd4 genes in Drosophila would be representative of a clinical phenotype without a previous validation work, dissecting the molecular functions of each one of them. Similarly, it is difficult to know a priori the interest of Drosophila to investigate Smyd3 class. It is possible that SMYD1 and SMYD2 carry out roles that are specific of vertebrates and cannot be modeled outside this group, and we don´t know if a Drosophila Smyd3 knock-out would be equivalent to a triple knock out in vertebrates of the whole class.From our results it is evident that in the case of ne Smyd1 . By direSmyd5, which seems to be more conserved in copy number and phylogenetic distribution. With some validation work, for example determination of the pattern of expression and mutant phenotype, and rescue of this phenotype with expression of human SMYD5, flies could be a good model if this gene proves to be of clinical interest. The biomedical interest of the SmydA group is probably null, but from a biological and evolutionary point of view, it would be extremely interesting to know what their relevance is in insect physiology, and whether they had an adaptive role in the diversification of this phylum.The situation is more straightforward with Smyd gene family is present in all eukaryotes, since the S. cerevisiae SET5 and SET6 proteins also contain a putative Zn finger similar to the MYND type. Metazoan Smyd genes can be grouped in three classes: Smyd3, Smyd4 and Smyd5. All three classes were present in metazoan ancestors, and at least Smyd3 and Smyd 4 are also found in plants. Smyd4 is the most variable class in animals, with large variations in copy number. Drosophila contains 4 Smyd4 genes, which are not redundant according to their expression patterns and effects of down-regulation. The phylogeny of Smyd genes should be taken into account to obtain information of biomedical interest using model organisms.The The experimental research performed in animals was approved by the Foundation Prince Felipe Research Center Ethics Committee for Animal Experimentation.−7) and continuous alignment within the core region containing the SET, MYND and post-SET domains. This search revealed proteins that were clearly homologous to one of the five query sequences, with homology beyond the SET-MYND core, and some other sequences that were only similar in this core, such as an arthropod-specific group. We performed further searches in all species with these novel sequences using the same criteria. Accession numbers of all the sequences are detailed in To build a database of Smyd proteins, we performed systematic BLASTP searches using as queries the sequences of the five human SMYD proteins. In organisms without a comprehensive protein database, additional TBLASTN searches against the genomic sequence were performed. The sequences were validated by reciprocal BLAST to check if they were most similar to the query sequence. We included in our database only those sequences with high similarity . Then, the alignment was edited with Jalview 2 [The resulting set of sequences was aligned using Clustal Omega  and the Maximum Likelihood (ML) methods, using the routines available in MEGA version 6.0 . Gaps wehttp://www.ncbi.nlm.nih.gov). For the prediction of PROSITE signatures we used the EXPASY server (http://prosite.expasy.org/); and for identification of PFAM signatures using hidden Markov models we used the HMMER platform (http://hmmer.janelia.org/search/hmmscan). Secondary structure predictions were done by using four different algorithms in the Max Plank bioinformatics server (http://toolkit.tuebingen.mpg.de/quick2_d).BLASTP and TBLASTN sequence searches were performed at the NCBI server .To compare the expression among the ion Data  and the ion Data  were dowThe anatomical expression data indicated in the graphics in The temporal expression data correspond to the following time points: embryo 0-2h, embryo 2-4h, embryo 4-6h, embryo 6-8h, embryo 8-10h, embryo 10-12h, embryo 12-14h, embryo 14-16h, embryo 16-18h, embryo 18-20h, embryo 20-22h, embryo 22-24h, larva L1, larva L2, larva L3 12 h old, larva L3 puffstage 1–2, larva L3 puffstage 3–6, larva L3 puffstage 7–9, white prepupae new, white prepupae 12hr, white prepupae (WPP) 24hr, pupae 2 day post-WPP, pupae 3 day post-WPP, pupae 4 day post-WPP, adult male 1 day, adult male 5 day, adult male 30 day, adult female 1 day, adult female 5 day, adult female 30 day.Drosophila Resource Center , CG7759 (stocks 100412 and 21052), CG8378 (stocks 40705 and 40706) and CG1868 (stocks 106709 and 25378); from this repository we also obtained UAS-Dcr2 to co-express the Dicer2 nuclease in order to increase interference. The GMR-Gal4, Mhc-Gal4, elav-Gal4 and Act5-Gal4 drivers were obtained from the Bloomington Drosophila Stock Center (http://flystocks.bio.indiana.edu/).Fly stocks were maintained at 25°C on standard corn flour medium. We obtained transgenic lines with inducible RNAi constructs from the Vienna drc.at/; ) for theAct5-Gal4 / Cyo; UAS-Dcr2 females and males of the homozygous RNAi stocks for CG14122 (stock 51783), CG7759 (stock 100412), CG8378 (stock 40706) and CG1868 (stock 106709). All the progeny carries the UAS-RNAi construct, and the numbers of flies carrying the Act5-Gal4 driver (knock-down) were compared to the number of siblings carrying the CyO chromosome (control), expecting a 1:1 segregation.For viability studies, we scored the offspring from two independent crosses of The human SMYD4 ORF was amplified by PCR from cDNA obtained from the human schwannoma cell line HEI-193 (ATCC no. PTA-4544). The primers containing restriction site adapters were:SMYD4_pEGFP_XhoI_F ggaaCTCGAGctATGGATCTGCCTGTGGATGAATGSMYD4_pEGFP_BamHI_R gcgcGGATCCAATGCAGGCCCTACAGGG)XhoI and BamHI sites of the pEGFP-N1 plasmid. The pEGFP-SMYD4 plasmid was transformed into a dam–/dcm−E. coli strain to avoid methylation of the XbaI site in the pEGFP-N1 plasmid. A SMYD4-GFP fragment was excised by digestion with XhoI and XbaI and cloned into the pUASt plasmid. The sequence of the construct was confirmed by sequencing on an ABI Prism 3130xl Genetic Analyser (Applied Biosystems). Transgenics were made by random integration method. Injections were performed by BestGene Inc. , and the selection of the transformants was done by us.The PCR product was digested and cloned between the Drosophila and mouse embryos, exon-containing gene regions were amplified using specific primers in polymerase chain reaction. As a template for amplification, we used genomic DNA of the y w strain for the Drosophila probes, and adult mouse brain cDNA for the mouse probe. The primer sequences were the following:To construct the plasmids used to synthesize the antisense RNA probes for in situ hybridization to CG14122-F TTGCGTGGACGTGCGTGATGCCG14122-R TGTCCTTGTGAGGCAGTGCAGCCG1868-F AACCTTCAAGTCATTGGCATCCCG1868-R TGCTCGCTAGCCAGATAATCCCG7759-F TGCAAGAGCGATGAGGAGCGCG7759-R AATCCTTGAGATGCTGTTGGCCG8378-F TGCAGGACTGGAAACTAATCGCG8378-R ATAGTCGTCACCGTATTGCGSmyd4-F AGTGCCTGAAGCTCTGAGTGCCSmyd4-R TGCCTGCAGATCAGTGACAACCDrosophila embryos of the Oregon-R strain, and in 15 micron cryostat sections of E14.5 mouse embryos of the C57BL/6J genotype.The PCR products were cloned into the pCR2.1-TOPO following the manufacturer’s protocol (Invitrogen). The vectors were linearized and used to generate a digoxigenin-labelled RNA anti-sense probe using the DIG RNA labeling kit (Roche) according to the manufacturer's directions. In situ hybridization was performed in whole mount Heads and thoraxes from adult flies were dissected and fixed in 4% paraformaldehyde in phosphate buffer saline (pH7.2) overnight at 4°C, followed by cryoprotection with 30% sucrose for 48 h at 4°C (heads were first pre-incubated with 10% sucrose for 2 h). Then they were embedded in OCT compound and cut in the cryostat (16 μm for thoraxes and 14 μm for heads). Cryosections were washed in PBT, blocked (PBS containing 5% BSA and 0.3% Triton X-100) for 45 minutes at room temperature and incubated with anti-GFP (Rockland) overnight at 4°C. After washes with PBT, the tissue was incubated with secondary antibodies. After washes with PBT, cryosections, the tissues were incubated with phalloidin-rhodamine diluted in PBT and then with DAPI. Sections were mounted with Aqua Poly/mount . Images were obtained with a Leica SP8 microscope.Scanning electron microscopy analysis of adult eyes was performed following the critical point drying method . BrieflyS1 FigMaximum likelihood and neighbor joining trees were constructed from the Clustal Omega alignment. The main branches are colored according to the classes shown in (TIF)Click here for additional data file.S2 FigSmyd4 class: Smyd4, Smyd4L and Smyd4I.These trees support the three main groups in the (TIF)Click here for additional data file.S3 FigA. thaliana and S. cerevisiae (highlighted in bold type).In addition to the sequences in (TIF)Click here for additional data file.S4 FigSecondary structures are coded blue E for β-sheet and red H for α-helix. The secondary structure for SMYD1 determined by X-ray crystallography is indicated at the top. For the putative Zn finger regions from the sequences in (TIF)Click here for additional data file.S5 FigL. gigantea 232186 and B. floridae 74594. The first one has high homology hits to its corresponding locus, to a downstream locus that seems to be a duplication comprising the last exon, and to a second locus in a different genomic scaffold which has not been annotated. The second one also has hits corresponding to its own locus and an additional one in a different scaffold. In both cases the second copy has the same putative intron/exon structure as the annotated gene. The images are screen captures of the results obtained in the NCBI server.TBLASTN searches were performed against the genomic contigs using as queries the protein sequences of (TIF)Click here for additional data file.S6 FigAt the top we show the sequence of the Zn fingers of all the proteins within each of the four classes. Two of the SmydA proteins have a second MYND Zn finger outside the Smyd core. At the bottom, we compare these sequences with the PROSITE consensus for the MYND domain, and shade those regions that depart from this consensus.(TIF)Click here for additional data file.S7 Figelav-Gal4  and in muscle under the control of Mhc-Gal4 (B-B”). In neurons, SMYD4-GFP is predominantly cytoplasmic (A), as revealed by lack of co-localisation with the nuclear stain DAPI (B). In muscle, SMYD4 co-localizes with the myofibrils  and is also present in the nuceus . Within the sarcomere, it is more abundant in the M lines (arrow) and weaker in the Z lines (arrowhead).The fusion protein was expressed in neurons under the control of (TIF)Click here for additional data file.S1 Filehttp://www.ebi.ac.uk/Tools/msa/mview/) and downloaded as an html file.The FASTA alignment was formatted in the mview server ((HTM)Click here for additional data file.S2 FileFormatted in mview.(HTM)Click here for additional data file.S3 FileFormatted in mview.(HTM)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammatory disorders, which require long term treatment to achieve remission and to prevent relapses and cancer. While current therapies are effective in most cases, they can have rare but serious side effects and are often associated with high costs. On the other hand, early discontinuation of an effective treatment may lead to a quick relapse and to complications at the restart of therapy. Therefore it is essential to determine the optimal duration of maintenance therapy, but clear guidelines are missing. The most important questions when deciding whether to continue or withdraw therapy in quiescent UC and CD patients are the efficacy of the continuous treatment to maintain remission in the long term, the frequency and severity of side effects, and the chance of relapse after discontinuation of therapy. This review summarizes the current knowledge on these topics with respect to 5-aminosalicylates, thiopurines, methotrexate, and biological therapies and collects information regarding when and in which specific patient groups, in the absence of risk factors, can withdrawal of therapy be considered without a high risk of relapse. Additionally, the particular aspect of colorectal cancer prevention by current therapies will also be discussed. Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), are chronic, immune-mediated disorders of the gastrointestinal tract. In Western countries the annual incidence rates can be as high as 6.3/100.000 for CD and 9.8/100.000 for UC, and the incidence in developing countries is also increasing . Pa. Pa11]. Whether continuous therapy can be replaced by intermittent treatment is also debatable. Fifty UC patients in remission for at least 1 month were enrolled in a 12-month clinical trial performed in Italy . Half ofEarly papers have already described side effects of sulphasalazine, the most frequent being gastrointestinal symptoms such as nausea, vomiting, or epigastric discomfort, which affect up to 45% percent of patients , 3. Cont6-Mercaptopurine and azathioprine are antimetabolite prodrugs, belonging to the group of thiopurines. It has been discovered early that these drugs work with a time lag of several months in IBD patients , and theCompared to aminosalicylates, more attempts have been made to clarify if thiopurines can be withdrawn in UC and CD patients in remission, mostly because the fear from their possible serious side effects makes physicians reluctant to prescribe these medications in the long term.The only placebo-controlled, double-blind withdrawal trial on UC patients was conducted in 1992  12]. Pa. Pa12]. The further available studies assessing the effect of thiopurine withdrawal in UC were retrospective, evaluating the outcome in patients, who stopped thiopurine treatment while in remission . A studyThe previously mentioned study from Oxford evaluated not only the long term efficacy but also the relapse rates in 222 IBD (143 UC and 79 CD) patients after stopping azathioprine treatment while being in remission  26]. No. No26]. In a study conducted in Italy, relapse rates after azathioprine withdrawal in steroid-free remission were high, similarly to previous studies  27]. In. In27]. In CD, the picture is more complex. An early double-blind withdrawal trial showed the efficacy of azathioprine maintenance therapy compared to placebo, irrespective of complementer anti-inflammatory drug use  13]. On. On13]. The efficacy of thiopurines in the long term was also confirmed by a follow-up study on 120 CD patients  28]. Th. Th28]. A retrospective study in France on CD patients showed no further benefit during and after the fifth year of remission in patients who continued to take azathioprine or 6-mercaptopurine compared to those who had stopped it  16]. Th. Th16]. A small randomized open withdrawal trial on CD patients in stable remission without steroids have confirmed the efficacy and need to continue azathioprine at least for 3 years, but did not give information on the previously questioned efficacy beyond 4 years  14]..14].A multicenter European retrospective study on UC and CD patients has shown that, in the first 4 years, thiopurines were equally efficient in the two diseases  29]. Lo. Lo29]. So far, the opinion that thiopurine therapy may lose its benefit after 4 years of treatment in CD patients was based only on retrospective results , 29 and The extension of this study was published four years later . All patA further study aimed to assess, whether the effect of azathioprine wears off after 4 years in CD patients . Namely,It is interesting to note that none of the above-mentioned studies have evaluated 6-thioguanine levels at the time of therapy withdrawal and the risk of relapse. 6-thioguanine nucleotides are the active metabolites of azathioprine, and their higher level in red blood cells in CD and UC patients is associated with an increased rate of steroid-free remission .5 to 10% of patients do not tolerate thiopurines due to idiosyncratic adverse events, most commonly fever, nausea, diarrhea, rash, abdominal pain, pancreatitis, and allergic reactions that mostly occur within the first 2-3 weeks of therapy , 54. MyeMethotrexate is generally reserved for CD patients with active or relapsing disease, who are refractory to or intolerant of thiopurines or anti-TNF agents . A recenAlthough some studies have demonstrated beneficial effects of methotrexate in the maintenance of remission in UC, contradictory results also exist . DiffereThere is very little data on discontinuation of methotrexate therapy in IBD patients on remission, as specific withdrawal studies are missing. A previously mentioned retrospective study reported an extremely high relapse rate after discontinuation of therapy  19]..19].Safety concerns of methotrexate have a large impact on its limited use in IBD patients. Studies report the incidence of side effects rising up to 22%, which lead to a discontinuation of treatment in 10–18% of cases . Short tα), and adalimumab, a fully human monoclonal IgG1 anti-TNF-α antibody, were both found effective as a maintenance therapy in UC and CD . 25% have stAccording to the Hungarian National Health Insurance Fund Administration reimbursement regulations, biological therapy must be discontinued after a 1-year treatment period in UC and CD patients who achieved remission. These regulations have created the basis to observe the effects of infliximab and adalimumab withdrawal in IBD patients , 24. In A study conducted in Spain has collected clinical data from three centers of the outcome after discontinuation of infliximab therapy in CD patients  20]. Th. Th20]. In a large observational study evaluating the long term effects of infliximab therapy in CD, treatment was stopped in 110 patients in remission  21]. Th. Th21]. In a multicenter prospective study called STORI, 115 CD patients were included, who were treated for at least 1 year with infliximab and an antimetabolite (methotrexate/azathioprine/6-mercaptopurine), and have been in corticosteroid-free remission for at least 6 months before inclusion  22]. Th. Th22]. n = 87) or adalimumab (n = 34)   23]. Si. Sin = 8α therapy, but the followup was only one year, and therefore no conclusions can be drawn concerning the long term effects [α treatment is demyelinating disease, which usually mimics multiple sclerosis [α agents, 6 cases of demyelinating disease were identified [Although infliximab and adalimumab are considered safe in the short term, many concerns exist regarding their long term use. A large case-control study with a mean followup of 1.9 years has shown no difference between the mortality rate of infliximab-treated and noninfliximab treated CD patients . Similar effects . In the  effects . At this effects . A furthclerosis , 83. In entified . FurtherIt is an important question whether a long remission while on therapy can predict a sustained remission after drug withdrawal. Results are controversial, and many of them point to the direction that the duration of previous remission does not predict the outcome after drug withdrawal . Time toMucosal healing is defined as the “complete absence of all inflammatory and ulcerative lesions in all segments of gut” at endoscopy . SeveralAs mucosal healing is a novel entity, its connection with a prolonged remission in earlier drug withdrawal studies could not been tested. In the study of Dissanayake and Truelove a sigmoidoscopy with rectal biopsy was performed at enrollment in the study, in case of a suspected relapse and at the end of the 6-month study period . Only paSimilarly, in case of thiopurines, no studies were designed specifically to answer if there is a link between the endoscopic-histological picture at therapy withdrawal and the risk of relapse. In the study of Hawthorne et al. a sigmoidoscopy was performed at the inclusion, and only patients with a mucosal appearance of grade 0 or 1  were eligible for the study . Howeverα therapy withdrawal in 41 CD and 22 UC patients  in UC has long been recognized. An early study performed on UC patients, who grew up in the era when effective treatment for IBD hardly existed, found a 20% risk of colon cancer per decade in children at risk, starting after 10 years of disease onset . It has α therapy has also been found protective against CRC [Several data support the hypothesis that the long term use of 5-ASA decreases the risk of CRC in UC, although the data are heterogeneous due to methodological differences . The ECCinst CRC . AltogetIt is well known from the clinical practice that the compliance of patients is rapidly decreasing after they have achieved remission, and if physicians are reluctant to reduce the number of tablets, the patients tend to do it themselves. Therefore it is essential to identify those patient groups where the maintenance therapy can be discontinued without a high risk of relapse.In case of 5-aminosalicylic compounds, many authors are unanimous in the opinion that 5-ASA should be continued lifelong in UC patients in remission unless side effects are present , 34. NevRegarding methotrexate, further studies on large number of patients are needed to determine the optimal therapy duration in IBD, but it seems that relapse rates after therapy withdrawal are outstandingly high.In case of azathioprine, for those patients who did not achieve remission within 6 months, there is probably no benefit in taking it . HoweverThese factors can be grouped in different categories shown in The ECCO states that, in case of CD patients in remission on azathioprine, “cessation may be considered after four years of remission. Benefit and risks of continuing azathioprine should be discussed with individual patients.”  In the sConsidering biological treatment, financial factors often influence the decision on the continuation or withdrawal of therapy. The introduction of biosimilars in the market will possibly reduce the present high costs and this may also be reflected in therapeutic decisions. Until clear guidelines for the optimal duration of therapy can be formulated, the opinions of physicians remain quite discrepant. In a questionnaire filled out by Canadian gastroenterologists, 77% would prefer to continue infliximab treatment indefinitely, if it is well tolerated and effective, while 12% opted to discontinue it after 1 year, and 2% would use it for a maximum of 6 months . One musAfter a relapse following a therapy withdrawal, the required treatment highly depends on the reason of the discontinuation of the drug. If the drug was discontinued due to side effects or intolerance, then new therapeutic solutions have to be found. Nevertheless, if the drug was well tolerated, the most obvious solution is the reintroduction of the previously efficacious therapy. Results from studies on azathioprine or anti-TNF medications show mostly excellent results with reintroduction of therapy. In the study of Treton and colleagues, 23 of the 32 relapsed patients were retreated with azathioprine alone or in combination with steroids, and among them, 22 have successfully achieved remission . The onlα within one year, which has successfully induced clinical remission in 54.7% of patients, while 9.1% underwent surgery [In case of anti-TNF treatment, many data are available concerning retreatment after withdrawal. In the study of Kim et al., out of 36 relapsing CD patients, 25 were retreated with infliximab, and 24 patients (96%) reached complete clinical remission . One pat surgery . In caseKnowledge on the outcome after withdrawal of different medical therapies in IBD patients who have successfully achieved remission is constantly growing but is still insufficient to create universal guidelines. As a final conclusion, the relapse rates after withdrawal of a well-established therapy in IBD patients are generally high, but in case of specific patients, the discontinuation of therapy can be considered. This decision must be made on an individual basis by the help of the predictive risk factors detailed above and must be discussed with the individual patient, after providing all the necessary information on the risks of the cessation and the continuation of therapy. More, well-designed prospective studies are needed to answer all the remaining questions concerning the optimal duration of therapy in IBD patients in remission.