MITF) and paired-box 3 (PAX3) are at the top of the cascade and regulate activities of many important melanogenic enzymes. Here, we report for the first time that cyclin-dependent kinase 5 (Cdk5) is an essential regulator of MITF and PAX3. Cdk5 knockdown in mice causes a lightened coat color, a polarized distribution of melanin and hyperproliferation of basal keratinocytes. Reduced expression of Keratin 10 (K10) resulting from Cdk5 knockdown may be responsible for an abnormal epidermal structure. In contrast, overexpression of Cdk5 in sheep (Ovis aries) only produces brown patches on a white background, with no other observable abnormalities. Collectively, our findings show that Cdk5 has an important functional role in the regulation of melanin production and transportation and in normal development of the integumentary system.The mammalian integumentary system plays important roles in body homeostasis, and dysfunction of melanogenesis or epidermal development may lead to a variety of skin diseases, including melanoma. Skin pigmentation in humans and coat color in fleece-producing animals are regulated by many genes. Among them, microphthalmia-associated transcription factor ( The skin serves as the first line of defense of the body and provides temperature regulation and some aspects of metabolism. Consequently, the color, thickness and secretions of the skin determine its ability to protect an individual from environmental and biological insults, such as ultraviolet radiation (UVR) and bacteria. Developmental defects and dysfunctions of the integumentary system are invariably associated with abnormal human and animal pathologies. Cutaneous melanoma, which arises from normal melanocytes or their precursors, is the most common form of skin cancer3, but the mechanism regulating epidermal development and skin pigmentation is still not fully understood.As the largest organ of the body, the integumentary system comprises the skin, hair, nails, and exocrine glands and plays multiple roles in homeostasis2. At the cellular level, melanocytes are functionally associated with fibroblasts and keratinocytes via dendrites to regulate the function and phenotype of the skin3. Melanin synthesis is restricted to melanocytes, where it plays a major role in antioxidant defense mechanisms6. In addition, it can also promote skin resistance to different types of cancer therapy, including chemotherapy or radiotherapy, in patients with metastatic melanoma6. Autocrine and paracrine regulation of melanin synthesis have been established7. For example, L-tyrosine and L-DOPA not only directly regulate melanogenesis but also indirectly mediate the process through specific receptor- or non-receptor-mediated processes8.Pigmentation of the skin, hair, and eyes depends primarily on the synthesis and distribution of melanin that is produced by melanocytes9. Although protein kinase C (PKC) plays a regulatory role in melanogenesis, cAMP is the most important biochemical regulator. Melanocortin receptor type 1 (MC1R) is one of seven transmembrane G protein-coupled receptors involved in regulation of melanogenic activity. In melanocytes, it can regulate melanogenesis, cell proliferation and survival8. It is activated upon binding of MSH or ACTH peptides (products of POMC processing) as a result of intracellular cAMP accumulation10. Among the transcriptional regulators, microphthalmia-associated transcription factor (MITF) and paired-box 3 (PAX3) are two major regulators of these processes14. MITF represents a melanocyte-specific communication hub that integrates intracellular signals and transcriptional responses in regulating cell differentiation, proliferation and survival15. MITF and PAX3 regulate the transcription of genes for three major pigmentation enzymes, namely, tyrosinase (TYR), tyrosine related protein-1 (TYRP1), and tyrosine related protein-2 )19. Furthermore, PAX3 regulates melanocyte specification, maintenance of the undifferentiated state, proliferation, and migration during embryonic development20. PAX3 directly interacts with the SRY-related HMG-box (Sox10) and can activate MITF expression in a synergistic manner21 while simultaneously competing with MITF for occupancy of an enhancer required for the expression of TYRP222. In addition, our previous investigations have identified approximately 105 miRNAs that are possibly involved in the regulation of coat color genes23. A further study showed that overexpression of miR-137 in transgenic mice results in altered coat color, thereby validating the functional role of miR-137 on MITF gene expression and indicating the complexity of the regulation mechanisms for melanogenesis and coat color gene expression post-transcriptionally24.At the molecular level, more than 125 genes have been identified as being involved in the regulation of melanocyte development and melanogenesis at different pathways, which act at transcriptional, translational, and posttranslational levels25 and two small RNA libraries from white and brown alpaca skin23. An analysis of the transcriptome profile of alpaca skin with different hair colors has revealed that cyclin-dependent kinase 5 (Cdk5) is a candidate gene for alpaca fleece quality, coat color and fiber growth and development25. Indeed, a subsequent study showed the localization of the Cdk5 protein to hair follicles, with higher expression levels in animals with a brown versus white fleece color26.Taking advantage of 22 natural coat-color variations in alpacas, we have constructed one cDNA library from white alpaca skin28. During neurulation in early development, NC stem cells arise from the dorsal neural tube and migrate throughout the body, where they give rise to critical components of numerous cell types, including melanocytes29. In melanocyte development, MITF is extensively involved and provides a central link between transcription factors and signaling pathways29. Despite the ubiquitous distribution of Cdk5 and its substrates30, Cdk5 activity has been mainly detected in the nervous system, where it is most abundantly expressed31. However, several recent studies have suggested that Cdk5 also plays a functional role outside of the nervous system, such as in muscle cells32, the lymphatic system33, the kidney34 and gonadal cells35. A functional role for Cdk5 in melanogenesis and epidermal development is still lacking.Cdks are serine/threonine kinases that are activated by binding to regulatory cyclins that play a key regulatory role in cell cycle progression. However, Cdk5 is unique and does not function as a cell cycle regulator. Cdk5 is best known for its role in post-mitotic processes and functions in the developing nervous system from the neural crest (NC)Cdk5 knockdown mice and Cdk5-overexpressing sheep to further confirm the functional role of Cdk5 on melanogenesis and epidermal development in vivo.In the present study, we first investigated the mechanism of action of Cdk5 in melanogenesis by determining the effect of Cdk5 on the expression levels of key enzymes in the melanogenesis pathway in cultured alpaca melanocytes. We then generated Cdk5 was induced in melanocytes by transfection of the pVAX1-Cdk5 vector. Induced overexpression of the Cdk5 gene in cultured melanocytes resulted in an increase in the expression level of Cdk5 mRNA , the ability of Cdk5 to interact with p35 was examined by co-immunoprecipitation (co-IP) experiments in the melanocytes. Using anti-Cdk5 and anti-p35 antibodies, the co-IP and reciprocal co-IP experiments showed that Cdk5 interacts with p35 in the cultured melanocytes  values of ≤0.01 and an RPKM (Reads Per kb transcriptome per Million reads) ratio of >2 were used in the analysis. The expression levels of the mRNA and proteins of a number of important hair color genes, namely, MITF, PAX3, MC1R, TYRP1, and TYRP2, were down-regulated in the Cdk5 knockdown mice  was thickened, due to more stratification, especially in the stratum spinosum, and the cells of all strata were enlarged  was identified as one of the targets that interacted with Cdk5. The interaction between the two proteins was confirmed with the co-IP experiments  , the epidermis of theged Fig. . Fontanaged Fig. . To undents Fig. . Consequ01) Fig. .Figure 6The polarized distribution pattern of melanin in the epidermis led to an investigation of the expression of membrane-associated transporter protein (MATP) in the skin. As shown in Fig. Due to the low efficiency of producing knockdown mice by microinjection of mouse zygotes, we chose to use testis germ cell transplantation to produce transgenic rams and offspring. The transplantation of 3 Dorset rams with testis germ cells transfected with a pLenti-Cdk5 construct resulted in all three producing transgenic sperm 41, mitogen-activated protein kinase (MAPK)42 and Wnt signaling pathways43. This study identified p35 as an activator of Cdk5 in melanocytes.The coat color of mammals is dictated by the balance of two kinds of melanins, namely, eumelanin and pheomelanin, which are produced in melanocytes45. PMEL fibrils have an amyloidogenic nature and share features with pathological amyloids46. The expression level of gp100 was reduced in the skin of the Cdk5 knockdown mice. Amyloid fibrils are formed by various proteins in the pathologies of different neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases47. Because melanogenesis can affect the immune responses of the skin to the different chemotherapies and radiotherapies used to treat melanomas48, several drugs, including BRAF, KIT and MEK inhibitors, are currently being evaluated with promising results49. Taken together, with the activation by p35, Cdk5 regulates melanin production via the control of MC1R and TYR expression both at transcriptional and translational levels in melanocytes. Therefore, it is reasonable to speculate that Cdk5 can become a therapeutic target for melanoma and even for neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases.Eumelanin is mostly synthesized in eumelanosomes, which have a characteristic ellipsoidal shape due to the accumulation of intraluminal fibrils (referred to as “the melanosome matrix”) and arise from the proteolytic processing of the premelanosomal protein in vivo knockdown of Cdk5 expression inhibits melanin production suggests that Cdk5 is a promising target gene for the production of various coat colors in fleece- producing species. In addition, the localized distribution of melanin to the upper dermal papilla in the hair follicles of knockdown mice is another important factor for the altered coat color. The transportation of melanin along the dendrites of melanocytes is responsible, at least partially, for the lightened coat color from the original black background. Indeed, the reduced expression of MATP in the skin of the knockdown mice suggests that Cdk5 regulates MATP expression in the hair follicles. With the prediction using RNA-Seq, we have also shown that Cdk5 regulates the expression of a master gene of melanocyte development and melanogenesis, MITF, in the skin of transgenic mice. However, based on our current results, we are unable to confirm whether the functional role of Cdk5 on MC1R, TYRP1, TYRP2 and PAX3 expression occurs via regulation of MITF or whether Cdk5 can also directly influence the expression of these important downstream enzymes of melanogenesis. The downstream target of Cdk5 in this process may be PAX3, which has a binding site in the MITF promoter sequence that is known to be critical for transcriptional activation of MITF50. Indeed, the expression for PAX3 in the skin of Cdk5 knockdown mice was significantly reduced at both the mRNA and protein levels. This pathway is followed during melanogenesis. Cdk5 knockdown induced a bright brown lightening of the new hair shafts that are produced during anagen, without inducing an ESR-detectable switch from eumelanogenesis to pheomelanogenesis. This phenomenon has also been reported as an effect of the oral zinc cations in C57BL/6 mice51. With the relative change in expression of TYRP1 and TYRP2, it is possible that eumelanin production in the hair of the Cdk5 knockdown mice was the main factor in the coat color phenotype change, although MITF expression was also decreased. This conclusion supports the hypothesis that MITF is not the only transcription factor for melanogenesis15. However, the ratio of the relative expression of TYRP2 and TYRP1 cannot only affect the actual proportion of eumelanin/pheomelanin but also influence DHICA/DHI in eumelanin. Thus, hair color may be variable due to the changes in either the quantity or type of the main eumelanin monomer51. Moreover, we also found that expression of another receptor, melanocortin (MC) MC4R, was decreased in the skin of the Cdk5 knockdown mice. It has been reported that MC4R in murine skin is tonically regulated by α-melanocyte stimulating hormone (α-MSH)52. Investigations of the regulation of Cdk5 on MC4R are underway.The finding that Cdk5 knockdown mice. Using co-IP followed by mass spectrometry, we identified K10 as one of the substrates of CDK5. In the skin of the Cdk5 knockdown mice, K10 was expressed significantly less than in the wild-type mice. It has been reported that the epidermis of adult Keratin 10 (K10)−/− mice displayed hyperproliferation of basal keratinocytes and an increased cell size53. Furthermore, reduction of Cdk5 activity via an inhibitor (roscovitine) in HaCaT cells resulted in reduced adhesion of the cells to fibronectin, which was associated with reduced active states of β1 integrin54. In addition, the hair cycle determines skin thickness, which is highest during anagen VI and lowest during telogen in mice55. The results of the Western blot analysis showed that the expression level of transforming growth factor-βRII (TGF- βRII) was higher in the skin of the Cdk5 knockdown mice, whereas the expression of interleukin (IL) -1RI (IL-1RI) was lower in comparison with the wild-type mice  in Cdk5 knockdown animals if they were allowed to live longer, but further investigation is required to validate this hypothesis.A striking finding of this study was the observation of abnormal epidermal structure in the Cdk5 knockdown mice, lambs with skin-specific overexpression of Cdk5 did not show any abnormalities. However, unlike the knockdown mice where the coat color changed evenly over the body, the transgenic lamb (#6281) only had a brown patch on the body and around one eye where a higher level of green fluorescence was observed. Although all the cells in the transgenic sheep were supposed to contain the transgene, the expression levels of ectopic Cdk5 might have been different due to epigenetic factors. We suspect that a phenotypic change in the skin color can only be observed when the expression of Cdk5 exceeds certain limits. Similarly, a transgenic sheep carrying a transgene for the overexpression of the solute carrier family 7 member 11 (SLC7A11) gene, which is implicated in pheomelanin production, also exhibited brown/yellow patches in a white background56. These results may imply that coat-color determination in sheep is more complex than in rodents and is controlled by other novel genes. Indeed, several such genes have been identified in sheep skin, two of which were highly abundant and exclusively expressed in black sheep57. Furthermore, our previous study of the skin transcriptome profiles associated with coat color in sheep revealed that Cdk5 was not included in the top 30 highly expressed genes. It is puzzling for us to find that the mRNA and protein expression levels of MITF and TYRP2 were significantly reduced in brown versus white skin in the transgenic lambs since our current knowledge suggests that MITF and TYRP2 increase melanin production in melanocytes7. However, our studies and those of others have previously shown differential expression of Endothelin3 (Edn3) in white and black skin of Friesian sheep58 and color markings in domestic cats59, suggesting that paracrine expression of Edn3 might coordinate localized color differences. Furthermore, the melanin content in the brown fleece was higher than in the white fleece, implying potentially different pathways for regulating melanin production and coat-color determination in sheep. This also confirms that coat-color determination in sheep is more complex and controlled through autocrine, paracrine and endocrine mechanisms.In contrast to Cdk5 as a promising target gene for the generation of transgenic fleece-producing species.In summary, our results 1) demonstrate that Cdk5 is essential for normal epidermal development and melanogenesis, 2) highlight the involvement of Cdk5 in the pathogenesis of skin diseases and the clinical relevance of pharmaceutical correction of Cdk5, and 3) suggest The housing and care of animals and the collection of skin samples for use in the indicated experiments were approved by the Animal Ethics Committee of Shanxi Agricultural University , and all experiments were performed in accordance with the relevant guidelines ([2010]088).Cdk5 coding sequence (CDS) was amplified by RT-PCR  and incubated at room temperature for 20 min to form DNA-liposome complexes. Eight hundred microliters of serum-free medium plus the pVAX1-Cdk5 DNA/liposome complexes was added to the cultured melanocytes and incubated at 37 °C for 3 days for use.A total of 1 × 10For each siRNA transfection, 6 μl of siRNA was added to 100 μl of the transfection medium  followed by addition of 6 μl of the siRNA transfection reagent (Santa Cruz Biotechnology). Melanocytes were seeded, cultured and transfected with the siRNAs and incubated for 6 h at 37 °C. The melanocytes were incubated for an additional 24 h for use.Total RNA was isolated using TRIzol reagent (Invitrogen). One microgram of total RNA per sample was reverse transcribed to cDNA. qRT-PCR analysis of mRNA abundance for genes was conducted using SYBR Green .Cells for immunocytochemical analysis were rinsed briefly in PBS for 5 min, fixed in 4% paraformaldehyde for 20 min at 4 °C. Cells were then incubated with primary antibodies for 1 h at 37 °C at the following dilutions: polyclonal rabbit anti-Cdk5 antibody , rabbit anti-MC1R  and rabbit anti-TYR . After incubation with appropriate secondary antibodies, images were obtained using a confocal microscope .The 4-µm paraffin skin sections were incubated in 3% hydrogen peroxide at room temperature for 30 min. After washing with PBS, the sections were blocked with 5% BSA at 37 °C for 30 min, followed by incubation with the anti-Cdk5 antibody , anti-MATP antibody  or anti-K10 antibody  at 4 °C for 14 h. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was used as the secondary antibody, and DAB was used as the chromogenic substrate.24 with the following primary antibodies: polyclonal rabbit anti-TYR antibody , polyclonal rabbit anti-β-actin antibody , polyclonal rabbit anti-K10 antibody , rabbit anti-TYRP1 , mouse anti-MITF , rabbit anti-PAX3 , rabbit anti-MC1R , polyclonal rabbit anti-Cdk5 antibody , rabbit anti-gp100  and polyclonal rabbit anti-MC4R antibody , rabbit anti-IL1R , rabbit anti-NASM antibody  and rabbit TGFβIIR . Immunoblots were scanned on a ChemiDOCTM XRS + imager , and protein levels were quantified using Image-Pro Plus software .Western blot analysis was performed as describedMelanocytes were lysed in lysis buffer, and anti-Cdk5 primary antibody was added to 200 μl of the cell lysate followed by incubation overnight at 4 °C. Twenty microliters of protein G agarose beads (50% bead slurry) was then added to the mixture and incubated for 2 h at 4 °C and then centrifuged at 3,000 g for 3 min to get the pellets, which were resuspended, heated at 95 °C for 5 min and loaded onto an SDS-PAGE gel (12–15% gradient). Western blot was performed using an anti-p35 antibody, anti-K10 antibody or anti-Cdk5 antibody. Reciprocal co-IP was performed using an anti-p35 antibody or anti-K10 antibody to co-precipitate Cdk5.http://www.ebi.ac.uk/IPI) was used for protein identification.Total proteins from mouse melanocytes and 30 µl of protein-A/G agar beads were mixed. The mixture was placed at 4 °C for 1 h and then centrifuged at 3,000 g for 3 min. The supernatant was obtained and mixed with an anti-Cdk5 antibody. After incubation overnight, 50 µl of the protein-A/G agar beads were added, and the mixture was incubated for 2 h followed by centrifugation at 3,000 g for 3 min. The precipitated proteins were then analyzed by LC-ESI-Q-TOF mass spectrometry. The International Protein Index (IPI) protein database  at a concentration of 3 ng/µl, it was microinjected into the male pronuclei of the zygotes of C57BL/6 J mice. Injected embryos were transferred into the oviducts of the pseudopregnant recipient females. The transgenic lines were maintained by backcrossing with C57BL/6 J mice, which were used as the control with a stable genetic background. Genotyping was performed by PCR using tail DNA as the templates.After 6) were transferred to T250 flacks and cultured at 37 °C in 5% CO2 overnight. Within 2 h, 150–200 × 106 cells in 5 ml DMEM/12 medium with 5% fetal bovine serum were transplanted into the rete testis of each testicle of the previously irradiated rams. After 13 weeks, the semen was collected from each recipient and genotyped for the transgene. Each of the Cdk5-positive rams was mated to 10 ewes. After the lambs were born, DNA was isolated from the skin biopsies, and the transgene was amplified by PCR using specific primers  was isolated for deep sequencing. The short cDNA fragments were produced and purified using the QiaQuick PCR extraction kit , and cDNA libraries were constructed after the fragment ends were repaired and tailed followed by ligation to sequencing adapters. Sequencing of the libraries was performed using the Illumina HiSeq™ 2000. A rigorous algorithm (http://www.genetics.cn) was used to identify differentially expressed genes in the skin between the Cdk5 knockdown and wild-type mice.Total RNA from the skin of the 60.Melanocytes were trypsinized, centrifuged at 10,000 g for 10 min and solubilized in 1 M NaOH. Hair was washed in mild detergent and then hydrolyzed in 1 M NaOH at 85 °C for 4 h. The concentrations of the alkali-soluble melanins (pheomelanins and some eumelanins) in the cell lysates or hair were calculated from spectrophotometric absorbance measurements at 475 nm, using a standard curve that was prepared with serial dilutions of a Sepia melanin suspension (1 mg/ml). Forty-milligrams of mice hair were collected, washed in xylene and dried at room temperature. Hair samples were placed into the glass tubes, and ESR signals were recorded using an ESR spectrometerDifferences in the mRNA, protein and melanin production abundances between the treatment groups (n = 3 replicates) were determined by analysis of variance using SPSS 11.5 software.Supplemental Info


Amaranthus caudatus and serves as a model for the family of amaranthin-like lectins. Though these lectins have been purified and characterized only from plant species belonging to the Amaranthaceae, evidence accumulated in recent years suggests that sequences containing amaranthin domains are widely distributed in plants. In this study, 84 plant genomes have been screened to investigate the distribution of amaranthin domains. A total of 265 sequences with amaranthin domains were retrieved from 34 plant genomes. Within this group of amaranthin homologs, 22 different domain architectures can be distinguished. The most common domain combination consists of two amaranthin domains followed by a domain with sequence similarity to aerolysin. The latter protein belongs to the group of β-pore-forming toxins produced by bacteria such as Aeromonas sp. and exerts its toxicity by making transmembrane pores in the target membrane, as such facilitating bacterial invasion. In addition, amaranthin domains also occur in association with five other protein domains, including the fascin domain, the alpha/beta hydrolase domain, the TRAF-like domain, the B box type zinc finger domain and the Bet v1 domain. All 16 amaranthin-like proteins retrieved from the cucumber genome possess a similar domain architecture consisting of two amaranthin domains linked to one aerolysin domain. Based on phylogenetic differences, four sequences were selected for further investigation. Subcellular localization studies revealed that the amaranthin-like proteins from cucumber reside in the cytoplasm and/or the nucleus. Analyses using qPCR showed that the transcript levels for the amaranthin-like sequences are typically low and expression levels vary among tissues during the development of cucumber plants. Furthermore, the expression of amaranthin-like genes is enhanced after different abiotic stresses, suggesting that these amaranthin-like proteins play a role in the stress response. Finally, molecular modeling was performed to unravel the structure of amaranthin-like proteins and their carbohydrate-binding sites. This study provided valuable information on the distribution, phylogenetic relationships, and possible biological roles of amaranthin-like proteins in plants.Amaranthin is a homodimeric lectin that was first discovered in the seeds of  Lectins are proteins with one or more non-catalytic domains that can recognize and bind reversibly with specific mono- or oligosaccharides . They arAmaranthus caudatus and represents a 66 kDa homodimeric protein further referred to as amaranthin. The three-dimensional structure of the A. caudatus agglutinin was resolved and revealed two carbohydrate-binding sites located at the interface of the two subunits (The first lectin from the amaranthin family was purified from the seeds of subunits . In receCucumis sativus L.) genome  fusion proteins allowed to study the subcellular localization for these AAT-like proteins in the cell. The transcript levels for these AAT-like genes were evaluated in several tissues during cucumber development under normal growth conditions as well as in the presence of several abiotic stresses or hormone treatments. In addition, the phylogenetic relationships between these AAT-like genes and other aerolysin-like genes were analyzed. Finally, molecular modeling was performed to give insight into the structure of amaranthin-like proteins and their possible carbohydrate-binding properties.The protein sequence for amaranthin (GenBank: AAL05954) was used to perform BLASTp searches against different plant genome databases   .The tandem arrayed amaranthin (AA) domain sequences from all identified amaranthin-like proteins in different plant genomes were used for the phylogenetic analysis of AA domains. Similarly, sequences encoding the toxin (T) domains were used for the phylogenetic analysis of aerolysin domains (Pfam: PF01117). Aerolysin domains from different cucumber AAT proteins as well as from proteins with similar domain architectures were included in the analysis: Hfr-2 (GenBank: AAW48295) and Dln1 (PDB: 4ZNR_A) from the Pfam database; aerolysin (GI: 113485), LSLa (GI:241161788), LSLb (GI:32261218), LSLc (GI:32261220) from Multiple sequence alignment was conducted with MUSCLE using the default settings . After t3.Reconciliation of the phylogenetic tree with the species tree was performed in Notung 2.9 . The speC. sativus L. cv. Vert Petit de Paris) except for AAT14, which was amplified from cDNA obtained from 8-day-old fruits. To obtain a complete attB PCR product, nested PCR was performed. The primers used for the PCR are shown in Supplementary Table Plasmids for expression of the amaranthin-like sequences N- or C-terminally fused to EGFP under the control of the CaMV 35S promoter were constructed using the Gateway Cloning technology (Invitrogen). Coding sequences were amplified as attB PCR products using cDNA obtained from RNA extracted from 9-day-old plant leaves from cucumber  were infiltrated by suspensions of Agrobacterium tumefaciens with different OD600 (0.05–0.2) containing EGFP fusion constructs. The tobacco leaves were checked 2 days after the infiltration by confocal microscopy. The images of tobacco leaves expressing the fluorescent proteins were acquired using a Nikon A1R confocal system, mounted on a Nikon Ti microscope body. Different fluorescent images were acquired along the z-axis to create a picture of the complete cell. The acquired images were analyzed by Fiji software4.The EGFP fusion constructs were transferred into C. sativus L. cv. Vert Petit de Paris) seeds were germinated on moist filter paper in a Petri dish for 2 days at 28°C in the dark. Germinated seedlings were transferred to pots containing commercial soil, and grown in a plant growth room at 28°C with a 16 h/8 h light/dark photoperiod. Samples of cotyledons, leaves, stems, roots, flower buds, and fruits  were collected from plants at different stages of plant development. All experiments were performed between April 2013 and September 2014.Cucumber  and treated with DNase I (Thermo Fisher Scientific) to remove any traces of genomic DNA according to the manufacturer’s instructions. The first strand cDNA synthesis was performed using M-MLV reverse transcriptase (Thermo Fisher Scientific). RNA concentrations were measured with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The quality of the cDNA was checked by standard RT-PCR using primers of reference genes.5 for amplification of a 100–200 bp fragment. The primers for target and reference genes (clathrin adapter complex subunit and Protein phosphatase 2A) for qPCR analysis are available in Supplementary Table Real-time quantitative PCR analyses were performed using Rotor-Gene 3000 using Rotor Discs . The qPCR reactions were carried out in a total volume of 20 μl containing 10 μl of SYBR Green PCR Sensi-Mix (Bioline), 1 μl of each primer (10 μmol/μl), 1 μl of cDNA template (20 ng/μl), and 7 μl of sterile distilled water. The conditions for qPCR reaction were 96°C for 10 min followed by 45 cycles at 96°C for 25 s, 58°C for 25 s and 72°C for 20 s. Gene-specific primers for qPCR were designed using Primer 3C. sativus L. cv. Vert Petit de Paris) seeds were germinated on moist filter paper in Petri dishes at 28°C in the dark. Afterwards, germinated seedlings were transferred to falcon tubes containing half-strength Hoagland solution and grown at 28°C with a 16/8 h light/dark photoperiod. Cucumber seedlings with two fully expanded leaves were used for abiotic stress treatments. For the low temperature treatment, the plants were placed at 4°C in dark. For salt, drought and abscisic acid (ABA) treatments, plants were kept in half-strength Hoagland solution containing 200 mM NaCl, 100 mM mannitol or 100 μM ABA, respectively. All stress treatments were applied for 1, 3, 6, 12, and 24 h. After treatment, the second expanded leaves from cucumber plants were harvested and immediately frozen in liquid nitrogen.Cucumber  according to manufacturer’s instructions. RNA concentrations were measured with Nanodrop 2000 spectrophotometer. The quality of the cDNA was checked by standard RT-PCR using primers of reference genes.Real-time quantitative PCR analyses were performed using CFX Connect Real-Time PCR Detection Systems (Bio-Rad). The qPCR reactions were carried out in a total volume of 20 μl containing 10 μl of SYBR Green Supermix (Bio-Rad), 1 μl of each primer (10 μmol), 1 μl of cDNA template (20 ng/μl), and 7 μl of sterile distilled water. The conditions for qPCR were 96°C for 10 min followed by 45 cycles at 96°C for 25 s, 58°C for 25 s and 72°C for 20 s. The clathrin adapter complex subunit gene and protein phosphatase 2A gene were used as reference genes for qPCR analysis. The stability of these reference genes was analyzed by qBase PLUS software . REST-38A. caudatus  (B. thuringiensis (4RHZ) as templates. Homology modeling of AAT4 was performed with the YASARA Structure program (The cucumber lectin AAT4 was modeled using the X-ray coordinates of amaranthin from de 2ZTB) , and the program , running6 , including 11 monocot plants and 20 dicot plants, as well as two other species . Details of the sequences are available from Supplementary Table S. moellendorffii, multiple sequences with different domain architectures were identified. Obviously, amaranthin domains are not ubiquitous in the plant kingdom. They are absent from more than half of the plant genomes studied (51 out of 84 plant species) and were not found in the six species belonging to green algae.The presence of amaranthin domains was investigated in 84 (nearly) completed plant genomes and is summarized in Figure 2). The most prevalent domain combination consists of two amaranthin domains in combination with one aerolysin domain (AAT) and was identified in 113 out of 265 identified sequences. Other protein domains identified in combination with an amaranthin domain include the fascin domain, the Bet v1 domain, the TRAF-like domain, an alpha/beta hydrolase domain and the B box type zinc finger domain.Sequence analyses revealed different domain arrangements among the sequences with amaranthin domains. Databases such as Pfam and Superfamily were also checked to obtain more comprehensive information about the protein domains. Based on all information retrieved, 21 types of domain architectures can be distinguished in all identified sequences  and Fragaria vesca (AAT domain architecture) were predicted to possess a transmembrane region, located at the C-terminal end of the unknown protein domain.Sequence analyses suggested that almost all amaranthin proteins are synthesized without a signal peptide and do not contain any transmembrane region. Only two amaranthin-like proteins from Not considering the unknown domains, the amaranthin family can generally be divided into six types of domain architectures. (1) Single-domain amaranthin-like proteins (A type); (2) proteins with a single amaranthin domain linked to another domain, e.g., aerolysin domain (A+ type); (3) double amaranthin domain proteins (AA type); (4) proteins with two amaranthin domains in combination with another domain (AA+ type); (5) proteins with triple amaranthin domains (AAA type); (6) proteins containing three amaranthin domains and one aerolysin domain (AAA+ type). Due the diversity in protein structures, different types of amaranthin-like polypeptides differ greatly in size, with calculated molecular masses ranging from 19.5 kDa (A type) to 69.7 kDa (AAA+ type).Figure 3, the top 4 plant genomes with the largest number of amaranthin-like sequences are flax (Linum usitatissimum), apple (M. domestica), papaya (Carica papaya), and cucumber (C. sativus). Some amaranthin domains from flax are linked to a Bet v1 domain or a TRAF-like domain, a combination that was not found in any other studied plant genome. The apple genome harbors the highest number of sequences with a single amaranthin domain and the cucumber genome has most sequences with an AAT domain organization.The differences in the abundancy of domain architectures between different plant species are remarkable. According to Figure 4). In general, amaranthin homologs clustered according to the species they were retrieved from. This dendrogram basically reflects the phylogeny of the plant species . Different conclusions can be drawn from the phylogenetic analysis. First, sequences from species belonging to the same plant family are often clustered together, such as, e.g., Cucurbitaceae  and Poaceae . Second, sequences with the AA domain architecture, including the amaranthin from A. caudatus, were mainly found in Caryophyllales, Rosales, and Poaceae and are mostly separated from AA sequences which are part of the AAT domain architecture. Third, several genes from distantly related species show higher sequence similarities than genes from more closely related species. For example, two genes from flax (Lus10029184.g and Lus10029186.g with AAB domain) are clustered with genes of AA domains from Rosales rather than other genes from flax. This indicated that flax genes with AAB domains might be evolved from genes with AA domains by obtaining an additional Bet v1 domain.Because of the low sequence similarity between single amaranthin domains, a phylogenetic analysis was conducted using 159 sequences containing two tandem arrayed amaranthin domains to investigate the evolutionary relationships between amaranthin-like proteins .The most prevalent domain architecture among all the plant species studied harbors two amaranthin domains linked to one aerolysin toxin domain (AAT), and was most abundant in the cucumber genome. In total, 16 sequences with an AAT domain architecture have been identified. Two of these sequences possess an N-terminal domain with unknown function. In our study, AAT genes of cucumber were numbered sequentially according to their location on the chromosome and domain architecture. Consequently, the AAT proteins from cucumber are referred to as AAT1 to AAT14, and NAAT1 and NAAT2 refer to the sequences containing an additional N-terminal domain. All 16 genes are localized in the first 7.1 Mb of chromosome 6 . Interestingly, these clades are in agreement with the intron/exon composition of the sequences. Seven out of 16 genes contain one or two intron sequences. The sequences AAT8, AAT9, and AAT10 contain two introns located in the first amaranthin domain and the aerolysin domain, respectively . The sequences AAT12 and AAT14 harbor a single intron in the middle of the second amaranthin domain  while AAT6 and AAT7 have one intron within the toxin domain . However, the position of the intron sequences is not conserved. All other AAT genes have no intron sequence. Our previous results have shown that tandem duplication is the main driver for the expansion of amaranthin-like genes in cucumber  representing one sequence from each major clade of the tree were selected for further analysis.N. benthamiana leaf epidermal cells. Fluorescence throughout the cells was analyzed using confocal microscopy.To investigate the subcellular localization of the amaranthin-like proteins from cucumber, EGFP fusion constructs were made and transformed into Figures 6A,B). Similar results were obtained for N- and C-terminal fusion constructs with EGFP. The expression of AAT4 in the nucleus and the cytoplasm agrees with the prediction that the AAT4 sequence does not contain a signal peptide or a transmembrane region. The two EGFP fusion constructs for AAT9 yielded similar results as for AAT4, with fluorescence in the nucleus and the cytoplasm . Z-stack images clearly showed that the NAAT1 is not expressed in the nucleus. DAPI counterstaining confirmed that the nucleus is free of fluorescence .Transient expression of the AAT4 constructs in tobacco leaves yielded strong fluorescence in the nucleus and the cytoplasm , plants with first true leaf (9 days), plants that start flowering (51 days), and fruits collected at different stages of maturation. For all samples, transcripts levels for the AAT genes were quantified and normalized against reference genes. Relative expression levels of AAT genes from vegetative organs  were compared to the expression level in cotyledons while relative expression levels of AAT genes from reproductive organs (flowers and fruits) were compared to the expression level in flowers.Figure 7 and Supplementary Figure Different expression profiles were observed for the different AAT genes under study (Plants at the two-true-leaf stage (approximately 2 weeks old) were subjected to cold, salt, drought stress, and ABA treatment. The transcript levels of four cucumber genes  were analyzed at different time points after treatment and compared to non-treated control plants . In order to restore functional carbohydrate-binding sites at the interface between two adjacent amaranthin domains, like in the amaranthin lectin from A. caudatus . However, the superimposition of the amino acid residues forming the carbohydrate-binding sites of amaranthin in complex with benzoyl T-antigen .The modeled AAT4 consists of two tandemly arrayed amaranthin domains, A1 and A2, linked to an aerolysin-like domain, T . The analysis showed that the AAT genes from cucumber are grouped together with plant sequences like Hfr-2 from wheat and FEM32 from R. acetosa. The LuAAL13 from flax is not close to other plant sequences and clustered together with the Dln1 sequence from zebrafish, the LSL sequence from mushroom and the aerolysin sequences from bacteria.To understand the evolutionary relationships of the aerolysin domains from cucumber AATs with other known pore-forming proteins, a phylogenetic tree was built with sequences encoding the aerolysin-like domain from different species. A selection was made for sequences encoding several aerolysin domains that are known to be linked with lectin domains . In contrast, amaranthin-like sequences are absent in Fabaceae and Asterids (only one sequence retrieved from 10 plant genomes). Furthermore, based on our analysis for 84 sequenced plant genomes, it is obvious that the majority of the amaranthin-like sequences contain multiple protein domains with known function, the amaranthin domain being at least one of them. The aerolysin domain was found most frequently linked with amaranthin domains. It has been reported that genes containing aerolysin domains in eukaryotes may result from the recurrent horizontal transfer of bacterial toxin genes , amaranthin domains were identified in almost all sequenced genomes with only a few exceptions  was found to be particularly enriched in floral tissues  has been reported in two other plant species, flax and  tissues  and the elopment . DespiteIn nature, cucumbers are very sensitive to low temperature , droughtMayetiola destructor), fall armyworm (Spodoptera frugiperda), and bird cherry-oat aphid , but little or no changes in expression levels were observed after wounding, virus infection or chemical treatments with salicylic acid and ABA  and its expression is up-regulated following infestation of virulent Hessian fly . However, since the amino acids in the putative carbohydrate-binding sites of AAT4 are changed compared to the binding site in the A. caudatus lectin it is impossible to draw conclusions. Obviously, other types of oligomerization between AAT monomers may also be hypothesized based on protein association similar to the aerolysin from Aeromonas sp. . These amaranthin domains specifically interact with T-antigen and N-acetylgalactosamine  upon binding of certain receptors on the cell membrane. After a conformational change, part of the aerolysin domain will be inserted into the cell membrane, resulting in pore formation in the target cell membrane and facilitating bacterial invasion .To our knowledge, this is the first study that describes the overall distribution of amaranthin-like genes and the diversity of their domain architectures. The investigation of amaranthin-like proteins from cucumber further expanded our knowledge on their subcellular localization, tissue-specific expression and their possible biological function. Considering the lack of research on this family of lectins, this work provided valuable information for future studies on amaranthin-like proteins and their physiological roles in plants.LD and EVD outlined and designed the study. LD performed the experiments, analyzed and interpreted the data, and prepared the first draft of the manuscript. PR conducted the molecular modeling. EVD conceived and supervised the experiments and critically revised the manuscript. All authors have read, revised, 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.


Many arthropod zinc-finger transcription factors contain a N-terminal domaincalled ZAD (Zinc-finger Associated Domain), which consists of four cysteinescoordinating a single zinc ion. Dimerization ability has been shown for severalZAD-domains. The functional role of this domain is poorly understood. In thispaper, we demonstrate that a point mutation within the ZAD-domain of the Zw5insulator protein disrupts its nuclear localization without affecting itsdimerization ability. The importance of the ZAD-domain for nuclear localizationhas also been shown for the Pita and Grauzone proteins. Therefore, one of theZAD-domain functions is control of the nuclear localization of transcriptionfactors. Drosophila melanogastercells contain over 90 transcription factorswith C2H2- and ZAD-domains . According to experimental data, subcellular localization oftranscription factors with a ZAD-domain is the subject of a complex regulation.For example, it has previously been demonstrated that the nuclearcytoplasmiclocalization of the Weckle protein changes during development .Our results show that nucleocytoplasmic distribution of ZAD-containingtranscription factors appears to have regulatory importance and that theZAD-domains are essential for this process.


NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs. Innate immunity is an ancient mechanism of defence common to both animals and plants, and comprises a large number of pattern recognition receptors (PRRs)  that senAll NLRs have a conserved tripartite domain architecture that consists of an N-terminally located effector domain, followed by a central NACHT domain and a C-terminally located ligand sensing domain . The effUntil recently, the mechanism underlying oligomerization of NLRs was thought to resemble that of the apoptosome, where interaction with cytochrome C induces oligomerization of Apaf1 molecules into a ring-shaped complex ,26. Howein vitro, MDP and ATP are sufficient to promote NLRP1 oligomerization /Vporod(BSA), Vporod of BSA was evaluated from BSA SAXS experiment using 66.2 kDa as the protein molecular weight (Mw(BSA)). The last approach produced values of 99000 Da and 91000 Da for the lower and higher concentration respectively. The three methods all produce values that are close to the molecular weight of the monomeric NLRP1 suggesting that in solution the protein is present as a monomeric species. This result agrees well with the size exclusion chromatography profiles reported in Figs Data collection details and parameters from the structural analysis of the scattering curves are reported in ab-inito low resolution molecular shape calculated by DAMMIF shows an elongated envelope where two lobes are clearly distinguishable  is represented by the structure of mouse NLRC4. Two possible conformations have been recently reported for NLRC4: a closed conformation bound to ADP (PDB 4kxf) [2 = 1.37) when compared to the closed conformation (χ2 = 1.97) suggesting that the NLRP1 construct used in this study adopts an extended conformation that is reminiscent of the open conformation of NLRC4 in the inflammasome complex. The same analysis carried out on the SAXS data obtained at higher concentration produced almost identical results (data not shown).The uishable , a largeDB 4kxf)  and an oan NLRP12–990 is rDB 4kxf) . Both stDB 4kxf)  shows thin vitro study carried out with a version of the enzyme that lacks the part of the C-terminus encoded by exon-14, suggests that the molecule oligomerizes into a ring-shaped complex upon activation by ATP and MDP [NLRP1 is one of the best studied NLRs and although a plethora of data on its biological function are available, many questions regarding its mechanism of activation remain unanswered. The only current activation model for NLRP1, largely based on an  and MDP . HoweverWhen we embarked on this project it quickly became apparent that full length NLRP1 is unstable, poorly soluble and has a tendency to aggregate, features that have heavily hampered biophysical studies so far and likely explain some of the current inconsistencies on the NLRP1 activation mechanism found in the literature. To overcome these challenges, we dissected NLRP1 into its constitutive domains in order to obtain protein constructs that could be purified to homogeneity. Interestingly, apart from the pyrin, CARD and LRR domains, for which structures are available, neither the NACHT domain, nor the FIIND domain could be produced in isolation. However, a construct containing both, the NACHT and the LRR domain, was found to be soluble when produced in insect cells, and could be purified to homogeneity. The observation that the NACHT domain can only be produced when the LRR domain is also present suggests that the two domains stabilise each other, probably by sharing an interface. This construct was found to be monomeric in solution, constitutively bound to ATP and devoid of any significant ATP hydrolysis activity. Moreover, when MDP and ATP were added in large excess to the recombinant protein no high molecular species were detected by size exclusion chromatography, suggesting that MDP is not able to promote oligomerization of the construct. Small-angle X-ray scattering data allowed us to obtain a low resolution three-dimensional model that suggests that the molecule in solution behaves as an elongated particle. The SAXS-derived envelope is not consistent with an NLRC4-like closed conformation where the NACHT domain is closed around the ADP nucleotide molecule and the LRR folds back onto it. In contrast, the SAXS data agree well with the structure of NLRC4 in an open conformation as observable in the NLRC4-inflammasome structure.Since our characterization suggests that NLRP1 shares some similarities with the open conformation of NLRC4 we analysed our data in light of the recently described NLRC4 oligomerization mechanism –29. NLRCC. elegans, a distant homolog of NLRP1 that plays a key role in programmed cell death, and only contains a CARD and a NACHT domain, is constitutively bound to ATP [A NACHT-domain-containing protein, constitutively bound to ATP and lacking ATP hydrolysis activity is not a novelty. In fact, CED-4 (Cell death protein 4) from d to ATP ,44. The d to ATP . CED-4 iAt present it is not known if any of the other domains present, especially the FIIND and C-terminal CARD that are specific to NLRP1, may contribute to ligand sensing and/or oligomerization. Before these questions can be answered it will first be necessary to develop novel methods that allow the production and stabilisation of larger fragments of NLRP1 and render this enigmatic protein accessible to a more detailed structural and biochemical characterization. In conclusion, our study provides new insights that deepen our understanding of NLRP1 and it sheds some light onto its mechanism of activation by revealing a new level of complexity that was unexpected on the basis of previous reports.S1 FigMus muculus NLRP1a and NLRP1b, Rattus morvegicus NLRP1 and the 21 remaining human NLRs were aligned using the Clustal Omega server . The alignment was displayed using Jalview  and the residues are shaded according to the percentage of identity. A red box indicates the region of conservation that common to all the sequences. This region spans the NACHT and the LRR domains, a green box indicates an extra region of conservation that is present in the C-terminal part of the NLRP1 sequences, and this region spans the FIIND and the CARD domain.Full length protein sequences for human NLRP1, (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This paper details the validation of a non‐conforming arbitrary Lagrangian‐Eulerian fluid‐structure interaction technique using a recently developed experimental 3D fluid‐structure interaction benchmark problem. Numerical experiments for steady and transient test cases of the benchmark were conducted employing an inf‐sup stable and a general Galerkin scheme. The performance of both schemes is assessed. Spatial refinement with three mesh refinement levels and fluid domain truncation with two fluid domain lengths are studied as well as employing a sequence of increasing time step sizes for steady‐state cases. How quickly an approximate steady‐state or periodic steady‐state is reached is investigated and quantified based on error norm computations. Comparison of numerical results with experimental phase‐contrast magnetic resonance imaging data shows very good overall agreement including governing of flow patterns observed in the experiment. Classification based on the nature of underlying meshes distinguishes immersed methods and moving domain methods. Immersed methods employ a fixed background mesh and a Eulerian description for the motion of the fluid over the flow domain, whereas the solid motion and deformation are described within a Lagrangian coordinate frame on an embedded mesh. The presence of the solid is accounted for either via adding a body force term to the fluid equations  that merge smoothly into a single outlet (diameter ⊘76.2 mm), with a solid (volume 11 × 2 × 65 mm3) attached to the wall in the merging section  of the experiment features two inlets (diameter The selected pump rates create steady (Phase I) and periodic (Phase II) inflow. This yields steady and time‐dependent periodic interaction between a moderately viscous incompressible fluid (aqueous glycerol solution) and an incompressible nonlinear solid .Experimental data were acquired using MRI techniques and are available for comparison with numerical results. The data include the geometry under zero inflow conditions , inflow boundary condition data and time‐resolved flow and deformation fields.v˜fI were defined on ΓfI with peak value Ty > 0) and lower (y < 0) inlet.For Phase I, parabolic inflow profiles )v˜z=630·4t3−8t2y>v˜fI were defined on ΓfI with peak values T fit to the experimental data, see Figure For Phase II, parabolic profiles Fluid material parameters and solid density are defined in the study of Hessenthaler et al2.2Ωf⊂R3×Ī and Ωs⊂R3×Ī , and its respective subdomain boundaries, Γf and Γs. The fluid domain boundary Γf=ΓfI∪ΓfO∪ΓfW∪ΓfC is partitioned into inlet ΓfI, outlet ΓfO, wall ΓfW, and coupling interface ΓfC subdomains, see Figure Γs=ΓsW∪ΓsC is partitioned into subdomains for the wall ΓsW and the coupling surface ΓsC. A third coordinate frame on the coupling domain Ωλ=ΓfC=ΓsC is introduced to enforce coupling of the fluid and solid equations. Further, Ωi0 denotes reference domain i (with respective boundary Γi0), and ni denotes the outward boundary normal.The 3D FSI domain arising from the definition of the 3D FSI experiment consists of the fluid and solid subdomains, Ωi0, to moving domains, Ωi(t), bijective mappings are introduced,wf is the fluid domain velocity (referred to as ALE velocity), ui is the deformation of the domain  ≠ 0 such that Ωi0≠Ωi(0) and Γi0≠Γi(0)), and X is the spatial coordinate of a point in the reference domain. More precisely, Equations Ωf0 to its counterpart on the moving domain Ωf, ie,x is the spatial coordinate of a point in the moving domain and likewise for functions given on Ωs0 and Ωs. Further, the Jacobian mapping of the domain displacement is given as Ji:=detFi, where Fi = ∇X(ui + X) is the deformation gradient tensor. In the following, the hat notation in Equation ∂t denotes the temporal derivative with respect to a fixed point in the reference domain  is a diffusion coefficient tensor, and vfd,wfd and tfn are Dirichlet and Neumann boundary condition (BC) data. In our case, vfd and wfd arev˜fI to the momentum balance in Equation y‐axis would yield flow in the outlet region due to violation of a zero traction assumption on ΓfO. On the other hand, the same conditions do not cause flow if a COV is employed.The Cauchy stresses in Equations 0σf:=μf∇xreal pressure is recovered by reversing the COV withΓfI∪ΓfO∪ΓfW) to the interior of the fluid domain, a mesh velocity wf is introduced that satisfies Equation Φ, to preferentially weight motion in the boundary normal direction. Specifically, the tensor Φ was defined as Φ = ∇XψT∇Xψ + I with ψ satisfying the Laplacian problem on Ωf0(with ψ = 0 on ΓfI∪ΓfO∪ΓfW and ψ = 80 on Ωλ). As the variable ψ yields a decaying diffusion field from the coupling interface, this introduces anisotropy in Φ preferentially distributing motion faster in the direction of ∇Xψ. The value set for ψ was selected based on simple test problems in three dimensions, illustrating preservation of the mesh quality over time.The 2.4tf:= σf·nf and ts:= σs·ns, and the no‐slip condition, as detailed in Equation λ, a Lagrange multiplier variable is defined as λ = tf =− ts, maintaining the interface conditions. Within the continuous weak form for both fluid and solid mechanical subsystems, the resulting boundary terms are substituted with λ, constraining the multiplier through the no‐slip interface condition. Finally, the respective fluid and solid models ∈S0h:=V0h×U0h×M0h×Wfh×Wsh and z∈W0h:Psn+θ=σsFs−T/Jsn+θ is the first Piola‐Kirchhoff stress tensor, un+θ=un+θΔtnvn+1, and the notation Yn + θ = θYn + 1 + (1 − θ)Yn.Find kth‐order piecewise continuous polynomials on respective discretized domains , where Pk are the polynomials of degree k,k⩾1. Finally, from the spaces,γΓf,hC is the trace operator on Γf,hC.Following the study of Nordsletten et al,2.4.3P1−P1 Taylor‐Hood elements for fluid velocity and pressure, P1 elements for fluid domain velocity and inf‐sup stable Q2−Q1 Taylor‐Hood elements for solid displacement and pressure. Further, P1 elements were employed for the Lagrange multiplier variable, that was again nested into the trace of the richest space on Ωλ, which was on the fluid side. The discrete solution at each step n in time can then be written as follows:Here, the cG(1)cG(1) scheme as given for the Navier‐Stokes equations in the study of Hoffman et alsn+1∈S˜Dh:=V˜Dh×UDh×M˜0h×Wfh×Wsh and wfn+1∈W˜Dh, such that for every d∈S˜0h:=V˜0h×U0h×M˜0h×Wfh×Wsh and z∈W˜0h:h is the mesh size and vmax is the expected peak velocity on Ωf,h0. Here, we replace Vh and M0h by V˜h and M˜0h, to use equal‐order interpolation for the fluid model. Then, V˜Dh, V˜0h, W˜Dh, and W˜0h are given similarly to Equations Find 2.5CHeart1http://cheart.co.ukMUMPSc = 100 is used for scaling the initial residual, γ = 3/4 requires a sufficient residual decrease, and the general notation,ϕ and ψ). Further, the residuals and Jacobians are denoted by wfd can be updated from the current solution of vf on ΓfC.Further, the Shamanskii‐Newton‐Raphson (SNR) method2.6z‐direction with z ∈ {3.5, 13.5, …, 93.5}mm and 5 mm spacing along the x‐ and y‐direction, as specified in the studies of Gaddum et alxi, i = 1, …, N: (i) box  x  x  mm3 and spacing 1 mm (in each direction) for the current fluid domain Ωf,h; and (ii) box  x  x  mm3 and spacing 0.5 mm (in each direction) for the solid domain in the reference configuration. We note that a mask is employed such that only points are considered that exist inside both fluid domains in the current configuration. For example, consider discretized domains Ωi,h(tp) and Ωi,h(tq) at times tp and tq. Then, the mask is given as, xi, consider an approximation w∈Rd and a reference v∈Rd at times tp and tq with d ∈ {1, 3}. Then, a scalar field is defined at each point xi by computing the Euclidean distance, ∥·∥2, between approximation w and reference v. To detect variation in the fluid and solid variables, we compute the maximum distance,Numerical results are compared with experimental data at equispaced data point positions at cross sections along the 3Numerical results for both phases of the 3D FSI benchmark were obtained using the inf‐sup stable Section  and cG1 schemes.3.1Ωf0, Ωs0, and Ωλ0 were selected accordingly /ρsg. Equation z‐direction , see Figure 1˜ was considered for the model given in Section ΓfC was discretized using triangle elements, such that a quadrilateral surface element on ΓsC would have two triangular surface elements as counterparts on ΓfC.For the model given in Section We note that the outlet of the computational domain was extended by 250 mm to regularize flow downstream. To study the necessity of such an elongation, a truncated domain Figure A in combΩf0 to define the mesh on Ωλ.The triangular surface mesh on the fluid domain's coupling boundary was extracted from the mesh discretizing 3.2t3 − 8t2) · g over 0.5 s and kept constant at 1 · g for t ⩽ 0.5 s. Because a steady‐state is expected,t = 1 ms, 60 s with Δt′ = 10 ms, and 138 s with Δt″ = 100 ms .Initially, the FSI system is at rest and subject to no body forces . Then, parabolic inflow is prescribed at both inlets as given in Equation Because of increased inflow and gravitational forces, the silicone filament moves into the way of the incoming flow jet. The impact causes strong disturbance in the flow field, which is then advected toward the outlet causing reflow regions at the outflow boundary. Here, backflow divergence is avoided effectively by employing outflow stabilization Equation .After a short transition phase, the silicone filament reaches a steady position and flow stabilizes. Figure vx and vy components is less pronounced  to simplify comparison of corresponding time steps of two different cycles, eg, cycles k and K with tkn and tKn.Initially, the FSI system is at rest with the initial configuration corresponding to the hydrostatic equilibrium . Parabolic inflow is prescribed at both inlets with peak values fit to experimental data Figure B. A totaresting position), the silicone filament is pushed downwards because of the impacting flow jet entering from the upper inlet , the instant when the maximum relative displacement is reached is predicted precisely and the swing at t ≈ 1.59 s governed . While the deflection pattern is consistently predicted by the inf‐sup stable scheme on all refinement levels (irrespective of fluid domain length), the cG(1)cG(1) scheme yields a slightly different relative displacement pattern with a lagging solid motion, thus the silicone filament reaching its resting position later , nλ, tλ, and sλ are linearly independent normal and tangent unit vectors on Ωλ. As Figure fy component is negative for downward motion of the silicone filament and positive when it starts moving upwards after the largest relative displacement. For the fx and fy components, the truncation of the domain does not induce significant differences; however, the maximum force in fz‐direction during cycle 10 is observed earlier. Forces predicted by the cG(1)cG(1) scheme are significantly different; however, match with observations of relative solid motion .Forces exerted onto the solid were computed by integrating the Lagrange multiplier variable cG(1) scheme Table , which a4An inf‐sup stable FSI scheme has been validated and compared with, and a stabilized cG(1)cG(1) scheme cG(1) scheme. On the other hand, numerical results for the Phase II experiment yield a good prediction of flow patterns cG(1) can be considered coarse.It is recommended to simulate Phase I for about 30 seconds and Phase II for at least 5 cycles to reach an approximate (periodic) steady‐state with sufficient accuracy . Further, computational cost can potentially be decreased by optimizing the length of the computational domain, using a coarser solid mesh resolution and making use of the symmetry in the Phase I setup.Future investigations on the considered 3D FSI benchmark will include, for example, studying temporal convergence, sensitivity to the selected solid material law, spatial mesh refinement for the cG(1)cG(1) scheme, a second‐order time‐integration scheme, and sensitivity to outflow stabilization (preliminary results with


Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in regulating neuronal and synapse development that have also been implicated in diverse brain dysfunctions, including autism spectrum disorders (ASDs). SALMs, also known as leucine-rich repeat (LRR) and fibronectin III domain-containing (LRFN) proteins, were originally identified as a group of novel adhesion-like molecules that contain LRRs in the extracellular region as well as a PDZ domain-binding tail that couples to PSD-95, an abundant excitatory postsynaptic scaffolding protein. While studies over the last decade have steadily explored the basic properties and synaptic and neuronal functions of SALMs, a number of recent studies have provided novel insights into molecular, structural, functional and clinical aspects of SALMs. Here we summarize these findings and discuss how SALMs act in concert with other synaptic proteins to regulate synapse development and function. Synaptic adhesion molecules play important roles in the regulation of various processes involved in synapse development and function, including early axo-dendritic contacts, maturation of early synapses, synaptic transmission and plasticity, and synapse maintenance and elimination , also known as leucine-rich repeat (LRR) and fibronectin III domain-containing (LRFN) proteins, are a family of synaptic adhesion molecules originally identified independently by three groups as novel cell adhesion-like molecules that bind through their C-terminal tails to the PDZ domains of PSD-95  domain, and a fibronectin type III (FNIII) domain in the extracellular side, followed by a transmembrane domain and a cytoplasmic region that ends with PDZ domain-binding motif Figure . The PDZOur previous review of SALMs summarized basic and functional characteristics of SALMs, including chromosomal locations of the corresponding genes and exon-intron structures, mRNA and protein expression patterns, protein–protein interactions, and involvement in regulating neuronal and synapse development , and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors  animals combined with high-quality antibodies, as well as advanced methodologies, such as proximity biotinylation and endogenous protein tagging using CRISPR/Cas9-mediated homology-independent targeted integration  LAR-RPTP binding to SALM3, Slitrks, IL1RAPL1 and IL-1RAcP  interactions between Slitrk1 and PTPσ . However, it remains unclear whether the SALM5–PTPδ interactions measured under the surface plasmon resonance condition involves the 2:2 stoichiometry.The 2:2 stoichiometry of SALM5–LAR-RPTP interactions that involves an antiparallel LRR dimerization, something that is not observed in other LAR-RPTP-related crystal structures  SALMs 1–3; (2) SALM4; and (3) SALM5.A careful examination of cis-interactions between SALMs may be more complex than previously thought.However, a recent study has complicated this picture, reporting that SALM4 can coimmunoprecipitate with SALM2 in the mouse brain , as well as altered failure rates with minimal stimulation of NMDA/AMPA-evoked postsynaptic currents (EPSCs). This suggests that many –/–Lrfn2 excitatory synapses are silent synapses, an immature form of excitatory synapse that harbors NMDARs, but not AMPARs , likely because silent synapses have more room to accommodate incoming AMPARs. In addition to these functional changes at excitatory synapses, –/–Lrfn2 mice show morphological changes, including reduced spine head size and increased spine length , and demonstrated that a missense mutation inhibits the association of SALM1 with PSD-95. Interestingly, –/–Lrfn2 mice show enhanced spatial learning and fear memory, consistent with the enhanced LTP observed in these mice and a report that some individuals with LRFN2 mutations show enhanced memory together with delayed speech development  have been used to investigate in vivo functions of SALM3  demonstrated that SALM4 has unexpected negative effects on the density of excitatory and inhibitory synapses  or a PSD-95-binding C-terminal tail (unlike SALMs1–3). However, it should be noted that SALM4 is immunodetected in neuronal synapses in addition to dendrites and axons  encompassing three genes, including LRFN2  of SALM3/5, resolving the crystal structures of SALMs in complex with LAR-RPTPs, elucidating the cis-interactions between SALMs in the brain, it remains unclear whether SALM family members other than SALM5 and SALM2 form dimeric structures. It is also unclear whether SALMs directly interact with NMDA/AMPARs and, if so, whether these interactions regulate receptor functions or synaptic adhesions in a reciprocal manner. Because SALM3 and -5 are part of many LAR-RPTP–interacting postsynaptic adhesion molecules, whether SALM3/5 has its own unique roles, or redundant functions, remains to be determined.For example, although SALMs can form heterodimers, and SALM dimers can explain the reported heteromeric In vivo functions of SALMs also require further exploration. It will be interesting to determine whether SALMs have distinct functions in different brain regions and cell types. Because individual SALMs have largely unique cytoplasmic regions, SALM-associated synaptic signaling pathways are likely to be quite diverse. Circuit mechanisms underlying various behavioral phenotypes of Salm-KO mice, in particular those associated with SALM-related developmental and psychiatric disorders, also need to be investigated. Given the rapid increase in information on the biology and pathophysiology of SALMs, the next 10 years are likely to witness a dramatic increase in our understanding of this interesting family of synaptic adhesion molecules.EL, YL, RK and EK wrote 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 ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways, where abnormal UPS function has been observed in cancer and neurological diseases. Many neurodegenerative diseases share a common pathological feature, namely intracellular ubiquitin-positive inclusions formed by aggregate-prone neurotoxic proteins. This suggests that dysfunction of the UPS in neurodegenerative diseases contributes to the accumulation of neurotoxic proteins and to instigate neurodegeneration. Here, we review recent findings describing various aspects of UPS dysregulation in neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Many neurodegenerative diseases are associated with aggregate-prone neurotoxic proteins (aggresomes or inclusion bodies) that perturb cellular homeostasis and neuronal function . These dAlzheimer’s disease is the most common form of progressive neurodegeneration and represents the main cause of senile dementia affecting about 10% of the population over the age of 65 and about 50% of the population over 85. AD can be divided into two forms: familial AD and sporadic AD. Familial AD accounts for 5%∼10% of AD patients, where inherited autosomal dominant mutations in three genes have been considered as the primary causes of familial AD: these include mutations in the genes encoding β-amyloid precursor protein (APP) and presenilins  is an autosomal dominant progressive neurodegenerative disorder, clinically characterized by cognitive impairment, chorea and dystonia, bradykinesia, uncoordinated movement, mood disorder and other psychiatric symptoms, and behavioral difficulties . As the A-voltage-dependent calcium channel (CACNA1A) in SCA6, Ataxin-7 in SCA7, Atrophin-1 in dentatorubral-pallidoluysian atrophy (DRPLA), and androgen receptor in SBMA , Ataxin-2 in SCA2, Atantin-3 in SCA3, α1 in SBMA . All pol in SBMA .Ubiquitin is an evolutionarily conserved 76-amino acid moiety covalently tandemly linked to target protein components for degradation by the ubiquitin-proteasome system (UPS), and is required for degradation of about 80% intracellular proteins in eukaryotes . The preFigure 1).Protein ubiquitination occurs through the coordinated activity of several enzymes, including an ubiquitin activating enzyme (E1 ligase), conjugating enzyme (E2 ligase), and E3 ligase. Initially, a single ubiquitin moiety is attached to an active-site Cysteine residue within the E1 ligase through a thioester bond in an ATP dependent manner. The activated ubiquitin is then transferred to an ubiquitin conjugating enzyme-E2 ligase. Finally, the E2 ligase will cooperatively transfer the ubiquitin chain with a specified E3 ligase to a particular substrate, whereby the 26S proteasome will target the polyubiquitinated protein for degradation .Ubiquitin E3 ligases determine the specificity of ubiquitination process. E3 ligases are grouped into three classes according to their unique domains, including Really Interesting New Gene (RING) or U-box domain-containing E3 ligases, Homologous to E6AP C-terminus (HECT) domain-containing E3 ligases, and RING-between-RING (RBR) domain-containing E3 ligases . RING E3The fate of an ubiquitin-linked protein is dependent on Lysine chain-specific polyubiquitination . For exaThe UPS is involved in protein quality control and removal of misfolded and aggregated proteins, and dysfunction of the UPS is implicated in the pathogenesis of neurodegenerative diseases .Figure 1). Pivotal roles for DUBs have been demonstrated in maintaining neuronal functions and regulating the pathogenesis of various neurodegenerative diseases  has been found to be important in maintaining synaptic function .Accumulating evidence indicates that dysfunction of the UPS is a key factor to initiate and aggravate the pathogenesis of neurodegenerative diseases. We will focus on the dysregulation of ubiquitination and proteasomal degradation in neurodegenerative pathology in AD, PD, and HD  ubiquitin E3 ligase, is reduced in AD brains . HRD1 inion ACR; . ACR proion ACR; .Polyubiquitinated tau has also been found in the brains of AD patients , where uPARK2  complex  is another polyQ repeat disease, which is caused by a polyQ-encoding CAG repeat expansion in the gene encoding Ataxin-3 . SCA3 isneration . Unlike neration .Although UPS-mediated protein degradation has been well-studied in the last few decades, regulation of ubiquitination and proteasomal degradation in normal brain function and pathogenesis of neurodegenerative diseases remains largely unknown. The regulation of polyubiquitination processes may vary at different physiological and/or pathologic conditions. This review surveyed dysregulation of UPS in several major neurodegenerative diseases, more research is required to understand how the UPS is disrupted in neurodegenerative pathogenesis. By enriching our understanding of protein degradation, we can begin to formulate strategies to treat neurodegenerative diseases remediating aspects of UPS dysregulation.QZ, LZ, and XW wrote the manuscript, TH, YZ, HL, and HX edited the manuscript. 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.


Plasmodium parasites, it is now possible to use comparative genomics to look for orthology across Plasmodium species and for species specific genes. This comparative analyses could provide important clues for the development of new strategies to prevent and treat malaria in humans, however, the number of functionally annotated proteins is still low for all Plasmodium species. In the context of genomes that are hard to annotate because of sequence divergence, such as Plasmodium, domain co-occurrence becomes particularly important to trust predictions. In particular, domain architecture prediction can be used to improve the performance of existing annotation methods since homologous proteins might share their architectural context.With the availability of complete genome sequences of both human and non-human Plasmodium genomes. Domain architecture reconstruction in Plasmobase relies on DAMA, the state-of-the-art method in architecture prediction, while domain annotation is realised with CLADE, a novel annotation tool based on a multi-source strategy. Plasmobase significantly increases the Pfam domain coverage of all Plasmodium genomes, it proposes new domain architectures as well as new domain families that have never been reported before for these genomes. It proposes a visualization of domain architectures and allows for an easy comparison among architectures within Plasmodium species and with other species, described in UniProt. Plasmobase is a unique database designed for the comparative study of Plasmodium genomes. Its graphical presentation of protein sequences, based on domain architectures, will hopefully be of interest for comparative genomic studies. It should help to discover species-specific genes, possibly underlying important phenotypic differences between parasites, and orthologous gene families for deciphering the biology of these complex and important Apicomplexan organisms. In conclusion, Plasmobase is a flexible and rich site where any biologist can find something of his/her own interest.Plasmobase is a valuable new resource for domain annotation in http://genome.lcqb.upmc.fr/plasmobase/. Plasmobase is accessible at  Plasmodium genus. The fully sequenced genomes of 11 species are accessible from PlasmoDB [Plasmodium species. This could be due (i) to the AT-richness of these genomes, (ii) to the specificity of a number of parasitic functional mechanisms evolved to evade host immune recognition, (iii) to the strong divergence of Plasmodium protein sequences that make homology detection a difficult task. These three reasons might have contributed to the development of new gene functions and changes in the parasite’s genome, occurring through gene acquisition and deletion [A large amount of genomic and post-genomic data is now available for the deletion .Proteins are composed of one or more “domains”, that is structural motifs that can evolve, function, and exist independently of the rest of the protein chain. Domains might be found in different combinations, and the arrangement of these domains in a protein forms the so called “protein architecture”. By focalizing on domain recognition, genome annotation can be highly improved. Several approaches and databases have been developed to detect and identify functional domains in a large number of proteins, including Pfam , SMART , and PROPlasmodium falciparum genome, CLADE outperforms the widely used tools based on a mono-source annotation strategy, HMMer and HHblits [P. falciparum 3D7 has been released with [DAMA can improve domain recognition methods such as HMMer , 10 used HHblits . The newsed with .Plasmodium genome, leading to an enrichment of 18–30% of the total number of domain families when compared to Pfam predictions (with an FDR <1%). In addition, Plasmobase is a unique platform for the comparative study of Plasmodium genomes. It proposes a visualization of domain architectures and it allows for an easy comparison among architectures within Plasmodium species and all other species in UniProt. A friendly interface allows users to interact with the platform to access new annotations and possibly detect annotation errors.Plasmobase, presented here, is a novel database reporting known and new protein domains identified by DAMA and CLADE on the 11 fully sequenced genomes in PlasmoDB , two macaques parasites (Plasmodium knowlesi and Plasmodium cynomolgi), one chimpanzee parasite (Plasmodium reichenowi), and five rodent parasites . All genomes were extracted from http://PlasmoDB.org, the official repository of the Plasmodium proteins used as a reference database by malaria researchers. For each species, the genome size and the number of proteins are shown in Table The 11 fully sequenced genomes present in PlasmoDB [ftp://ftp.ebi.ac.uk/pub/databases/Pfam/releases/Pfam27.0/swisspfam.gz. Within each taxon group, sequences are grouped according to the name of their phylogenetic clade. A generic clade named “others” collects all clades with less than 2% of domain architectures. A reference list of clades has been extracted from NCBI (http://www.ncbi.nlm.nih.gov/taxonomy) and used for organizing the species where similar architectures are found.In order to display the proportion of proteins with similar architecture to a given query protein, all proteins  with known Pfam domain architectures are extracted from UniProtKB  and orgaThe gene ontology initiative , besidesPlasmodium organisms, CLADE to identify potential domains and DAMA to reconstruct protein domain architectures. Both tools were run with default parameters, corresponding to a FDR smaller than 1%. The two tools are briefly described below. For the FDR estimation and other details refer to the original articles.CLADE  and DAMACLADE is a computational approach that highly increases the sensitivity of domain prediction. It is a multi-source approach where several hundred probabilistic profiles are used to represent each domain, instead of one as for mono-source strategies, employed by methods like HMMer , 10 and e−3 by default. CLADE uses this PSI-BLAST default E-value (1e−3) and sets the number of PSI-BLAST iterations to 5. On average 161 CCMs were constructed to represent each Pfam domain. These models span regions of protein sequence space that are not well represented by Pfam consensus models, and they highlight motifs, structural characteristics or physico-chemical properties that are shared by similar homologous sequences. Hence, if the original set of sequences for a domain is made of very divergent homologs, clade-centred models, are expected to describe properties that could be missed by the original Pfam model, representing the global consensus.The second class of profiles is constituted by hundreds of probabilistic models, associated to each Pfam domain. They are constructed starting from homologous sequences spreading a large panel of taxonomic origins, to guarantee that the phylogenetic tree is well represented. For each selected homologous sequence, a specific profile, named “clade-centred model” (CCM), is constructed. To construct a CCM, the selected homologous sequence is used as a query to search for close homologs within the non-redundant protein database (NR) with PSI-BLAST . The CCMhttp://www.lcqb.upmc.fr/CLADE.The CLADE library, made of Pfam consensus models and clade-centred models, is composed of 2,404,066 profiles. This library is used to identify potential domains in query proteins. Potential domains are then filtered by using support vector machines (SVM) combining various optimisation criteria, ultimately converted into a score, to specifically deal with false positives and discriminate domains. The SVM selects most probable predictions among domain hits displaying a small E-value, a sufficiently long domain hit, the phylogenetic proximity between the taxon of the sequence to be annotated and the reference species generating the probabilistic profile, and the agreement among models leading to the prediction. This filtering step is fundamental in domain prediction. Once domains are filtered, CLADE calls DAMA to find the most probable architecture for a given query sequence. CLADE can be downloaded at http://www.lcqb.upmc.fr/DAMA.Since homologous proteins might share their architectural context, domain architecture prediction are used to improve the performance of CLADE annotation. The problem can be complex when a query sequence matches several probabilistic models, producing a set of conflicting predictions with overlapping domain boundaries. To address this problem, DAMA combines a number of criteria including multi- (possibly pairwise-) domain co-occurrence and domain overlapping. Domain co-occurrence is expected to enhance the level of confidence in a prediction  mainly be−10) and (2) they do not overlap an existing domain in the architecture. By setting this parameter, one can increase the number of predicted domains and takes into account the identification of new domain architectures  they present a significant E-value , the start and the end position of the hit either in the domain sequence used to construct the clade-centred model or in the Pfam consensus model, the clade name (if any) and the organism name of the sequence originating the clade-centred model used for the domain identification. Note that if a protein sequence is annotated by several domains, the file contains several rows, one for each domain annotation.Plasmobase website provides access to downloadable xls files containing the full list of annotations for the 11 Plasmodium genomes.Plasmobase is a platform for the exploration of protein architectures and their comparison across species. Its features and its multiple ways to analyse a predicted architecture or to explore potential architectures, are presented together with some global statistics on new domain annotations for the 11 Plasmodium database Plasmobase is a friendly interface allowing users to search by domains (with a Pfam accession number or a Pfam description), proteins (with a protein accession number or a protein annotation) or domain architectures (with a list of Pfam accession numbers) on all Plasmodium species or on a specific one. As a result, it provides the list of corresponding proteins with their accession number, Plasmodium species name, PlasmoDB annotation, a list of domains forming the predicted architecture (where new domains are highlighted), and the accessibility (“Look up” link) to a graphical interface providing comparative information on the predicted architecture. Each protein in the list is linked to its PlasmoDB description, and each domain forming the protein architecture is linked to its Pfam description.The P. falciparum protein PF3D7_1369500, identified by PlasmoDB as a “conserved Plasmodium protein, with no known function” and no predicted domain. It is accessible through the “Look up” link. On the top, the graphical interface shows the protein details: PlasmoDB accession number, protein length, and PlasmoDB text annotation. Then, it displays the predicted architecture constituted by three new domains , and the GO terms associated to the domains (accessible by clicking “GO Terms show”). For each predicted domain, there is an interactive legend (appearing when the cursor passes over the domain icon) with the description of the domain . The “Pfam-27 architecture” box shows the annotation proposed by the Pfam database version 27, where domains are predicted by HMMer and the architecture is obtained by employing a simple strategy that considers highest confidence domains without overlapping. For PF3D7_1369500 no domain was predicted by HMMer, and an empty box is shown. This display is accessible by clicking on the “show” link.  and explore for their co-occurrence either in Plasmobase Fig. a or in UPlasmodium species is possible by clicking on the “show” link at the bottom of the page  [P. knowlesi H to up to 30% in P. falciparum 3D7, and the number of proteins with no domain annotation largely decreased in all organisms. Compare the percentages of proteins with unknown function in Plasmobase and in PlasmoDB reported in Table Table hmmscan) , 10 usinPlasmodium species: P. falciparum 3D7, P. vivax Sal-1 and P. yoelii yoelii 17XNL. It uses hmmscan with permissive E-values to generate a set of potential domains, CODD [Plasmodium species while EuPathDomain considers just 3 organisms,considers 11 proposes a visualization of the predicted architecture while EuPathDomain displays the list of predicted domains found within the sequence,Plasmodium or in UniProt species, while EuPathDomain does not. Note that in Plasmobase, the user can explore domain architectures formed by any combination of CLADE domains, belonging to the proposed architecture or simply detected as potential hits of the sequence,compares architectures in a species with architectures in other searches proteins by keywords  and not only by Pfam identifiers and genome accession numbers, as EuPathDomain,allows searching for architectures described by multiple domains, while EuPathDomain only considers a single domain,displays domain architectures for orthologous groups, while EuPathDomain does not.Both systems provide GO functional annotation and, for each predicted domain, they indicate the other domains occurring in the architecture that are known to co-occur with it. Note that EuPathDomain displays Interpro domain predictions but it does not use Interpro database to predict new domains. Only Pfam domains are used, like in Plasmobase.The EuPathDomain  is a prons, CODD  is then All information contained in the database can be downloaded in xls files, one for each species, permitting the user to process the information in alternative ways.Plasmodium species. Plasmodium clade contains particularly AT rich genomes  are used to reconstruct the domain architecture of a large number of sequences presenting no domain annotation in Pfam_27 in order to annotate all m genome . Here, COn false discovery rate in Plasmobase Plasmobase provides multiple pieces of information to help users to evaluate a domain prediction: E-value, SVM probability, co-occurrences, and the possibility to compare predictions in orthologous genes. Indeed, one of the purposes of Plasmobase is to highlight several evidences to believe in the proposed domain annotation. Yet, as for any domain prediction tool, there exists the possibility that a domain is falsely predicted by CLADE. In this respect, several statistical tests to estimate CLADE false discovery rate (FDR) have been reported in [e−3, that is, 1 in 1000 predictions are expected to be a false domain.orted in , where iPlasmobase contribution to new protein annotations In Plasmobase, the user can explore and compare a large number of new domain annotations helping to the identification of a protein function. The protein PF3D7_1369500 illustrated in Fig. e−21, 1e−19 and 1e−8 and known to co-exist within protein sequences of metazoan, fungi and viridiplantae. In Plasmobase, this information, coming from UniProt, is accessible. The 96 UniProt metazoan sequences can be listed and their UniProt description can be looked up by clicking the protein accession number. By so doing, the user discovers that for these sequences, the architecture suggests the involvement of the protein in RNA metabolism and its role of RNA binding protein, but that its annotation goes from “uncharacterized protein” to “nuclear cap binding protein sub unit 1 (NCBP1)” inferred by homology, by experimental evidence at transcript level (in Mustela pitorius furo et Xenopus laevis) and by experimental evidence at protein level (in Mus musculus and Homo sapiens). This finding is supported by inspecting the 26 UniProt viridiplantae sequences. The majority of them was not studied but the Arabidopsis thaliana Q9SIU2 sequence was also annotated as NCBP1 by experimental evidence at protein level. From this analysis, one can infer that the annotation of NCBP1 is not solely a putative one because a biological validation was carried out in several studies conducted in mammals, Xenopus and Arabidopsis. Waiting for the biological validation in Plasmodium, this annotation could be proposed for the Plasmodium protein. This annotation is missing in PlasmoDB.e−60.Another example is the PBANKA_0110700 protein with unknown function and no identified domain in Pfam. CLADE identified a peptidyl-tRNA hydrolase domain in it, with the extremely low E-value 2Also, CLADE is helpful to confirm annotations identified with a low confidence. For instance, protein PF3D7_1452000 has no Pfam nor InterPro annotation but it is annotated as a rhoptry neck protein 2 (RON2) in PlasmoDB. In contrast, CLADE identifies the CLAG domain with an E-value 0 in PF3D7_1452000 based on a “clade-centred model” (in short CCM), that is a CLADE probabilistic model see "". Note tPlasmobase contribution to new protein architectures The detection of new architectures is fundamental to the understanding of genome evolution. In this respect, DAMA was designed by trying to minimise the effect of prior knowledge on known architectures. This was done in two ways. First, DAMA combines information coming from different known architectures. This allows to identify new architectures with coexisting domains possibly belonging to different known ones. Second, DAMA searches for extra domains (not belonging to known architectures) that have no overlapping with those identified by exploiting known architectures, and that have a sufficient good score compared to the other predicted domains. These extra domains, satisfying the required conditions , are added to the predicted architecture based on prior knowledge and enrich it. These two properties of the DAMA design assure the possibility to identify innovative architectures.Plasmodium genomes. All Plasmodium species reach a very large improvement in domain annotation, as reported in Table Plasmodium sequences are also annotated for the first time. Plasmobase graphical presentation of protein sequences, based on predicted domain architectures, is of easy exploitation for comparative genomic studies. Plasmobase is expected to help to discover species-specific genes, possibly underlying important phenotypic differences between parasites, and orthologous gene families for deciphering the biology of these complex and important Apicomplexan organisms. The interactive nature of the platform allows the user to easily learn plenty of information on a given protein, on its protein family, on its orthologs in other Plasmodium species, and in other eukaryotes. This is especially important for those highly AT-rich proteins whose annotation by comparison to eukaryotic proteins is particularly difficult, due to sequence divergence.Plasmobase is a valuable new resource for domain annotation in