The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing , antibody humanization, and antibody engineering in relation with effector properties.At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. Under these HGM auspices, IMGT The creormatics . The varensional D structuensional D structu12 different specificities per individual. Owing to the particularities of their synthesis that involve DNA rearrangements, there was a need for a systematic and coherent numbering of the amino acids and codons, whatever the molecule, configuration or chain type. The IMGT unique numbering was therefore a breakthrough in immunogenetics and immunoinformatics when it was defined for the first time in 1997 for the variable (V) domain [® databases, tools and Web resources (http://www.imgt.org) [The antigen receptor IG and TR variable domains form a huge repertoire of 2.10) domain ,5,6. The) domain ,5,6. Botmgt.org) .® multiple alignments of the germline IG and TR genes and, for the rearranged CDR3-IMGT, on statistical analysis of the IG and TR rearrangements [The IMGT unique numbering was created by taking into account the high conservation of the structure of the V domain and by integrating the knowledge acquired by the analysis of multiple sources: alignment of more than 5000 sequences, literature data on the framework (FR) and complementarity determining regions (CDR), structural data from X-ray diffraction studies and characterization of the CDR hypervariable loops ,5,6. Thengements ,5,6. Thengements , was rapngements . Based ongements . ® positions and lengths, based on the IMGT unique numbering for V domain (V-DOMAIN of IG and TR and V-LIKE-DOMAIN) [The V domain strands and loops and their IMGT-DOMAIN) , are shoThe V domain (V-DOMAIN and V-LIKE-DOMAIN) is composed of the A-STRAND of fifteen (or fourteen if gap at position 10) amino acids (positions 1 to 15), the B-STRAND of eleven amino acids (positions 16 to 26) with the first conserved cysteine (1st-CYS) at position 23, the BC-LOOP , the C-STRAND of eight amino acids (positions 39 to 46) with the tryptophan (CONSERVED-TRP) at position 41, the C’-STRAND of nine amino acids (positions 47 to 55), the C’C”-LOOP , the C”-STRAND of nine (or eight if gap at position 73) amino acids (positions 66 to 74), the D-STRAND of ten (or eight if gaps at positions 81 and 82) amino acids (positions 75 to 84), the E-STRAND of twelve amino acids (positions 85 to 96) with a conserved hydrophobic amino acid at position 89, the F-STRAND of eight amino acids (positions 97 to 104) with the second conserved cystein (2nd-CYS) at position 104, the FG-LOOP  and the G-STRAND of eleven (or ten) amino acids (positions 118 to 128) . In the In the IG and TR V-DOMAIN, the structurally conserved antiparallel beta strands are also designated as framework regions (FR-IMGT) whereas the loops are designated as complementarity determining regions (CDR-IMGT) . StrandsIMGT anchors belong to the strands (or FR-IMGT) and represent ‘anchors’ supporting the three BC, C’C” and FG loops (or CDR-IMGT). V domain anchor positions are positions 26 and 39, 55 and 66, and 104 and 118, shown in square in IMGT Colliers de Perles. In a V-DOMAIN, the 2nd-CYS at position 104 (F strand) and J-PHE or J-TRP at position 118 (G strand) are anchors of the FG loop (or CDR3-IMGT) [The loop length (number of amino acids (or codons), that is number of occupied positions) is a crucial and original concept of IMGT-ONTOLOGY . The len®http://www.imgt.org, IMGT Scientific chart > Numbering > IMGT unique numbering for V-DOMAIN and V-LIKE-DOMAIN).IMGT gaps are shown by dots in IMGT Protein displays ,2,9,10 a®http://www.imgt.org, IMGT Scientific chart > Numbering > IMGT unique numbering for V-DOMAIN and V-LIKE-DOMAIN).For CDR3-IMGT (FG) loop longer than 13 amino acids, additional positions are created, between positions 111 and 112 at the top of the loop, in the following order: 112.1, 111.1, 112.2, 111.2, 112.3, etc.   , are shoThe C domain (C-DOMAIN and C-LIKE-DOMAIN) is composed by the A-STRAND of fifteen (or fourteen if gap at 10) amino acids (positions 1 to 15), the AB-TURN , the B-STRAND of eleven amino acids (positions 16 to 26) with the 1st-CYS at position 23, the BC-LOOP , the C-STRAND of seven amino acids (positions 39 to 45) with the CONSERVED-TRP at position 41, the CD-STRAND of one to nine amino acids , the D-STRAND of eight (or seven if gap at 82) amino acids (positions 77 to 84), the DE-TURN , the E-STRAND of twelve amino acids (positions 85 to 96) with a conserved hydrophobic amino acid at position 89, the EF-TURN , the F-STRAND of eight amino acids (positions 97 to 104) with the 2nd-CYS at position 104, the FG-LOOP , and the G-STRAND of eleven (or less) amino acids (positions 118 to 128) [IMGT anchors belong to the strands and represent, for the C domains, anchors for the BC and FG loops and by extension to the CD strand (as C domains do not have the C’-C’’ loop) . Anchor ®http://www.imgt.org, IMGT Scientific chart > Numbering > IMGT unique numbering for C-DOMAIN and C-LIKE-DOMAIN).The A-STRAND and B-STRAND of the C domain are similar to those of the V domain . The lonIMGT gaps are shown by dots in IMGT Protein displays and by hatched circles or squares in IMGT Colliers de Perles for C domain and correspond to unoccupied positions according to the IMGT unique numbering for C domain . The longest BC-LOOP of the C domain have 10 amino acids . For BC loops shorter than 10 amino acids, gaps are created from the apex in the following order 34, 31, 35, 30, 36, etc. The FG-LOOP of the C domain is similar to that of the V domain. Gaps for FG loops shorter than 13 amino acids and additional positions for FG loops longer than 13 amino acids, are created following the same rules as those of the V domain.Additional positions in the C domain define the AB-TURN, DE-TURN and EF-TURN . For AB-Homo sapiens V-REGION and J-REGION. The objective of antibody humanization is to graft at the DNA level the CDR of an antibody V domain, from mouse (or other species) and of a given specificity, onto a human V domain framework, thus preserving the specificity of the original (murine or other species) antibody while decreasing its immunogenicity . IMGT/DoAnalyses performed on humanized therapeutic antibodies underline the importance of a correct delimitation of the CDR and FR. As an example, two amino acid changes were required in the first version of the humanized VH of alemtuzumab, in order to restore the specificity and affinity of the original rat antibody. The positions of these amino acid changes (S28>F and S35>F) are now known to be located in the CDR1-IMGT and should have been directly grafted, but at the time of this mAb humanization they were considered as belonging to the FR according to the Kabat numbering . In contHomo sapiens IGHJ2, IGHJ3, IGHJ4, IGHJ5 or IGHJ6  Overview).IMGT Colliers de Perles from crystallized 3D structures in IMGT/3Dstructure-DB ,10,11 hiThe amino acids of the V-DOMAIN CDR-IMGT involved in the contacts with the antigen can be visualized in IMGT/3Dstructure-DB Contact analysis ,10,11 wh®http://www.imgt.org, IMGT/3Dstructure-DB > Query 3ixt > Paratope and epitope). The IMGT Colliers de Perles show that eight (out of the eight VH positions of the paratope) belong to the VH CDR-IMGT: A35 (CDR1-IMGT); W58, D59, and K64 (CDR2-IMGT); I109, F110, N112 and F113 (CDR3-IMGT), and that similarly seven (out of the seven V-KAPPA positions of the paratope) belong to the V-KAPPA CDR-IMGT: G37 and Y38 (CDR1-IMGT); D56 (CDR2-IMGT); G107, S108, G109, and Y114 (CDR3-IMGT) [The IG paratope of 3ixt (motavizumab Fab) comprises AA of the VH (3ixt_H chain) and of the V-KAPPA (3ixt_L chain). Fifteen AA of the IG, eight from VH and seven from V-KAPPA, form the paratope . S40 > G (+ + -) means that the two AA involved in the change (S > G) at codon 40 belong to the same hydropathy (+) and volume (+) classes but to different physicochemical properties (-) classes [The number of amino acid differences in the FR-IMGT and CDR-IMGT is one of the criteria to evaluate the potential immunogenicity. The framework of a VH domain comprises 91 positions , whereas the framework of a VL domain comprises 89 positions    > 2D and 3D structures > CDR1-IMGT (summary) and correspondence with "canonical structures": human (Homo sapiens) and mouse (Mus musculus) Immunoglobulins; ibid CDR2-IMGT). Indeed, (1) most identified (15 out of 19) canonical structures correspond to a given CDR-IMGT length, (2) only two CDR-IMGT lengths have two canonical structures , (3) canonical structures have not been identified for every CDR-IMGT length, (4) many ‘variants’ are described in the literature, based only on sequences and without experimental evidence, (5) canonical structures cannot be identified for CDR3 owing to their diversity in lengths and sequences and to their flexibility, and (6) canonical structure identification is reliable only if 3D structures are known [For V-DOMAIN comparison including sequences and structures, the CDR1-IMGT and CDR2-IMGT lengths are more informative than the "canonical structures"  and in Camelidae in general: the conventional IG  and the ‘only-heavy-chain’ IG  [Camelus dromedarius in the 6-letter species abbreviation) IGHV3 genes belong to two sets based on four amino acid changes which are characteristic of each set [Two IgG antibody formats are expressed in the dromedary or Arabian camel (ing CH1) . The Cameach set . The fireach set . The fouCamelus bactrianus) of Central Asia and in the llama (Lama glama) and alpaca (Vicugna pacos) of South America. The genetic event (splicing defect) responsible for the lack of CH1 occurred in their common ancestor before the radiation between the ‘camelini’ and ‘lamini’, dating approximately 11 million years (Ma) ago. The respective heavy gamma2 and gamma3 chains are both characterized by the absence of the CH1 domain owing to a splicing defect . It is tH when they have to be distinguished from conventional VH . The term ‘nanobody’ initially used for describing a single-domain format antibody is not equivalent to VHH, as it has been used for V domains other than VHH and for constructs containing more than one V domain (VH and/or VHH)   antibody synthesis; ibid. IMGT/mAb-DB > caplacizumab; ibid. IMGT/mAb-DB > ozoralizumab).The V domains of Camelidae only-heavy-chain antibodies have characteristics for potential pharmaceutical applications . They are designated as VH®http://www.imgt.org, IMGT Education > Tutorials > Molecular defects in Immunoglobulin Heavy Chain Diseases (HCDs))The camelidae only-heavy-chain antibodies synthesis is remarkably reminiscent of what is observed in human heavy chain diseases (HCD). These proliferative disorders of B lymphoid cells produce truncated monoclonal immunoglobulin heavy chains which lack associated light chains. In most HCD, the absence of the heavy chain CH1 domain by deletion or splicing defect may be responsible for the lack of assembly of the light chain . SimilarGinglymostoma cirratum, ‘Gincir’ in the 6-letter species abbreviation) IgN antibodies  [®http://www.imgt.org, IMGT Repertoire (IG and TR) > Protein displays: nurse shark (Ginglymostoma cirratum) IGHV).A convergence mechanism in evolution is observed in nurse shark (ceptor’)  which ar®http://www.imgt.org, The IMGT Biotechnology page > Glycosylation (IMGT Lexique)).A N-linked glycosylation site is present in the CH2 domain of the constant region of the human IG heavy chains of the four IgG isotypes. The N-linked glycosylation site belongs to the classical N-glycosylation motif N-X-S/T  and is defined as CH2 N84.4. As shown in the IMGT Collier de Perles, this asparagine is localized at the DE turn. The IMGT unique numbering has the advantage of identifying the C domain  and, in the domain, the amino acid and its localization  which can be visualized in the IMGT Collier de Perles and correlated with the 3D structure ,26,27 .The knobs-into-holes methodology has been proposed for obtaining bispecific antibodies . The aim domains , were se domains ,10,11. T domains  .Amino acids in the IGHG constant regions of the IG heavy chains are frequently engineered to modify the effector properties of the therapeutic monoclonal antibodies. Amino acids changes are engineered at positions involved in antibody-dependent cellular (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), half-life increase, half-IG exchange, and B cell inhibition by coengagement of antigen and FcγR on the same cell (IMGTHomo sapiens IGHG1v1), and then the domain(s) with AA change(s) defined by the letter of the novel AA and position in the domain . The IMGT engineered variants are classified by comparison with the allele *01 of the gene and, if the effects are independent on the alleles, as a reference for the description of the amino acid (AA) changes for the other alleles. In those cases, the same variant (v) number is used for any allele of the same gene in the same species. The IMGT engineered variant nomenclature  has been®, created in 1989 with the official recognition of IG and TR genes, is at the origin of immunoinformatics [http://www.imgt.org/IMGTrepertoire/2D-3Dstruct/2D-representations/mouse/IG/E5.2Fv/ighV-D-J_E5_2Fv.html).IMGTormatics . The conormatics . Interes® databases, tools and web resources have been built to manage immunogenetics knowledge and immunoinformatics, based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts [® provides standardized and integrated databases, tools and web resources for IG and TR, from gene to structure and function [gnathostomata), from fishes to humans, providing a unique resource whatever the antigen receptor, the chain type and the taxon, for study of the adaptive immune response [® standardization, developed originally to handle the huge diversity of the immune repertoires, more needed as ever. Since October 2010, the IMGT/HighV-QUEST web portal has been a paradigm for the characterization of the V domain diversity and expression and the identification of the IMGT clonotypes (AA) [The IMGTconcepts . Nowadayfunction ,41,42,43pes (AA) ,45,46. Spes (AA) ,48. NGS pes (AA) ,50,51.The therapeutic monoclonal antibody engineering field represents the most promising potential in medicine. Standardized genomic and expressed sequence, structure and interaction analysis of IG is crucial for a better molecular understanding and comparison of the mAb specificity, affinity, half-life, Fc effector properties, and potential immunogenicity. IMGT/3Dstructure-DB provides a standardized description and antibody structure/contact analysis characterization, at the V and C domain level, at the chain level (with the ‘chimeric’ and ‘humanized’ added as ‘taxon’), and at the receptor level. Amino acids (or codons) changes . This modular characterization per domain (and/or region) provides a great flexibility and is applicable to any novel format of antibody engineering ,57,58,59


BU while the final viscosity ranged between 594 and 709 BU. The flour blends had promising dynamic rheological characteristics; at 15% incorporation of defatted mango kernel flour, G’ was greater than G’’ while a stage was reached when G’ was equal to G’’ at 25% incorporation. At 50% incorporation, G’’ was greater than G’. The cookies produced showed high (>70%) quality in terms of physical properties, color, and sensory evaluation at 25% incorporation of defatted mango flour.Defatted mango kernel seed flour was added to wheat flour for the production of cookies. The protein contents of the wheat‐mango kernel flour blend were optimized using optimal mixture design of response surface methodology. The farinograph analysis had acceptable mixing profile at 25%‐defatted mango kernel flour incorporation, after which the mixing profile was adversely affected. The pasting temperature was between 65.8 and 75.6°C; peak viscosity ranged between 448 and 521  Traditionally, baked products are produced from wheat. With the awakening consciousness of the need to consume health‐promoting foods by consumers worldwide, consumption of junk foods is being discouraged while researches are being focused on the development and production of functional baked products that are health‐promoting , and they have been identified as serious causes of environmental problems and economic losses if not utilized effectively  is one of the most important fruits marketed in the world with global production exceeding 26 million tons in 2004  polyphenols exhibit anti‐inflammatory and cancer‐cytotoxic properties in multiple cancer types, including malignancies of the colon and breast  to 5% (ethanol extraction)  by Avent Agro Pvt. Ltd, Narela, Delhi 110040, India, was sourced from Mysuru, India. Shortening , sugar powder, common salt, vanilla essence (Bush Boake), and corn flour were procured from local markets.2.2The experimental design for the optimization of the protein content was carried out using optimal mixture design . The independent variables were wheat flour (0–100 g/100 g) and mango kernel flour (0–100 g/100 g), while the dependent variable was the protein content. The number of replicates is shown in Table 2.3Protein content of the composite flour was determined using Flash 2000 N/Protein Analyzer (Thermo Scientific). About 3.5 mg sample was analyzed for the determination of the nitrogen content. The protein content was automatically evaluated by assuming a conversion factor of 6.25.2.4Blends of wheat flour and defatted mango kernel flour were prepared in the ratios 100:0 (W), 85:15 (X), 75:25 (Y), and 50:50 (Z) by replacing WWF with DMKF.2.4.1The farinograph evaluation was carried out according to the standard AACC  methods.2.4.2The pasting characteristics were determined by using Micro‐Visco‐Amylograph  according to the standard AACC  methods.2.4.3The dynamic rheology for wheat flour‐defatted mango kernel flour blends was carried out by using modified method of Demirkesen et al.  using Mo2.5The method of Sudha et al.  was used2.62.6.1Method of Sudha et al.  was used2.6.2The color  of the cookies was measured using Hunter Lab's Color Flex EZ spectrophotometer (Color Flex EZ 45/O).2.6.3The sensory evaluation was carried out according to Sudha et al. . Cookies2.7The analysis of variance (ANOVA), the multifactor analysis of variance, and the multiple range test were done using the Statgraphic Centurion XVI, version 16.1.11 (StatPointInc). All analyses were carried out in triplicate. The results were expressed as mean ± standard deviation. Duncan's test at significant level of p ≤ 0.05 was applied to the data to establish the significance of the differences between the samples.33.12) were significant (p ≤ 0.0001) while the model term (AB (A−B)) was significant (p = 0.0003). The R2 and adjusted R2 values were 1.000 and 0.9999, respectively. The implication of these results is that the composite flour consisting wheat and mango kernel flours were good sources of protein. The final equation showing the effect of the independent variables on the protein content is given in Equation The actual and predicted protein contents of wheat‐mango kernel flour blends are presented in Table 2) had the highest positive coefficient, followed by sample B (wheat flour) and sample a (mango kernel flour), respectively. The highest coefficient of the mixture is a proof that wheat protein content was actually increased by the inclusion of mango kernel flour.From the equation, the mixture of wheat and mango kernel flours at the quartic level (AB (A−B)3.23.2.1The results of the farinograph which describe the mixing properties of the flour blends as shown in Figure A dough development time (DDT) of 2 min was also reported for refined wheat flour  had maximum cohesiveness and elasticity at the peak consistency, which reduced with increasing mixing time. Refined wheat flour also had farinogram similar to the whole wheat used in this study  followed by sample Y, X, and Z in that order. The peak viscosity represents the pastes strength from gelatinization; it is the maximum viscosity attained during cooking  and high mechanical shear stress , loss modulus (G’’) and complex viscosity are shown in Figure Increase in the incorporation of mango seed flour 50%), however, reversed the dynamic rheology trend observed in 100% wheat flour. An equal G’ and G’’ were obtained as the angular frequency increased in sample Z Figure c, while 0%, howev3.3The results of the physical characteristics of the cookies are presented in Table The sensory evaluation Table  showed t4Incorporation of defatted mango kernel flour into wheat flour would be suitable for production of baked products. The protein content was nutritionally sufficient while the rheological evaluation showed that wheat‐defatted mango flour blends up to 25% defatted mango flour addition were adequate; they displayed acceptable dough mixing and viscoelastic behavior. In fact, addition of defatted mango flour enhanced the elastic characteristic of the flour blends. The pasting properties of the blends were similar to 100% wheat flour, indicating a good starch heating properties. The cookies at 25% incorporation of defatted mango kernel flour showed an excellent (>70%) physical and sensory properties as well as acceptable measurement. The overall cookie quality acceptability at 25% defatted mango kernel flour incorporation was over 70%.Authors declare there is no conflict of interests.This study does not involve any human or animal testing.


Aluminum matrix (Al99.5) syntactic foam containing expanded perlite particles was produced using the pressure infiltration technique. The dominant deformation mechanisms during compression of this foam were determined by sequential k-means analysis of the acoustic emission data. Since the different deformation mechanisms were concurrently active even at small strains, successive unloading and reloading measurement was proposed for cluster identification. The repetitive unloading and reloading allowed us to identify two mechanical parameters, namely the unloading modulus and the loss for unloading-reloading cycles. Based on the correlations among the strain localization within the specimen, the acoustic emission results, the changes in these mechanical parameters, and the transition from quasi-elastic deformation to plasticity were revealed in this material. Metal matrix syntactic foams (MMSFs) are extensively studied as novel materials ,2,3,4. T3) and high energy absorption efficiency [In recent years, the effects of different macroscopic and microscopic parameters on the mechanical properties of perlite particles-filled aluminum syntactic foams have been extensively investigated ,10,11,12ficiency . The other advantage of the pressure infiltration process is that it results in an isotropic structure. Manufacturing processes can introduce anisotropy to the cell structure  that results in direction sensitivity of mechanical properties. This phenomenon is well-known and widely investigated in the literature ,15. Howe*X and X, respectively, β and n are constants related to the shape and the character (closed or open cells) of the cells, respectively, and The investigations have so far revealed that the foam density ,10, the g effect . 3. In samples with lower density, the deformation is localized in large individual shear bands at an angle of approximately 45°–65° with respect to the compression axis [It was further reported that the deformation mechanisms can be influenced by changes in the foam density  or localThe deformation mechanisms and failure during compression can be effectively investigated by means of acoustic emission (AE). AE represents transient elastic waves that originate within the material due to sudden localized structure changes, such as collective dislocation motion or crack initiation and propagation. Thus, AE yields information on the deformation mechanisms during the deformation of the material. It has been proven that applying the recently developed adaptive sequential k-means (ASK) clustering method on the raw AE data allows separating AE events originating from different sources . In this method, the AE signal is first sectioned into time-windows. Clustering is based on the statistical parameters of power spectral density functions (like energy and mean frequency), which are then determined for each time-window. For details on the algorithm, see Reference . HoweverThe aim of this work was to find mechanical parameters that can help predict the progress of failure in EP syntactic foams. These foams are particularly a good choice for this kind of investigations since the EP particles have low strength and they are brittle, so that the matrix material deforms the particles crumble, enhancing the failure progress. Successive unloading and reloading measurements were proposed to monitor the evolution of failure in the EP syntactic foam since both the unloading modulus and the area of the hysteresis loops of the stress-strain curve  can characterize the failure state of a syntactic foam ,19. Furt2, 12 wt% Al2O3, 3 wt% Na2O, 4 wt% K2O, 2 wt% CaO, and 1 wt% MgO as the main constituents, provided by the supplier) with a diameter between 1 and 2 mm were poured into a closed-profile crucible with a cross-section area of 50 mm × 50 mm and were slightly compacted. Thereafter, a stainless steel mesh was fixed on top of the particles to avoid the movement of the EP particles during the infiltration. A block of commercially pure Al (Al99.5) was placed on the mesh and the closed-profile crucible was enclosed by welding. During heating, the interior of the closed-profile crucible was kept in helium atmosphere using an inlet, which was welded to the closed-profile crucible. The assembly was heated up to 750 °C and held at this temperature for 30 min. Then, 1 bar overpressure of helium gas was applied for 2 min to complete the infiltration. Finally, the crucible with the infiltrated EP particles was quenched into water at ambient temperature. Skin with a diameter of 2 mm was removed at the sides and the bottom of the syntactic foam block. The perlite-aluminum syntactic foam was produced by the pressure infiltration technique at the Eötvös Loránd University, Budapest. First, EP particles . Slices for the microscopy were cut from the deformed sample with a diamond disc cutter at a low speed to minimize the damage of the PE particles. In order to reveal the strain distribution during deformation, a digital image correlation (DIC) analysis was performed using the Ncorr scripts implemented in Matlab . th and 7th cycles of loading and unloading.) The compression tests were stopped at 2200 N which was the force limit of the testing machine. In this way, we monitored the damage mechanisms at low strains (<12%). In this deformation range, the stress-strain curve consists of two stages: a quasi-linear stage and a plastic region. In order to avoid recording of spurious AE signals originating from the machine at the strain direction reversal occurring at zero stress, the unloading was terminated approximately at 200 N.A typical time-stress curve with an AE response of an EP aluminum syntactic foam during repetitive loading and unloading and the corresponding deformation curve can be seen in The AE activity was observed practically as soon as the compression started. In the quasi-linear stage, the AE activity increases with increasing stress and it reaches its peak intensity approximately at the end of the quasi-linear stage. This behavior is similar to that of other aluminum matrix syntactic foams ,23. Even−3) at the beginning of the deformation  and it increases with increasing applied strain. We found that the rate of the rise of the unloading modulus changes and the loss value changes during deformation Since the AE measurement was launched before starting the deformation test, the first cluster naturally belongs to the noise. This cluster mostly consists of AE signals with low amplitude cf. a. The si• Cluster 2—Friction inside the syntactic foam (color code: green)This cluster is active during both unloading and reloading. It is not active in the quasi-linear stage; however, it becomes active as the formation of the deformation band begins . The AE • Cluster 3—Cracking of EP (color code: blue)Cracking of EP particles is the most dominant AE source in the quasi-linear stage. The burst character of the signals with a short rise time is typical of fracture c. The bu• Cluster 4—Plastic deformation and crack initiation and propagation in the matrix (color code: black)Cluster 4 is active only during reloading when the stress exceeds the maximum stress value of the previous cycle a,b. ThisThe ASK analysis revealed that, even at small stress, the matrix of the EP aluminum syntactic foam yield and, simultaneously, the cell walls of the EP particles are being crushed. To confirm the EP cracking as a possible AE source, sections of deformed samples were investigated by SEM. The deformed sample was compressed at the same conditions as other samples. The compression was stopped at 1 MPa in the quasi-elastic stage (the stress at the end of the quasi-linear stage is about 5 MPa). The inspection of the SEM images of the deformed samples showed that in the EP particles two types of cracks can be distinguished. One of them is straight, canyon-like and can be seen on the SEM images of both the deformed and undeformed foam and originates from the foam production , i.e., tThe changes in the unloading modulus as a function of strain can be seen in th unloading), The AE measurements revealed that plastic deformation takes place already during the quasi-linear stage, but both the cumulative energy and the number of element in cluster 4 (matrix plasticity) are much lower than in the plastic stage. This means that the quasi-linear stage can be characterized mostly by microplasticity, i.e., only a small volume suffers plastic deformation, while in the plastic stage macroplasticity occurs. This is clearly visible both on the deformation of the surface We identified three main failure processes during the compression of an EP aluminum syntactic foam: plastic deformation of the matrix material, and cracking of walls of the EP particle and friction of these broken walls. These mechanisms start almost at the same time at very low stresses; however, the dominant deformation mechanism changes with the deformation. First, the cracking of the EP is the dominant deformation mechanism. At the end of the quasi-linear stage, the formation of a deformation band starts and, as a consequence, the effect of friction becomes more significant.(2)We found that the end of the quasi-linear stage and the beginning of macroplasticity can be indicated by two mechanical parameters, namely the unloading modulus and the area of the stress-strain curve upon unloading and reloading (loss). The changes in these parameters predict the changes in failure mechanism.


Breadfruit flour B had the lowest (p < 0.05) bulk density, while banana flour had the greatest (p < 0.05) bulk density. The swelling power of the tropical flours was greater (p < 0.05) than the wheat flour. The viscosity of the tropical flours was higher than wheat flour but decreased significantly when temperature was held at 130 °C. These results indicated that the two breadfruit flours and banana flour have great potential for application in processed food products, and have similar compositional attributes to a more traditional flour.The objective of this study was to compare the compositional and functional properties of tropical flour sources (two breadfruit flours (type A and type B) and a banana flour) with a more traditional flour source (wheat flour). Macro-nutrient composition, pH, water and oil holding capacity, bulk density, particle size, solubility, swelling power, pasting properties, and thermodynamics (gelatinization and retrogradation) were determined. All flours evaluated were similar in their composition with high levels of carbohydrates (greater than 82.52 g/100 g on a dry-matter basis), with most of the carbohydrate content comprised of starch (greater than 67.02 g/100 g). The tropical fruit flours had greater ( Starches are an important ingredient used for many purposes in the food manufacturing industry. Common sources of starches include those from some of the most commonly grown staple crops in the world like wheat ,2. ThereArtocarpus altilis origin. The banana flour was provided by LiveKuna and was manufactured by Kunachia LLC . The banana flour was of the Musa acuminate origin. Wheat flour was obtained commercially from a local ingredient supplier Bulk Barn Foods, Aurora, ON, Canada) located in Guelph . The flours were used in their native state. Moisture, lipid, protein, and ash content were determined according to the AOAC methods 925.10, 920.85, 992.15, 923.03, respectively [v/v) to remove the D-glucose and stirred with 2M KOH at approximate 4 °C (ice/water bath) to pre-dissolve the resistant starch. Tropical flour sources evaluated in this research included two types of breadfruit flour and one type of banana flour. The first type of breadfruit flour (Breadfruit flour A) was provided by Natural Foods International . The second type of breadfruit flour (Breadfruit flour B) was provided by Jungle Project/Tropico Agroforestal S.R.L. . Both breadfruit flours were of the ectively ,17,18. Tectively . Freeze-Carbohydrate content was calculated according to an equation provided by the Food and Agriculture Organization of the United Nations  with minCalorie and energy content were calculated according to the following equation :CalorieTen g flour samples (in triplicate) were homogenized in 50 mL of distilled water for the determination of pH. pH values were quantified using a benchtop pH meter  following calibration with buffer solutions of pH 4.0 and pH 7.0.g on a bench centrifuge . The clear supernatant was carefully removed and discarded. Water holding capacity was expressed as the weight (in g) of water bound by 1 g of dried flour sample. Oil holding capacity was expressed as the weight (in g) of oil bound to 1 g of dried flour sample.Fifteen mL of distilled water  was added to 1 g of the flour sample in a weighed centrifuge tube. The contents were mixed using a vortex mixer for 2 min, and then centrifuged for 20 min at 6000× 3) was calculated as the weight of sample per unit volume of sample.Fifty g of flour samples was placed in a 100 mL measuring cylinder. The cylinder was gently tapped on a laboratory bench several times to ensure a constant volume. Bulk density . Particle size distribution of flour samples was measured using a Malvern Master Sizer laser diffraction analyzer  at room temperature (approximately 21.5 °C) according to the methodology of Roman et al.  with modzero hermetic aluminum DSC pans (TA Instruments). The sample pan was then hermetically sealed. The sealed samples were placed at room temperature (approximately 21.5 °C) for 24 h to equilibrate. An empty pan was used as reference. The first run was used to measure the gelatinization process: at a heating rate of 10°C/min over the temperature range from 25 °C to 100 °C. After the first heating run, the sample pans were stored at 4 °C for 7 days. After the 7-day period, the second run was conducted for the measurement of the retrogradation process: at a heating rate of 5 °C/min over the temperature range from 20 °C to 100 °C. Measurements were performed in three replications and results were presented as mean values. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy (ΔH) of gelatinization and retrogradation were obtained by integration of the endothermic peak from DSC thermograms.The gelatinization and retrogradation process of the flours were characterized calorimetrically using a differential scanning calorimeter  equipped with a refrigerated cooling system  and nitrogen purge gas. An amount of 6 ± 0.1 mg of each flour sample was accurately weighed and mixed with 18µL distilled water in Tg for 10 min. The supernatants were carefully poured into weighed aluminum dishes and evaporated at 100°C using a drying oven  for 24 h. The weight of dry solids was determined for the calculation of solubility. The weight of wet sediments in the centrifuge tube was determined for the calculation of swelling power:One wt% aqueous suspension of each flour sample was heated to pre-determined temperatures  for 1 h with constant stirring using a magnetic stir bar and a stirring hotplate . Samples were poured into a weighed centrifuge tube and centrifuged at 3000× Pasting properties of the flour suspension were analyzed by two different methods. The first method was conducted with a Rapid Visco Analyser , where the maximum temperature was 95°C. It is noteworthy that some starchy flours, such as banana starch, has been reported to exhibit an underdeveloped pasting profile or, more specifically, a lack of breakdown and setback upon heating . For thiWith both methods, the moisture of flour was first measured by a moisture meter  to obtain the correct flour sample weight and amount of water required for the test. The flour suspension  was poured into an aluminum sample cup and the cup, with a plastic stirring paddle, was inserted into the RVA machine. For the first method, the sample temperature was held at 50 °C for 1 min, increased to 95 °C, held at 95 °C for 2.5 min, lowered to 50 °C, and held at 50 °C for 2 min. For the second method, the sample temperature was held at 50 °C for 1.5 min, increased to 130 °C, held at 130 °C for 3 min, lowered to 50 °C, and held at 50 °C for 2 min.p ≤ 0.05. Proximate composition of flours was conducted in duplicate for each flour type. All other analyses were conducted in three replications for each treatment (flour type). Statistical analyses were performed with SAS . Data  were analyzed with PROC GLIMMIX of SAS with fixed effects of flour type and a random effect of replication. Data for solubility and swelling power were analyzed with PROC GLIMMIX of SAS with fixed effects of flour type and temperature, and a random effect of replication. Least square means were separated using the PDIFF option with a Tukey-Kramer adjustment. Differences were considered statistically different at The main compositional component of the two breadfruit flours, banana flour, and wheat flour was carbohydrates (which ranged 82.52 to 91.68 g/100 g on a dry matter basis), with most of the carbohydrates comprised of starch  pH value and breadfruit flour A having greatest (p < 0.05) pH value  water holding capacity, followed by banana flour and breadfruit flour A, while wheat flour had the least (p < 0.05) water holding capacity. Similarly, breadfruit flour B had the greatest (p < 0.05) oil holding capacity, followed by breadfruit flour A and wheat flour, while banana flour had the least (p < 0.05) oil holding capacity. Overall, the water and oil holding capacity of wheat flour was less (p < 0.05) compared with the breadfruit flours. Water and oil holding capacity are based on several factors such as: flour carbohydrate contents, particle size, amount of damages starch, the ratio of amylose to amylopectin in starch, and intra and inter molecular forces [4,3) which indicated the central point of the volume distribution of the particles was presented. The particle size of breadfruit flour B (220.94 μm) and banana flour (222.89 μm) were similar to those reported by Roman et al. [p < 0.05) than that of breadfruit flour A (138.73 μm) and wheat flour (132.54 μm). As depicted in the plotted particle size distribution profile, bimodal distributions were observed for breadfruit flour A and breadfruit flour B, which likely indicated the distinct separation of particle size between starch granules and flour particles with these flours. Several previous research studies have described that differences in processing techniques affected particle size distribution, which may have meaningful effects on a variety of chemical and physical properties [Water holding capacity is desirable in most food processing systems to improve yield and provide the appropriate organoleptic properties that make foods unique and acceptable to consumers . Oil holr forces , which wn et al. . These voperties ,32,33. Tp < 0.05) bulk density, while banana flour had the greatest (p < 0.05) bulk density.Bulk density is important for determining packaging and material handling requirements in the food industry. As an example, this includes the design of metering equipment such as particulate solid feeders. Bulk density is generally affected by flour density . Greaterp < 0.05) T0, Tp, and Tc when compared with wheat flour. This could be the result of greater heat stability of double helices comprised by longer amylopectin chains. In fact, Roman et al. reported greater gelatinization temperatures for banana starch, which was attributed to the longer population of short chains  [The melting of double helices of starch chains was evaluated through DSC . Flours  chains) . These r chains) .p < 0.05) soluble at all the evaluated temperatures (from 30 °C to 90 °C) when compared with the other flour sources (with the exceptions of no significant difference at 70 °C and 90 °C between the breadfruit flour A and wheat flour). Solubilized amylose molecules leach from swelled starch granules [Solubility is determined as the percentage of dissolved flour from a heated solution. The solubility generally increased when the temperature increased from 30 °C to 90 °C for each type of flour . Breadfrgranules . Lower sp < 0.05) than the tropical flours at from 30 °C to 70 °C. At 70 °C, two breadfruit flours had greater (p < 0.05) swelling power compared with banana flour and wheat flour indicating the overall weaker granule structure of the former flour types. Swelling power is a measure of hydration capacity and when flour is heated to above the gelatinization range in excess water, hydrogen bonds stabilizing the structure of the double helices in crystallites are disrupted and are embraced with water, thus leading to flour swelling and increased overall volume [Swelling power in reference to flours is the ability of the flour to imbibe water when heated in an aqueous suspension and is defined as the swollen sediment weight per g of dry flour. Generally, for each type of flour, the swelling power increased when the temperature increased from 30 °C to 90 °C . Swellinl volume .p) and conclusion (Tc) temperatures for the melting of retrograded amylopectin in tropical fruits compared to that from wheat, indicating that the amylopectin in tropical fruits is formed by longer A and B1 chains [During storage, amylopectin chains tend to re-associate with other amylose and/or amylopectin chains through the formation of double helices or aggregates of double helices . The the1 chains . The wid1 chains . Likewis1 chains .The absence of a well-developed peak was found in the pasting results of the first RVA method (temperature was increased up to 95 °C) , which ip < 0.05) compared with wheat flour (p < 0.05) than that of the wheat flour. However, pasting temperature did not statistically differ (p = 0.46) among treatments. Moreover, the viscosity of all the flours decreased during temperature holding at 130 °C (p < 0.05) than wheat flour. The peak viscosity and the breakdown were much greater than those of using the first RVA method. The differences between using the first and the second RVA method may be caused by different components such as protein and fat were attached to starch granules, which leads to uncompleted gelatinization process of the flours under 100 °C. The flours might need higher temperature to become fully gelatinized. It is interesting to note that the tropical flours had extraordinary swelling power, especially breadfruit flour, although they did not resist heating and the viscosity decreased significantly.For the pasting results of the second method (temperature was increased to 130 °C), the peak viscosity, trough viscosity, and final viscosity of the tropical flours, was greater  had greater water holding capacity and oil holding capacity compared with the traditional flour source (wheat flour). A high propensity of tropical fruit flour amylopectin re-associated during storage since tropical fruit flours exhibited a dramatically greater retrogradation enthalpy compared with wheat flour. The tropical flours had greater the peak viscosity, trough viscosity, and final viscosity compared to wheat flour. Moreover, the tropical flours did not resist heating but had extraordinary swelling power. Based on the results of flour composition, functional properties, and pasting properties, it is reasonable to conclude that both of these sources of flour (breadfruit flour and banana flour) may serve as useful ingredients and substitute the traditional flour such as, wheat flour in food products  with improved functional properties . The next stage of this research would be to investigate the use of these sources of flour in several different food applications.


UBE3A), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of Ube3a in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics.Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A  mice with a Ube3a-YFP fluorescent tag on the paternal allele (Ube3am+/pYFP mice) or mice that lack the maternal Ube3a allele and hence model AS (Ube3am−/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay.We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of Ube3a. Several compounds exhibited favorable paternal Ube3a unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax  and EC50 .All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal Ube3a unsilencers. All 13 tested compounds were effective at unsilencing paternal Ube3a, although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of Ube3a unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal The online version of this article (10.1186/s13229-018-0228-2) contains supplementary material, which is available to authorized users. UBE3A) gene, which is genomically imprinted. Only the maternally inherited copy is expressed in neurons [UBE3A is biallelically expressed in most other tissues. This neuron-specific imprinting provides insight into why deletions or mutations in the maternal copy of UBE3A primarily impact brain function and cause AS. However, the paternal UBE3A allele is intact, as demonstrated by biallelic expression in other tissues, raising the possibility that AS could be treated by unsilencing the dormant paternal UBE3A allele in neurons.Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by developmental delay, intellectual disability, speech impairment, seizures, and ataxia –5. AS ha neurons , whereasUBE3A. In a previous study, we developed a high-content assay to identify small molecules that could unsilence paternal Ube3a in mouse primary neurons. In that screen, we used knock-in mice carrying a yellow fluorescent protein (YFP)-tagged Ube3a reporter, allowing us to visualize maternal- or paternal-specific expression of Ube3a-YFP in cultured neurons. As expected, Ube3a-YFP was expressed in cultured neurons when inherited maternally but was not expressed (silenced) when inherited paternally. We found that topoisomerase I (Top1) inhibitors  could effectively unsilence paternal Ube3a in mice [Ube3a in vivo in mice, and unsilenced paternal UBE3A in induced pluripotent stem cell-derived neurons of AS patients [This led us to try pharmacological approaches to identify small molecules capable of unsilencing the dormant copy of  in mice , raising in mice  could bepatients .Topotecan is FDA-approved for the treatment of cancer and is well tolerated in adult and pediatric cancer patients –15. It iUbe3a unsilencing as many of these compounds produce particularly stable Top1 cleavage complexes [Ube3a unsilencing [↓C-- vs. topotecan --T↓G--) [Indenoisoquinoline-derived Top1 inhibitors offer a promising class of compounds for paternal omplexes –25, whicilencing . Over 30ilencing , 27–31. ilencing –25, 32. --T↓G--) –25, 32.Ube3a, with the expectation that some of these compounds might prove to be safe and have favorable CNS bioavailability. All of the tested compounds showed a capacity to unsilence the paternal Ube3a allele, with several of the compounds exhibiting unsilencing efficacy similar to topotecan. Excitingly, two of the tested indenoisoquinoline derivatives, indotecan (LMP400) and indimitecan (LMP776), are already in clinical trials [The goal of this study was to establish indenoisoquinoline derivatives that could effectively unsilence paternal l trials , 34. TheUbe3am−/p+) were generated by crossing Ube3am+/p− females with wildtype males. Paternal YFP-tagged mice [Ube3a+/pYFPm) were generated by crossing heterozygote Ube3a-YFP males with wildtype females. Mice were housed at 12 h:12 h LD and given ad libitum access to water and food. Both male and female mice (embryos) were used in all studies.All animal experiments were handled with an Institutional Animal Care and Use Committee (IACUC) protocol approved by the University of North Carolina School of Medicine. AS model mice   and indimitecan (LMP776) were obtained from the Developmental Therapeutics Program (DTP) branch, National Cancer Institute. Eleven structural analogues of indotecan and indimitecan, all indenoisoquinoline derivatives, were provided by Dr. Mark Cushman at Purdue University. Syntheses of 12 indenoisoquinolines have been previously described: indotecan (LMP400) and indimitecan (LMP776) , DB-III-analogue . These cUbe3a-YFP (Ube3a+/pYFPm) or maternal deletion of Ube3a (Ube3a−/p+m). Isolated cortical neurons were plated onto 384-well plates  for high-content imaging and onto 6-well plates  for Western blot analysis. Cultured neurons were initially incubated for 7 days, replacing culture medium every 3–4 days. Drugs were freshly prepared in DMSO as a 10-mM stock, unless further dilution in DMSO was necessary. On day 7 (DIV 7), the indicated compounds  and YFP-positive cells, and the number of YFP-positive cells was divided by the total number of cells. Fluorescence intensity was measured in neurons expressing unsilenced paternal Ube3a-YFP and normalized to vehicle control.We followed the protocols for fluorescent immunostaining and high-content imaging of cortical neurons as previously described , 38. BriToxicity of the compounds was tested in cultured cortical neurons in vitro. Using Cyto Tox-Glo assay (Promega), we followed the manufacturer’s protocol to measure luminescence proportional to the number of dead vs. live cells. Briefly, 72 h after drug treatment, we directly added AAF-Glo substrates into the drug-treated (or 0.1% DMSO vehicle-treated) neurons and incubated them at room temperature for 15 min. We then measured luminescence produced by dead-cell protease activity.We followed the same procedures of Western blot analysis as previously described , 38. In Ube3a-YFP or Ube3a, followed by Dunnett’s multiple comparison test. Two-way ANOVA was performed to determine changes in EC50, Emax, and LC50 . Moreover, indotecan exhibits favorable CNS penetration [The goal of this study was to identify novel Top1 inhibitors as potential AS therapeutics. We chose to focus on the compounds indotecan LMP400), indimitecan (LMP776), and their analogues because indotecan and indimitecan recently completed phase I clinical trial testing at the National Institutes of Health . On the other hand, topotecan (positive control) unsilenced paternal Ube3a-YFP as previously reported [Ube3a-YFP in our reporter mouse  DMSO-treated neurons expressed Ube3a-YFP above threshold  number of neurons expressed paternal Ube3a-YFP when cultures were treated with topotecan (33.0 ± 2.56%), indotecan (37.1 ± 5.19%), indimitecan (23.9 ± 1.72%), DB-IV-58 (17.7 ± 3.87%), or DB-V-37 (14.1 ± 3.55%) , fewer neurons treated with indimitecan, DB-IV-58, or DB-V-37 expressed paternal Ube3a-YFP compared to topotecan-treated neurons (p < 0.05 compared to topotecan-treated neurons).As we anticipated, paternal reported  , we performed Western blot analysis using primary cultured cortical neurons. No UBE3A-YFP protein was expressed in wildtype neurons (negative control), whereas paternal UBE3A-YFP was marginally detectable in DMSO-treated cells from Ube3am+/pYFP mice, possibly due to contamination from glial cells that express Ube3a biallelically or to very modest expression of paternal Ube3a expressed during early stages of development. Topotecan, indotecan, indimitecan, DB-IV-58, and DB-V-37 treatments led to paternal UBE3A-YFP protein production   neurons. Topotecan, indotecan, indimitecan, DB-IV-58, and DB-V-37 treatments result in a high level of paternal UBE3A protein compared to DMSO-treated neurons. These data confirm the ability of the tested indenoisoquinolines to unsilence paternal Ube3a.Although unlikely, we wanted to rule out the possibility that the unsilencing was an artifact of the 50) of indotecan being significantly better than topotecan (Table p < 0.05). This suggests indotecan may have potential as a possible AS therapeutic. Indimitecan is less likely as a candidate therapeutic, because although it exhibited good potency in a nanomolar range and had similar efficacy as topotecan , it likely has a low therapeutic index (discussed below). We also examined 11 structural analogues. Among 11 compounds, we found two of them, DB-IV-58 and DB-V-37, with similar efficacy and potency to topotecan , suggesting that it might have a low therapeutic index. Lastly, we also tested dose dependency of nine additional analogues  share similar pharmacological profiles, as their efficacy and potency were similar. However, their effectiveness of unsilencing paternal Ube3a-YFP was less than our lead compounds. Six compounds  unsilenced paternal Ube3a-YFP to a certain degree; however, they showed a very limited range of doses that produce Ube3a-YFP unsilencing before showing toxicities . Because of the limited dose ranges, their EC50 values were not clearly determined  in the cultured neurons revealed that the cytotoxicity of the first three drugs  was similar to that of topotecan. On the other hand, indimitecan exhibited toxicity at a significantly lower concentration than topotecan  as a potential AS therapeutic agent that warrants further examination in vivo for CNS bioavailability and safety.The goal of this study was to explore indenoisoquinoline derivatives as possible AS therapeutics by characterizing their effects on UBE3A governed by genomic imprinting provides a therapeutic opportunity for AS by reactivating the paternal UBE3A allele [UBE3A allele, providing the first proof of concept of pharmacological reactivation of paternal UBE3A as a potential therapeutic intervention for AS [Ube3a because of their similar ability to effectively inhibit almost 100% of Top1 enzymatic activity [ClinicalTrials.gov ID: NCT01051635).The unique expression of A allele , 9, 11. n for AS , 41, 42 n for AS , while an for AS . Howevern for AS , 18. Then for AS . Of manyactivity  and receUbe3a unsilencers, with different unsilencing properties. Of the compounds we tested in vitro, indotecan (LMP400) [Ube3a unsilencing properties than topotecan. While both topotecan and indotecan exhibit CNS penetrance [UBE3A unsilencers. Although indotecan exhibits better efficacy and potency than topotecan, the similar cytotoxicity of the two drugs must be considered for in vivo applications. Importantly, the DNA cleavage complex patterns of indotecan are different from topotecan in a manner that may confer some important advantages for clinical use. Indenoisoquinolines such as indotecan produce more stable cleavage complexes than camptothecins such as topotecan [Ube3a unsilencing [Ube3a unsilencing as we observed. Moreover, after drug removal, the Top1-DNA complexes induced by indenoisoquinolines persist under conditions where camptothecin-induced Top1-DNA complexes completely reverse [http://sea.bkslab.org) enables us to predict off-targets. SEA analysis revealed that indotecan possesses 55 potential off-targets, including aurora kinase A, aurora kinase B, and acetylcholinesterase. Regardless of off-target effects, clinical trials have demonstrated that, at least at the concentrations examined, indotecan is well tolerated in a clinical population [50 of ~ 26 nM to produce Ube3a unsilencing.Here, we demonstrate that indenoisoquinoline-derived Top1 inhibitors are potent LMP400) , 34 appenetrance , 40, the0 , 34 apnetrance , suggestnetrance . Moreovenetrance  appears netrance , 23. Theopotecan , which, ilencing , should  reverse . This ob reverse , 46, 47.Ube3a, and these compounds could be roughly categorized based on their ability to inhibit Top1 in cell-free assays: those compounds  that have 100% of the ability of camptothecin to stabilize the ternary drug-DNA-Top1 cleavage complexes [50 values were mainly due to limited effective dose ranges. Although we did not test their cytotoxicities in our cultured cortical neurons, we expect that they are more toxic than topotecan because we could not measure the fluorescence intensity in unsilenced UBE3A-YFP protein, possibly due to cell death produced at concentrations > 1 μM. On the other hand, we observed similar efficacy and potency of three compounds  with between 75 and ~ 100% Top1 inhibitory activities relative to camptothecin, but their effectiveness seems to be less than those of indotecan or topotecan. Interestingly, although DB-IV-26 and DB-V-41 have ~ 100% Top1 inhibitory activity, their EC50 values were also ambiguous due to limited effective dose ranges. Since the cleavage complexes are critical for producing paternal Ube3a unsilencing [Ube3a unsilencing. More importantly, the various hydroxylated side chains contribute to differences in the pharmacological action in Ube3a unsilencing. For example, the compounds with lower Top1 inhibitory activities  either lack the hydroxylated side chains that potentially serve as hydrogen-bond acceptors/donors that enable Top1 inhibitory activities and cytotoxicity at physiological pH [Ube3a.We tested 11 structural analogues of indotecan/indimitecan for their ability to unsilence paternal omplexes , those complexes . We alsogical pH  or the hUbe3a unsilencers will require optimization of Top1 inhibition and cytotoxicity through modulating chemical characteristics, including the length of the side chains. Although in vivo assays are necessary to further evaluate the unsilencing effects of indenoisoquinolines, this study provides a framework for developing novel AS therapies using different classes of Top1 inhibitors.Taken together, our study suggests that clinical development of paternal UBE3A could be one promising approach. Pharmacological inhibition of topoisomerase I (Top1) leads to re-expression of the dormant UBE3A allele. Here, we provided pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as Ube3a unsilencers in mouse neurons to identify potential AS clinical candidates. Our data suggest that a number of indenoisoquinolines, and in particular indotecan (LMP400), are potent unsilencers of the paternal Ube3a allele and should be further assessed in vivo for their translational potential.Angelman syndrome is a neurodevelopmental disorder without effective therapeutic interventions. However, pharmacological restoration of the epigenetically silenced copy of Additional file 1:Chemical structures of nine analogues of indotecan/indimitecan and their pharmacological properties in unsilencing of paternal UBE3A-YFP in vitro. A. DB-III-17, B. DB-IV-26, C. DB-IV-50, D. DB-IV-56, E. DB-V-41, F. DB-V-46, G. DB-V-47, H. MJ-II-66A, I. MNR-IV-64. Estimated potencies and efficacies of the drugs are summarized in Additional file Additional file 2:Image analysis in CellProfiler. Morphological changes in the neurons were assessed by nuclear structure of Hoechst-stained neurons. A Immunofluorescence images of Hoechst-stained neurons obtained using a BD Pathway 855 high-content imager (top). The size (between 18 and 40 pixel units) and intensity (threshold range between 0.005 and 1) of stained nuclei were used to segregate putative viable cells recognized as objects  from clumped or dead cells . Recognized objects were further processed based on size, intensity, and shape (round) . No objects were identified in the neurons treated with 10 μM indotecan, as no nuclei met the criteria of immunofluorescence size and intensity. B Quantitative analysis of identified objects. The average numbers of objects were comparable between neurons treated with DMSO (0.1% vehicle control) and indotecan (0.01 μM and 0.3 μM)-treated neurons. (PDF 338 kb)Additional file 3:Table S1. Potency and efficacy of nine analogues (PDF 270 kb)


Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure.Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented  Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low.Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae-related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure. Rhipicephalus microplus ticks in China , and Amblyomma javanense ticks  originating from Guinea; in Ugandan primates  were also reported previously , with Amblyomma testudinarium ticks from Lao People's Democratic Republic (Lao PDR), and with Ixodes ricinus ticks from metropolitan France, as well as in urine of Pteropus lylei bats from Cambodia and (ii) by using luciferase immunoprecipitation system (LIPS)-based serological screening of humans and cattle exposed to tick bites in France, Guadeloupe, and Lao PDR to determine the prevalence of JMTV-like infection in asymptomatic humans and cattle.In this study, we aimed at increasing the knowledge of the host range and geographical distribution of Jingmenviruses (i) by reporting the identification and full-genome sequencing of JMTV-like viruses associated with sensu stricto was first identified in various arthropods  , which probably reflects cofeeding of these two tick genera on the same cattle host, as previously suggested .Jingmen tick virus (JMTV)  Finland –7, 9, 11Finland –, 6). In uggested . Therefo10.1128/mSphere.00645-19.1FIG S1FIG S1, TIF file, 0.1 MB.Agarose gel electrophoresis of PCR targeting putative JMTV endogenous viral elements. “DNA” refers to total nucleic acids without an RT step; “RNA” refers to total nucleic acids with an RT step. Arrows highlight expected size bands. Download Copyright © 2019 Temmam et al.2019Temmam et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Flaviviridae. Similar genomic organization and expression patterns were reported for phylogenetically distant Jingmenviruses described in fleas, mosquitoes, crickets, and aphids by Ladner  the segmentation; (ii) the monocistronic or bicistronic expression patterns; and (iii) the presence of two key nonstructural viral proteins that are shared with y Ladner  and Shi y Ladner  Fig. 2)Flavivirius clade . The latRhipicephalus microplus  for the tick part and of JMTV strains isolated from Microtus obscurus Chinese rodents and Ugandan primate for the mammalian counterpart. The Laotian A. testudinarium strain of JMTV belongs to clade A, as does a Chinese A. javanense JMTV isolate. Interestingly, two sequences of Chinese tick-borne JMTV are embedded in the subclade of rodent-borne JMTV, suggesting frequent events of transmission between ticks and rodents and a possible role of reservoirs for rodents. Human JMTV-related viruses originating from Kosovo and tick-borne JMTV originating from French Antilles and from Trinidad and Tobago belong to clade B. Finally, clade C is composed of JMTV strains from I. ricinus originating from metropolitan France and Finland for the invertebrate part and of Chinese human Alongshan Jingmenvirus for the mammalian counterpart. Surprisingly, the Cambodian bat-associated strain is placed at different positions of the tree depending on the segment. For example, it is located at the root of clades A and B in segments 1 and 3 , whereas it is located at the root of all clades in segment 2. Similarly, bat-borne JMTV is located at the root of clade C in segment 4 . As for its flavivirus counterpart, NS5-like genes of Jingmenviruses code for the RNA-dependent RNA polymerase (RdRP) and the methyltransferase, as previously described (Tick-borne Jingmenvirus genome organization is well conserved between strains belonging to the three subclades . Segmentescribed . Segmentescribed . Segmentescribed . Interesluciferase immunoprecipitation system (LIPS). Results are presented in There is mounting evidence that Jingmenviruses constitute novel tick-borne arboviruses. We therefore analyzed two tick/human interfaces and one tick/cattle interface for the presence of specific JMTV antibodies in mammals by the use of a Ixodes ricinus ticks to French human populations exposed to tick bites was evaluated with LIPS assay targeting the two predicted external domains of JMTV glycoprotein (GP). None of the serum samples exceeded the positivity threshold when the second domain (ORF2) was used as the antigen. One slightly positive French human serum sample and a significant difference in light unit (LU) means (P = 0.00005) were observed between the two groups when GP ORF1 was used as the antigen , suggesting that the conditions for sustained tick-borne JMTV transmission to cattle are not present in Guadeloupe.The tick/human interface in Lao PDR mediated by ulations . SimilarEmerging infectious diseases are described as infections that are newly appearing in a population or that have existed but are rapidly increasing in incidence or geographic range . Among tFlaviviridae-related viruses with segmented genomes. The origin of this segmentation has been extensively described elsewhere . Thelsewhere , 17, thelsewhere . Indeed,lsewhere . Togethelsewhere , rodentslsewhere , or domelsewhere  that coupus bats , 22, indpus bats .At a more local scale, the ecological cycle of Jingmenviruses seems to be permissive to different arthropod hosts. Indeed, the wide range of distribution of arbo-Jingmenviruses between multiple tick species could be explained by frequent cofeeding of different tick species on the same mammalian host. This mechanism was described previously as “nonviremic transmission” of arboviruses by Labuda et al. . Those aIxodes ricinus JMTV was detected. Although this result needs to be further confirmed  to exclude the possibility of cross-reactions, the phylogenetic proximity of French JMTV to Alongshan virus human pathogen raised the issue of the circulation of Jingmenviruses in French human population. We observed that the seroprevalence of Jingmenvirus infection in asymptomatic populations was very low, as described previously in other studies  as previously published  were sampled in Guadeloupe and a total of 285 adult ticks  in Martinique between February 2014 and March 2015. Ticks were processed as previously described  collected in Lao PDR were homogenized in 400 μl of 1× phosphate-buffered saline (PBS) containing lysing matrix A beads . Ticks were ground for 6 min at 25 Hz in a TissueLyser II system (Qiagen). Homogenates were centrifuged at 14,000 × g for 3 min at 4°C, and 100 μl of supernatant and TRIzol LS (Invitrogen) were used for total RNA extraction. After extraction, residual DNA was digested with 20 U Turbo DNase (Invitrogen). RNA was purified with a RNeasy minikit (Qiagen), analyzed using a Agilent Bioanalyzer, and used as the template for library preparation using a SMARTer stranded total transcriptome sequencing (RNA-Seq) kit–Pico input mammalian kit , according to the manufacturer’s instructions. Library sequencing was performed on an Illumina NextSeq sequencer in paired-end 2-by-75-bp format outsourced to DNAVision.(ii) Processing of bat samples. A total of 481 Pteropus lylei bats were sampled during monthly captures performed between May 2015 and July 2016 in Kandal Province, Cambodia. Bats were captured using mist nets; handling and sampling were conducted following FAO guidelines . After extraction, DNA was digested with 20 U Turbo DNase (Invitrogen), and RNA was purified by the use of an RNeasy minikit (Qiagen), analyzed on a Agilent Bioanalyzer, and used as the template for library preparation using a SMARTer stranded total RNA-Seq kit–Pico input mammalian kit (Clontech). Library sequencing was performed on an Illumina NextSeq sequencer in paired-end 2-by-75-bp format outsourced to DNAVision.idelines  with thede novo assembly, ORF prediction, and BLASTP-based similarity search against the protein Reference Viral Database (RVDB  2017.97.NW).A total of 70 French human serum samples were collected in Alsace from persons with recorded tick bites. Ethics approval was obtained from the Comité de Protection des Personnes d’Ile-de-France VI in 2014, from the National Information Science and Liberties Commission in 2016, and from the French Ministry of Research .A total of 178 cattle sera collected in Guadeloupe in 1994 to 1995 and 22 cattle sera collected in 2019 were tested with the LIPS technology. These sera were obtained from other surveillance campaigns approved by the animal owners and the local representative of the French Ministry in charge of agriculture and fisheries.The LIPS antigens were designed as previously described . Extracet test (IC 95%).Significant differences between exposed and nonexposed groups of human sera tested by LIPS were calculated using the Student MN095519 to MN095534.Complete coding sequences of the four segments of tick-borne and bat-borne Jingmenviruses were deposited into the GenBank database under accession numbers


In practice, field evaluation of vaccine efficacy in individual herds is often based on a historical comparison of productivity data following initiation of vaccination. Being biased by time, this study design highly contrasts the more controlled, parallel-group design used for most initial vaccine efficacy studies but offers the possibility of including a larger number of animals and herds. As an important add-on to previous findings in controlled studies, the objective of this study was to evaluate the field efficacy of the ready-to-use combination vaccine Porcilis® PCV M Hyo  by an observational historical study design using routinely generated herd productivity data.p = 0.014), + 5 g for average daily weight gain (p = 0.555) and − 0.06 feeding units(FU)/kg for feed conversion rate (p = 0.074) in nursery herds and − 0.5 percentage points for mortality (p = 0.012), + 34 g for average daily weight gain (p < 0.001) and − 0.04 FU/kg for feed conversion rate (p = 0.133) in finishing herds. Even though some nursery and finishing herds also previously vaccinated against PCV2 and/or Mycoplasma hyopneumoniae, this did not significantly affect the results. For finishers, these results were obtained when difference in arrival weights between the periods and shared ownership of the herds were additionally taken into account.Data on mortality, average daily weight gain and feed conversion rate were collected as yearly averages for one year prior to and one year after implementation of Porcilis® PCV M Hyo vaccination from 20 nursery and 23 finishing herds. When comparing pre- and post-vaccination periods, the average improvements in productivity data amounted to − 0.4 percentage points for mortality  contains supplementary material, which is available to authorized users. Mycoplasma hyopneumoniae (M. hyopneumoniae) was first described [M. hyopneumoniae vaccines concerning significantly reduced enzootic pneumonia-like lung lesions [Since the safety and efficacy of Porcilis® PCV M Hyoescribed , the vacescribed –3. Some  lesions  and PCV2 lesions –5) and p lesions  Usually,M. hyopneumoniae generally originate from a cost/benefit assessment. While this is ultimately true for finishing herds, nursery herds might also vaccinate due to a request from the purchaser of growers. In the Danish production system, the nursery period lasts from weaning at 6–7 kg to around 30 kg and the finishing period from 30 kg to around 110 kg live weight.In Denmark, Porcilis® PCV M Hyo became commercially available in November 2014. Reasons for initiating a vaccination program against PCV2 and Herd identification was done in cooperation with swine veterinarians in order to find herds which had been vaccinating with Porcilis® PCV M Hyo according to label during minimum 1½ years and which routinely generated herd productivity reports. Furthermore, their herd health status had to be unchanged  during tM. hyopneumoniae and, to a varying degree, other respiratory pathogens such as Porcine Reproductive and Respiratory Syndrome (PRRS) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotypes 2, 6 and 12 [All herds were positive for 6 and 12  (Additio6 and 12 .M. hyopneumoniae (2 nursery and 4 finisher herds) to multiple different combinations of different PCV2 vaccines .Vaccination strategies in the different herds prior to initiating Porcilis® PCV M Hyo vaccination differed substantially /kg gain, where one FU = 7.38 MJ ) were coP-values < 0.1 were considered tendencies.Evaluation of Porcilis® PCV M Hyo vaccination efficacy was done herd-wise by calculating the change in each of the productivity parameters  by subtracting the herd average in Period 1 from the herd average in Period 2. The resulting units for the changes were therefore percentage points, g and FU/kg, respectively, and herd was the statistical unit of observation. This was done for nursery and finishing herds separately. As the changes in productivity parameters were all normally distributed, one-sample Student’s t-tests were used to determine whether the change in each parameter for nursery and finishing herds, respectively, was significantly different from zero. However, the significance level was adjusted from 0.05 to 0.017 (Bonferroni method) as three comparisons  were done for each dataset. M. hyopneumoniae as well as yearly national improvements merely due to genetics were relevant to consider for both nursery and finishing herds. As a proxy for the effect of yearly national improvement, the year of initiated vaccination with Porcilis® PCV M Hyo was used. The obtained changes were also compared to the national improvements during the same period , year of initiated vaccination and difference in arrival weight for inclusion as fixed effects with owner included as random effect to account for shared ownership. Model selection consisted of a standard stepwise selection procedure based on AIC (Akaike’s Information Criterion) where fixed effects with a p-value< 0.05 were retained in the models. Assessment of residuals for normality was done visually. All statistical analyses were done in R [For the change in each of the finisher productivity parameters, three linear mixed models were built testing previous vaccination strategies . All of these provided data on mortality, 19 herds on average daily weight gain and 17 herds on feed conversion rate.Table p = 0.168 , p = 0.286 (average daily weight gain), p = 0.793 (feed conversion rate)). At a national level, productivity parameters during the same period (2014 compared to 2016) increased 0.2 percentage points for mortality and 2 g for average daily weight gain, whereas feed conversion rate decreased by 0.04 FU/kg , + 34 g average daily weight gain (p < 0.001) and − 0.04 FU/kg feed conversion rate (p = 0.133). None of the variables explaining earlier vaccination strategy (against PCV2 or M. hyopneumoniae) or year of initiated vaccination were retained in the final models for any of the finishing herd productivity parameters. Neither did the difference in arrival weights significantly impact the changes in mortality or feed conversion rate.Results from the linear mixed models are displayed in Tables If the national changes in finisher productivity parameters from 2014 to 2017  were also found. When evaluating the size of the productivity changes, it is important to note that some of these also previously vaccinated against PCV2 and/or M. hyopneumoniae and that the improvements in productivity parameters for both nursery and finishing herds exceeded the improvements due to genetics in the same period. However, as data were collected retrospectively, the level of PCV2 and M. hyopneumoniae infections prior to initiation of Porcilis® PCV M Hyo vaccination was unknown, which is a possible confounder. Furthermore, due to the absence of non-vaccinated control herds, the study results cannot be considered directly applicable to herds in general. In future studies, a pre-vaccination diagnostic work-up in the herds would be valuable for later interpretation of results.This historical comparison of productivity data for 20 nursery and 23 finishing herds following initiation of Porcilis® PCV M Hyo vaccination demonstrated a significant improvement in average daily weight gain amounting to 34 g in finishing herds, confirming previous findings in randomized, controlled trials –5. In adAdditional file 1:Description of data collection regarding definition of Period 1 and 2, herd health status, previous vaccination status and productivity data. (DOCX 32 kb)Additional file 2:Overview of nursery and finishing herd details and national developments in productivity data during the study period . (DOCX 4


Cyclin-dependent kinase 5 (CDK5) is a unique member of the cyclin-dependent kinase family. CDK5 is activated by binding with its regulatory proteins, mainly p35, and its activation is essential in the development of the central nervous system (CNS) and neurodegeneration. Recently, it has been reported that CDK5 plays important roles in regulating various biological and pathological processes, including cancer progression. Concerning prostate cancer, the androgen receptor (AR) is majorly involved in tumorigenesis, while CDK5 can phosphorylate AR and promotes the proliferation of prostate cancer cells. Clinical evidence has also shown that the level of CDK5 is associated with the progression of prostate cancer. Interestingly, inhibition of CDK5 prevents prostate cancer cell growth, while drug-triggered CDK5 hyperactivation leads to apoptosis. The blocking of CDK5 activity by its small interfering RNAs (siRNA) or Roscovitine, a pan-CDK inhibitor, reduces the cellular AR protein level and triggers the death of prostate cancer cells. Thus, CDK5 plays a crucial role in the growth of prostate cancer cells, and AR regulation is one of the important pathways. In this review paper, we summarize the significant studies on CDK5-mediated regulation of prostate cancer cells. We propose that the CDK5–p35 complex might be an outstanding candidate as a diagnostic marker and potential target for prostate cancer treatment in the near future. Drosophila neurodegenerative model . Se. Se40]. Androgen deprivation treatment (ADT) is an essential therapeutic option for prostate cancer. Many prostate cancer patients are receiving standard ADT, and this treatment shows a better outcome for patient recovery within three years ,100. HowCDK5 is an essential kinase in postmitotic neurons and participates in the development of the central nervous system. CDK5 hyperactivation is important to the development of neurodegenerative diseases. CDK5 is involved not only in the central nervous system, but also in various biological and pathological processes such as gene expression, migration, apoptosis, and proliferation of prostate cancer, which is the most common type of cancer in men. The activator protein of CDK5, p35, is necessary to the activation of CDK5 protein in both the central nervous system and cancer tissues. The latest findings show that CDK5 is related to the pathophysiology of cancer-related pain  and type