The flour ratios for acha and pigeon pea were 100:0 (ACC), 80:20 (APC1), and 70:30 (APC2), respectively. The developed cracker biscuits were evaluated for chemical acid compositions, antioxidant, as well as antidiabetic properties. Protein contents of the formulated crackers increased with increase in supplementation with pigeon pea flour. The antinutrient content of the formulated snack was low hence may not adversely affect nutrient bioavailability. Glutamic and aspartic acids were the predominant amino acids while methionine and lysine significantly increased as a result of supplementation with pigeon pea flour. The biscuit exhibited good antioxidant properties indicated by its strong ability to scavenge hydroxyl, superoxide, DPPH radicals, and reduced Fe3+ to Fe2+. The formulated snack especially APC2 possessed low glycemic index (47.95%) and significantly inhibited the key digestive enzymes (α‐amylase and α‐glucosidase). All parameters evaluated indicated that APC2 could serve as a functional snack in the management of hyperglycemia (diabetes) and prevention of associated degenerative diseases.Pigeon pea was treated by blanching and used to supplement  It is a unique cereal with relatively high sulfur amino acid (methionine and cystine) content, and it has been shown to have a low glycemic index  on the other hand is an underutilized legume rich in protein, minerals, and other phytonutrients. This study was carried out to develop ready‐to‐eat snacks (crackers) from the blends of acha and pigeon pea flours as well as to evaluate the functionality of crackers from the flour blends and its potential in the management of hyperglycemia.Cereals and legumes play important roles in human nutrition. Recent reports have shown that they contain constituents with demonstrated health benefits for humans, such as antioxidant and antidisease factors  was purchased from Minna central market, Minna, Niger State, Nigeria, and pigeon pea (Cajanus cajan) seeds (TCc‐AO/TB78‐9) were obtained from the Gene Bank of the International Institute of Tropical Agriculture, Ibadan, Oyo State, Nigeria. Ammonia, sodium metabisulfite (SMB), lecithin, yeast, and enzymes were obtained from a biscuit company in Lagos, Nigeria. All other reagents used were of analytical grade and purchased from renowned chemical stores in Akure, Ondo State, Nigeria, and Canada. Acha and pigeon pea seeds were processed into flours by sorting, dehulling, drying, milling, and sieving according to the methods of Olapade, Aworh, and Oluwole  and SMB were mixed together and hydrated with water. It was subsequently added to the cream and mixed for about 5 min to obtain a hard‐extensive dough texture. The dough was allowed to ferment for 3 h and thereafter kneaded to a thickness of 1 mm. It was cut using a circularly shaped biscuit cutter, and docking was carried out to create air spaces. The baking was carried out at 150–200°C for 15 min.The cracker biscuits were produced as described by Han, Janz, and Gerlat , with sl2.1The moisture content (hot air oven method) and fat content (using soxhlet extraction method) were determined as described by Pearson  while as2.2The amino acid profiles were determined using the HPLC Pico‐Tag system according to the method previously described by Bidlingmeyer, Cohen, and Tarvin  after sa2.3g for 20 min, and the supernatant was filtered and stored at 4°C for further analyses.Aqueous extract of the biscuit was obtained by milling and hydrating 10 g of the milled biscuit in 100 ml of double distilled water for 24 h using a stirred plate. The suspension was thereafter centrifuged at 9000 2.3.1The scavenging activity of the biscuit extract against the DPPH radical was determined using the method described by Girgih, Udenigwe, Li, Adebiyi, and Aluko . Samples2.3.2The method described by Xie, Huang, Xu, and Jin  was used2.3.34. To initiate reaction in the wells, 50 μl of hydrogen peroxide (H2O2) solution was added to the mixture, which was then covered and incubated at 37°C for 1 h with shaking. Thereafter, the absorbance of the mixtures was measured at 536 nm every 10 min for a period of 1 h. The hydroxyl radical scavenging activity was calculated as follows based on change in absorbance (ΔA):The hydroxyl radical scavenging assay was modified based on a method described by Girgih et al. . An aliq2.3.43. 6H2O, at the ratio of 5:1:1 (v/v/v), respectively. Solutions of FeSO4·7H2O in the range of concentrations of 0.0625–1.0 μmol/μl were used for the calibration. The samples (40 μl) were placed into a clear 96‐well plate, and 200 μL of the FRAP reagent heated to 37°C was added. The reaction was carried out at 37°C, and the absorbance was read at 593 nm. The results were expressed in mmol of Fe2+ per mg of sample based on the calibration curve.The reducing power of the biscuit samples was determined according to the modified method of Benzie and Strain . Sample 2.42.4.1The inhibitory activity on α‐amylase was determined according to a modified version of an assay from the Worthington Enzyme Manual , the resulting supernatant was filtered using 0.45‐μm syringe filter. Samples were hydrated in 0.1 M sodium phosphate buffer (pH 6.9), acarbose served as the positive control. Sample aliquot (50 μl) was pipette into 96‐well clear plate, 50 μl of α‐glucosidase enzyme was added and incubated at 37°C for 10 min. After preincubation, 100 μl of 5 mM ρ‐nitrophenyl‐glucopyranoside solution was added to each well. The reaction mixtures were incubated at 37°C for 30 min, and readings were recovered every 5 min. Before and after incubation, absorbance was read at 405 nm and compared to a control which had 50 μL of buffer solution in place of the extract. The α‐glucosidase inhibitory activity was expressed as inhibition% and was calculated as follows:α‐Glucosidase assay was carried out according to the method of Kwon, Apostolidis, Kim, and Shetty  with a s2.4.3In vitro starch hydrolysis rate and hydrolysis index were determined according to the method of Gooni, Garcia‐Alonso, and Saura‐Calixto . SampleseGI = 39.71 + 0.549 HIwhere GI = Glycemic Index (%); and HI = Hydrolysis Index (%).2.5The in vitro protein digestibility was estimated using a multienzyme technique described by Hsu, Vavak, Satterlee, and Miller . Sample x = pH of protein suspension after 10 min of multienzyme digestionY = IVPD (%); 2.6Data generated was subjected to analysis of variance (ANOVA) using Statistical Package for Social Sciences (SPSS) V. 17.0. The means were separated using Duncan's Multiple Range Test (DMRT) at 95% confidence level.33.1p < 0.05) in the fat contents of the formulated snacks in spite of the fact that equal amount of fat (margarine) were measured for crackers production. The variation could be due to differences in the oil absorption capacities of the different flour blends. Protein contents (10.47–19.18%) increased with increase in percentage substitution with pigeon pea flour from 0% to 30%. Addition of pigeon pea flour in crackers has great potential in overcoming the common menace of protein‐energy malnutrition. Similar result of increased protein content from 5.0% to 14.2% was reported for biscuits from wheat–soybean flour . This could be due to the fact that the biscuit is composed mainly of acha cereal which has high carbohydrate content. Carbohydrate contents of formulated biscuits decreased from 65.42% to 54.59% as level of acha flour substitution increased. This implies that increased supplementation with legume and reduced percentage of cereal flour resulted in a reduction in carbohydrate contents and consequent increase in protein contents. Supplementation with pigeon pea flour contributed to reduced caloric contents; thus, the biscuit may be useful to consumers with health challenge requiring low carbohydrate content foods.The chemical compositions of crackers formulated from different flour blends are presented in Table 2+, hence, affect their absorption. Significantly higher phytate contents (Tables acha). The biscuits can be regarded as safe for consumption without a negative effect on mineral bioavailability  as the oxalate content is less than the recommended 50–60 mg per day consumption  than those of the cereal‐only biscuits (BCC and ACC) which had 0.04 and 0.11 mg/g TI, respectively. TI content was observed to increase with increase in supplementation with pigeon pea flour, which agrees with the report that legumes contain more trypsin inhibitors than cereals . However, antinutrient contents of the formulated crackers are low, hence high possibility of nutrient  bioavailability for consumers of the snack.The presence of antinutrients in food reduces nutrient bioavailability when such food products are consumed. Phytic acid is an antinutrient capable of binding to divalent minerals such as Ca, Mg, Zn, Fes Tables  were obss Tables . APC1 ans Tables . Sparvols Tables  also repacha flour is the main source of mineral in the snacks. The iron contents of the formulated biscuits were significantly higher than those of control (BCC) (p < 0.05). Iron is known to play an important role in nutrition. Potassium has a beneficial effect on sodium balance, and a high dietary intake has been shown to protect human from conditions affecting cardiovascular function. A Na/K ratio less than one is recommended in the diets for regulating blood pressure. The Na/K ranged between 0.77 and 3.38; however, the ratio for APC1 and APC2 was <1; hence, its consumption will not contribute to the sodium level of the consumer and suggests that the biscuits could be suitable snacks for hypertensive and diabetic patients. Healthy biscuits with low Na/K ratio of about 0.5 were reported by Mousa . Results obtained showed that the snacks are very important as protein sources due to the presence of sulfur‐containing amino acids, especially methionine ranging between 1.62 and 4.96 g/100 g with 100% acha cracker (ACC) having the highest. The formulated snack also exhibited increased lysine content with a supply of balanced amino acid superior to market sample which served as control (BCC). The biscuits showed a good profile of essential amino acids, which exceeded the FAO/WHO  and APC2 (55.92%) also exhibited significantly high radical scavenging activities. The formulated biscuits  were observed to scavenge the DPPH• better than market sample (BCC). The result suggests that they may easily donate free hydrogen atoms to electron deficient DPPH• thereby terminating the radical chain reaction and produce less harmful or unharmful products. The results obtained in the present study are higher than those reported by Adefegha and Oboh (Legumes are noted for their high levels of bioactive compounds which can influence glucose metabolism. Incorporation of legume (pigeon pea) flour into cereal flour for biscuits formulation can contribute to the bioactivity of the biscuits. Different mechanisms such as free radical scavenging activities and reducing properties are used by food to exert its antioxidant properties. Free radicals are generated by aerobic organisms during normal metabolic processes. They are highly reactive; oxidize protein, lipids, DNA, and consequently result in oxidative damage. Antioxidants play vital role in defending the human body against generated free radicals. The results of antioxidants activities of the biscuits are presented in Figure and Oboh  for whea3+ (ferricyanide complex) to Fe2+ in the presence of reductants (antioxidants). The Fe2+ was monitored by measuring the formation of Perl's Prussian blue at 700 nm  exhibited higher FRAP values than non‐supplemented biscuits (BCC and ACC). The ability to reduce Fe3+ to Fe2+ by the cracker biscuits increased with increase in supplementation with pigeon pea flour which may be attributed to the ability of incorporated pigeon pea flour to form reductants that could react with the free radicals thereby stabilizing and terminating the radical chain  higher antioxidant properties observed in the formulated biscuits compare with market sample (BCC) may be attributed to the cereal choice as well as legume flour incorporation which contributed potential bioactive properties to the snacks. Overall, the results of the antioxidant activities of the cracker biscuits suggest that the formulated snacks may be practical radical scavengers, capable of combating key degenerative diseases associated with free radicals as well serving as a functional snack for dietary intervention.Ferric Reducing Antioxidant Power (FRAP) is based on the reduction of Fe3.4acha‐pigeon pea cracker is presented in Figure acha cracker) had the least inhibitory activity (23.59%). Percentage α‐amylase inhibition increased with increase in supplementation with pigeon pea flour with APC1 showing inhibitory activity of 48.26% while APC2 exhibited the highest α‐amylase inhibitory activity (54.22%). There was no significant difference between the percentage α‐amylase inhibitory activities of BCC and APC2. The results obtained are, however, lower than that of acarbose (62.86% at 0.003 mg/ml), a potent antidiabetic drug for the treatment of type II diabetes. This discovery in this study agrees with previous reports which ascertained that plant phytochemicals and underutilized legumes inhibited salivary and pancreatic α‐amylase activities  refers to the actual amount of protein absorbed into the body relative to the amount that was ingested or consumed  is not sufficient to predict its bioavailability upon consumption. The protein digestibility profile of the formulated biscuits is presented in Table IVPD of the crackers decreased with increase in substitution with pigeon pea flour. The formulated crackers (ACC) with the least protein content (10.47%) had the highest IVPD (78.67%) while the crackers with the highest protein content (19.18%) showed the lowest IVPD (71.43%). There was no significant difference in the IVPD of BCC (72.34%) which had the least protein content (9.32%) and APC2 (71.43%) that showed the highest protein content (19.18%). This could be as a result of the high content of phytate in BCC which may bind to protein thus reducing nutrient availability, digestibility, and utilization. Also, the result suggests that high protein content does not directly imply high digestibility of the protein when ingested. The presence of antinutrients such as trypsin inhibitors has been reported to cause a reduction in protein digestibility, although the amount of trypsin inhibitors in the biscuit samples was quite low to account for the consequently observed decrease in IVPD of cracker biscuits containing pigeon pea flour. The observed decrease in IVPD may be as a result of nonenzymic browning reaction between flour protein and sugars resulting in possible nonreversible formation of compounds leading to reduction in the protein available for digestion. The results of Ayo, Ayo, Nkama, and Adewori  of the crackers ranged from 47.95% to 69.73%. According to GI classification of foods, values <55 are low GI foods, >55 but <70 are intermediate GI foods and >70 are high GI foods  showed the highest inhibitory activity against potent digestive enzymes (α‐amylase and α‐glucosidase) responsible for breakdown and absorption of carbohydrate. It equally exhibited the lowest glycemic index which may be relevant in lowering postprandial hyperglycemia. From the foregoing, the formulated crackers especially APC2 may be potential snacks for management of hyperglycemia and may serve as functional foods for the prevention of degenerative diseases.Increased supplementation of The study did not involve human and animal testing.The authors declare that there is no conflict of interests.


TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene  and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities.Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in  Within the last years, phenotypically and biologically coherent modules within this large and heterogeneous group have been increasingly delineated, indicating that overlapping phenotypes are often caused by mutations in genes involved in the same molecular networks5. This has been demonstrated mainly for well-defined pathways or complexes such as the RAS-MAPK-pathway4 or the SWI/SNF chromatin remodelling complex6, but is less characterized for disease genes involved in a broader biological context or process such as transcriptional regulation. Identifying and characterizing such connections and correlations contributes to a better understanding of the complex mechanisms and pathomechanisms in neurodevelopment and its associated disorders.Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous, and currently more than 1,000 genes have been implicated (SysID database# 610954), MEF2C-related intellectual disability , Mowat-Wilson syndrome , ATRX-related intellectual disability  and Angelman syndrome  represent a particular group of syndromic NDDs. They are important differential diagnoses towards each other and share phenotypic characteristics such as severe intellectual disability, epilepsy, postnatal microcephaly and some facial features7  models as done e.g. for autism spectrum disorders22 or to validate newly identified NDD-associated candidate genes by establishing biological links to phenotypically overlapping, known NDD-associated genes in terms of a chromatin-modification module5.Drosophila melanogaster as a model to screen for genetic interactions, we identified specific functional links between several genes in the fly, most stringent between Ube3a and Mef2. This interaction was furthermore confirmed in a human cell line using co-immunoprecipitation experiments. These molecular commonalities might contribute to the clinically overlapping features of the investigated disorders.By utilizing Drosophila melanogaster as an in vivo model system and tested genetic interaction between the fly orthologues Mef2 (MEF2C), Zfh1 (ZEB2), Daughterless (Da) (TCF4), XNP (ATRX) and Ube3a (UBE3A).To systematically investigate functional links between these genes of interest, we used Zfh1  or overexpression (five genes) either ubiquitously or in several different tissues and evaluated parameters such as viability, morphological alterations, synapse development and gross neurological behaviour  confirmed knockdown (KD) to 35–70% residual levels and 3 to 8.5fold overexpression (OE) for all used lines  and overexpression of all five tested genes resulted in lethality. Wing specific knockdown resulted in morphological phenotypes for two genes , and wing specific overexpression resulted in  lethality or morphological wing phenotypes for all five genes , and eye-specific overexpression resulted in lethality or morphological phenotypes for four of the genes  (Table Ubiquitous knockdown of two of the four tested genes Mef and Da aes Table . Table 11) Table .Figure 3Zfh1, Da or Ube3a or glial overexpression of either Zfh1, Da, XNP or Mef2 resulted in variable lethality phenotypes  additive, when the combined phenotype represented the sum of the two single phenotypes, b) as antagonistic, when the combined phenotype was milder than the more severe of the two single phenotypes, and c) as synergistic, when the combined phenotype was different or more severe than what was expected from the sum of the two single phenotypes.In the evaluation of viability and gross morphologic eye and wing phenotypes upon ubiquitous and tissues specific dosage manipulation we observed several modified phenotypes in the pairwise combinations Table . We consUbe3a and Mef2. Combined knockdown in either wing  and therefore in an improvement of the phenotype. In contrast, simultaneous knockdown of Ube3a and overexpression of XNP in the wing led to a more severe phenotype in the females, with stronger disorganization of cross veins compared to the phenotype in the single lines . Pan-neuronal overexpression of Ube3a in combination with knockdown of XNP resulted in worsening of incompletely penetrant lethality in females  and complete lethality in males .Also for combined dosage manipulation of nes Fig. . Pan-neuDa;OE_XNP construct compared to the respective single conditions, which, however, might represent an additive effect of both single gene conditions. No other consistent modifications of the observed single phenotypes were observed . Analysis of NMJ morphology and bang sensitivity upon pairwise dosage manipulations did not reveal any phenotypic modifications .Pairwise manipulations of other tested genes did not result in multiple or consistent phenotypic modifications. When we evaluated the climbing assay upon pairwise dosage manipulations, we observed a significantly increased climbing impairment of the OE_24 when simultaneously overexpressed in HeLa cells, indicating that a physical interaction is possible  or on a more systematic, large scale but consequently less detailed18 level. We now investigated possible functional links between TCF4, MEF2C, ZEB2, UBE3A and ATRX which are all implicated in clinically overlapping, severe human neurodevelopmental disorders. To do this, we utilized Drosophila melanogaster as a model system to systematically investigate functional links between the orthologues of these genes/proteins  in vivo.Though it has increasingly been acknowledged that similar clinical neurodevelopmental phenotypes are caused by mutations in genes/proteins connected in common networks and processes, this has mostly been characterized for specific complexes or pathways  glia development and maintenance has been reported or discussed43, it has not yet been characterized in detail. Transcriptome analysis on cell populations from mouse brain and on human brain tissues summarized in the Brain RNA-Seq database indicates expression of all five genes not only in neurons but also in astrocytes, oligodendrocytes and microglia, with a higher expression of TCF4, UBE3A and ATRX in fetal compared to mature astrocytes45. Our observations therefore might suggest not only a role of neuronal but also of glial defects that might contribute to the neurodevelopmental phenotypes in humans with mutations in either of these genes.Contrary to our expectations for NDD-relevant genes, we did not observe consistent synaptic or behavioural phenotypes upon pan-neuronal manipulation in the utilized assays. Although all of the investigated genes are associated with epilepsy in humans, neither pan-neuronal knockdown nor overexpression of any single condition or combination resulted in bang sensitivity, a 46, corresponding experimental follow-up of possibly underlying molecular commonalities is largely lacking. According to the mouse brain atlas47, there is overlapping expression of orthologues of UBE3A, MEF2C, ATRX, ZEB2 and TCF4 in several neuronal subtypes including excitatory neurons of the cerebral cortex and various cell types of the hippocampus. This would be in line with molecular commonalities in the pathomechanisms of the associated NDDs. However, according to the STRING database48 (status July 2019), there is no experimental evidence on interaction between the human proteins so far, and available data is restricted to co-expression or interaction in other species. In our genetic interaction screen, we observed modification of phenotypes upon combination of several manipulated genes. Severe phenotypes such as severe and fully penetrant lethality upon Zfh1 overexpression precluded further evaluation for genetic interaction. For all other combinations, we observed modified phenotypes which might point to a functional link between these proteins in terms of genetic interaction  and synergistic effects . Additionally, we confirmed a physical interaction between human UBE3A and MEF2C in an independent cell-based system. To our knowledge, this is the first evidence for a functional link between UBE3A/Ube3a and MEF2C/Mef2 that might contribute to the phenotypic overlap between Angelman syndrome and MEF2C-associated intellectual disability. Apart from that, variants in either UBE3A or MEF2C might also represent modifiers for the phenotypic expression/severity of the respective other condition as discussed for CNV models22.The most stringent evidence for a functional interaction was detected between Ube3a and Mef2. Several lines of evidence point to a true genetic interaction: a) we observed modified phenotypes in several tissues and in various knockdown/overexpression combinations, b) additive effects only can be excluded as some phenotypes occurred only upon pairwise manipulation  or were different or much more severe than expected from both single conditions (e.g. eye phenotypes), and c) we observed both antagonistic , and contributing to protein homeostasis by being involved in tagging substrate proteins with ubiquitin which are then degraded in the proteasome9. MEF2C (myocyte enhancer factor 2) belongs to the subfamily of MADS (MCM1-agamous-deficiens-serum response factor) transcription factors, whose transcriptional activity relies on the recruitment of and cooperation with other transcription factors as well as on translational and posttranslational modifications49. MEF2C might therefore be a transcriptional regulator of UBE3A expression. This might be supported by the identification of MEF2 binding sites in the Ube3a promoter50. Vice versa, also for UBE3A a transcriptional co-activation function has been reported51. However, a transcriptional regulation mechanism appears unlikely as in flies expression levels of Mef2 were unaltered upon Ube3a knockdown and vice versa, and as in a human cell line UBE3A knockdown did not change MEF2C expression levels. Additionally, stem cell-derived neurons modelling Angelman syndrome, did not show significant transcriptional changes of other genes (compared to 15q duplication neurons)52. As the most likely hypothesis, we suggest that UBE3A might regulate MEF2C activity and levels by ubiquitination, leading to subsequent degradation in the proteasome. UBE3A has been found to be located in the neuronal nuclei in discrete hotspots over euchromatin, thus well-positioned to regulate active genes24, and such a ubiquitin-ligase-dependent regulation has been reported for other transcriptions factors before in mouse models, e.g. for SOD153. Interestingly, only very recently, the critical importance of the nuclear isoform of UBE3A for the Angelman syndrome phenotype was characterized. Mice lacking the nuclear isoform but not mice lacking the cytosolic isoform displayed all major behavioural phenotypes and synaptic deficits also seen upon complete UBE3A knockout in the previous Angelman syndrome mouse model54. The hypothesis of ubiquitin-ligase dependent regulation of a putative nuclear substrate such as MEF2C by UBE3A is also supported by our genetic interaction findings. Pairwise knockdown of Ube3a and Mef2 in the fly resulted in antagonistic genetic interaction with ameliorated eye and wing phenotypes. This might be explained by knockdown of Ube3a leading to decreased ubiquitination and degradation of Mef2. Subsequently increased Mef2 levels might result in a partial compensation of the Mef2 knockdown phenotype. All other combinations resulted in synergistic genetic interaction, i.e. more severe phenotypes. This would be in line with a) overexpression of Ube3a resulting in increased ubiquitination and degradation of Mef2 and thus in a further decrease of its already low knockdown levels and b) knockdown of Ube3a resulting in decreased ubiquitination and degradation of Mef2 and thus in increased Mef2 abundance even above its overexpression levels. The observed interaction of MEF2C and UBE3A in co-immunoprecipitation experiments supports such a link. However, specific experimental proof of UBE3A regulating MEF2C in an ubiquitin-dependent fashion is still required.Taking into account the diverse functional roles of UBE3A and MEF2C, there are different possibilities conceivable how their interaction or mutual regulation might work. Observations from the genetic interaction and expression studies might already provide some insights into the possible nature of these interactions. The Drosophila melanogaster as a model to screen for genetic interactions and by subsequent co-immunoprecipitation in a human cell line, we identified a robust interaction between UBE3A/Ube3a and MEF2C/Mef2. These molecular commonalities might contribute to the clinically overlapping features of the associated disorders.By using Drosophila orthologues of TCF4 (daughterless [CG5102]), ZEB2 (Zfh1 [CG1322]), MEF2C (Mef2 [CG1429]), ATRX (XNP [CG4548]), and UBE3A (Ube3a [CG6190]) were identified with DIOPT55. Manipulation of Da/TCF4 in Drosophila has been established as a specific model for PTHS previously56.57. GAL4-driver stocks and transgenic lines for overexpression of Zfh1 and XNP, respectively, were obtained from the Bloomington Drosophila Stock Center. UAS-Mef2 was obtained from the Zurich ORFeome Project (FlyORF)31, and the UAS-Da line was a gift from Pascal Heitzler (IBMP Strasbourg). For generation of the UAS-Ube3a lines, the gene was amplified from fly cDNA  and cloned into a pUAST fly expression vector58. After verification of the sequence, the vector was sent to BestGene Inc. for the creation of a stable transgenic line. For a complete list of used Drosophila lines see Supplementary Table 58 and confirmed by quantitative RT-PCR  were obtained from the Vienna Drosophila Resource Center (VDRC)GMR-GAL4 (eye), ms1096-GAL4 (wing), repo-GAL4 (glia cells), elav-GAL4  and Actin-GAL4 . Resulting offspring were counted and analysed with a Carl Zeiss™ 2000C stereo microscope for gross morphological abnormalities. If the driver line contained a balancer chromosome, offspring with balancers were counted, too. In crosses with double OE/KD constructs retaining balancers, the expected ratio of balancer to non-balancer offspring may deviate from 50%. Wings and eyes were analysed per fly, and a phenotype was counted when it occurred in at least one wing or eye per fly, respectively. Wings were visually evaluated for parameters such as shape, degree of unfolding and wing vein structure. Eyes were visually evaluated for parameters such as ommatidial structure, bristles and gross size and shape. If applicable, p-values were determined using Fisher’s Exact test.RNAi- and transgenic overexpression lines were crossed to five different driver lines: 60 upon pan-neuronal manipulation (elav-GAL4). In brief, offspring were collected within 72 h of eclosion under CO2 anaesthesia in groups of ten . After 24 h, flies were transfered to the testing vial, tapped to the bottom and filmed for 30 s while climbing up. Time until 70% of the flies had crossed line at 8.8 cm height was measured from the videos. If less than 70% of flies from a vial managed to cross the line within the videotaped interval, time was considered to be 30 s. P-values were determined using the Wilcoxon-Mann-Whitney test, and Bonferroni correction was applied for multiple testing. For testing bang sensitivity, flies were vortexed for 10 s and filmed while recovering. The fraction of flies within a vial displaying spasms 5 s after vortexing was determined from the videotapes.Climbing behaviour and bang sensitivity was performed as described elsewhere60. Male L3 non-GFP larvae upon pan-neuronal manipulation  were dissected in PBST, fixated in 4% paraformaldehyde and stained with nc82 and anti-discs large antibodies . Secondary antibodies used were Alexa 488 labeled anti-mouse antibody and the Zenon™ Alexa Fluor™ 546 Mouse IgG1 Labeling Kit (ThermoFisher Scientific). Images were taken with a Zeiss Axio Imager Z2 microscope in z-stacks and analysed in ImageJ61. NMJ area and length, as well as the number of active zones, synaptic islands, branches, and synaptic boutons were determined from the image stacks. Per genotype, at least 11 NMJs from at least four independent larvae were analysed. P-Values were determined using the Wilcoxon-Mann-Whitney test, and Bonferroni correction was applied for multiple-testing. Upon Mef2 overexpression, we observed an additional signal in cross-segmental neurons with Dlg staining (red channel). This signal was also present in the parental Mef2 line, in another line from the same background, and was also present without Dlg staining  of muscle 4 was performed as previously describedFor RNA sampling from flies, whole larvae (~5), adult flies (~4), heads (~10) or larval brains (~40) were collected and frozen at −80 °C for at least one hour. Total RNA was isolated with the RNeasy Lipid Tissue Mini Kit (Qiagen) using TRIzol™ (ThermoFisher Scientific) instead of QIAzol and QIAshredder columns (Qiagen) for homogenization. DNAse digestion was performed on-column with the RNAse-free DNAse kit (Qiagen). Reverse transcription of RNA into cDNA was performed using the SuperScript™ II reverse transcriptase (ThermoFisher Scientific). RNA from HEK293 cells was isolated using the RNeasy Minikit. DNAse digestion and reverse transcription was performed as described above.Actin or Tubulin for Drosophila experiments or B2M for experiments on human cells. Relative expression levels were obtained using the ∆∆Ct method with isogenic background lines (Drosophila) or cells transfected with scrambled siRNA as references. Results were confirmed in at least a second biological replicate.Expression analysis was performed using the ABsolute QPCR SYBR Green ROX Mix (ThermoFisher Scientific) and (whenever possible) exon spanning primers (sequences available on request) on a QuantStudio™ 12 K Flex System (Life Technologies). Reactions were performed in quadruplicates, and Ct values were normalized to those of the endogenous controls MEF2C and UBE3A and respective negative control plasmids were obtained from Sino Biologicals  and used for transient transfection. Transfected HeLa cells were grown on poly-lysine coated coverslips, fixated with 4% paraformaldehyde in PBS for 10 minutes and stained with anti-Myc  and anti-HA  and with Alexa Fluor™ 488 goat anti–mouse and Alexa Fluor™ 488 donkey anti–rabbit antibodies . Nuclei were counterstained with DAPI (Serva). Images were taken with a Zeiss Axio Imager Z2 Apotome microscope with a 63x objective and analyzed in ImageJ.Expression plasmids expressing human HEK293 cells were transiently transfected with a combination of UBE3A-Myc and MEF2C-HA or the respective negative controls and harvested 48 h post transfection. Cells were scraped from the culture dish in lysis buffer . Immunoprecipitation was performed with Protein A Mag Sepharose bead suspension , incubated with the sample and anti-Myc or anti-HA antibodies  at 4 °C overnight. Subsequently, beads were washed in lysis buffer, and samples were eluted with 1x Lämmli buffer.Proteins were separated in stain-free 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad), blots were stained with anti-Myc and anti-HA antibodies and imaged on a ChemiDoc™ Touch Imaging System (Bio-Rad).UBE3A (Qiagen) were transiently transfected into HEK293 cells using jetPrime (Polyplus) according to the manufacturer’s instructions. 48 h post transfection, RNA for expression analysis (see description above), was harvested.Two different siRNAs against Supplementary Information.Supplementary Table S2.


Branchiostoma belcheri and the Florida amphioxus, Branchiostoma floridae has led to the conclusion that the rate of creation of novel domain combinations is orders of magnitude greater in lancelets than in any other Metazoa, a finding that contradicts the notion that high rates of protein innovation are usually associated with major evolutionary innovations. Our earlier studies on a representative sample of proteins have provided evidence suggesting that the differences in the domain architectures of predicted proteins of these two lancelet species reflect annotation errors, rather than true innovations. In the present work, we have extended these studies to include a larger sample of genes and two additional lancelet species, Asymmetron lucayanum and Branchiostoma lanceolatum. These analyses have confirmed that the domain architecture differences of orthologous proteins of the four lancelet species are because of errors of gene prediction, the error rate in the given species being inversely related to the quality of the transcriptome dataset that was used to aid gene prediction.Lancelets, extant representatives of basal chordates, are prototypic examples of evolutionary stasis; they preserved a morphology and body-plan most similar to the fossil chordates from the early Cambrian. Such a low level of morphological evolution is in harmony with a low rate of amino acid substitution; cephalochordate proteins were shown to evolve slower than those of the slowest evolving vertebrate, the elephant shark. Surprisingly, a study comparing the predicted proteomes of Chinese amphioxus,  Cephalochordates (lancelets), the basal group of extant chordates, diverged from other chordates (urochordates and vertebrates) about 550 Mya ,2. LanceBranchiostoma, Epigonichthys and Asymmetron), are widely distributed in tropical and temperate shallow seas inhabiting inshore waters protected from wave action. Phylogenetic analyses based on amino acid sequences of protein-coding genes of amphioxus species have revealed that the Asymmetron clade diverged first from the common ancestor of extant lancelets, followed by the Epigonichthys and Branchiostoma clades [Lancelets, consisting of three genera , develop through embryonic and larval stages, has provided the most extreme example of hybridization that has ever been demonstrated among multicellular animals as it occurs between species that diverged ~120–160 million years ago. Wilson and coworkers were the first to point out that the chief molecular barriers to interspecific hybridization are the regulatory system differences between the maternal and paternal genomes, which must function in concert if an interspecific zygote is to develop [B. floridae and A. lucayanum can give rise to interspecific hybrids also supports the notion that the rate of regulatory evolution is extremely low in the cephalochordate lineage. The recent observation of Holland et al.  that egg develop ,11. As a develop ,11. Bird develop ,11,12. ACephalochordates are thus prototypic examples of phenotypic stasis; practically no morphological change over very long evolutionary periods. The underlying process causing phenotypic stasis is that populations well adapted to their local environment are subject to stabilizing selection as long as the local environment remains essentially unchanged, that is, the populations occupy relatively stable niches ,14,15. FB. floridae and A. lucayanum have shown that strong purifying selection holds for the majority of genes and that these proteins are evolving more slowly than in the case of the slowest-evolving vertebrates [Studies on the proteomes of lancelets have provided evidence compatible with the notion that they are subject primarily to stabilizing selection; analyses of the sequences of protein-coding genes of tebrates ,17. B. belcheri, and the Florida lancelet, B. floridae, has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in any other Metazoa [Surprisingly, a study comparing the proteomes of the Chinese lancelet,  Metazoa . In this Metazoa . B. belcheri and B. floridae were compared is not accompanied by a high amino acid substitution rate. In agreement with the data of Delsuc et al. [Interestingly, the unusually high rate of domain architecture differences observed when the proteomes of c et al.  and Putnc et al. , the autc et al. ,20.B. belcheri and B. floridae might reflect gene prediction errors rather than true innovations [In an earlier work, we have suggested a possible explanation for these contradictions: the differences in domain architecture of orthologous proteins of ovations . The plaovations . Furtherovations .Asymmetron lucayanum [B. lanceolatum [B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, reflecting differences in the information-content of their transcriptome datasets.In the present work, we have extended our studies to include the genomes and transcriptomes of two additional lancelet species, ucayanum ,24 and Bceolatum . Furtherceolatum , we haveB. belcheri from the dataset Branchiostoma.belcheri HapV2_proteins.fa deposited on the website of the genome data of Chinese lancelet [We have randomly selected one hundred proteins of lancelet  as descrlancelet . B. belcheri proteins were downloaded from the Chinese Lancelet Genome website [HOX, OTX, KROX, and PBX genes that are crucial for determining anteroposterior patterning. In the present work, we have also selected key developmental genes that are known to be critical for determining anteroposterior (AP) and dorvosventral (DV) patterning, and left-right (LR) asymmetric development of amphioxus ,28,29,30 website . The genB. belcheri, the National Center for Biotechnology Information’s (NCBI) non-redundant database of B. floridae proteins, the transcriptomes of A. lucayanum and B. lanceolatum, as well as the human section of the high quality, manually curated UniProtKB/Swiss-Prot database. As the mean identity of orthologous proteins of the Chinese and Florida lancelets was calculated to be 81.2% [We have used the reciprocal best-hit method to identify orthologs of the selected proteins, using the Branchiostoma.belcheri HapV2_proteins.fa dataset for be 81.2% , we haveThe domain architectures of proteins (defined as the linear sequence of Pfam-A domains) were determined using Pfam . MultiplB. belcheri, B. floridae, B. lanceolatum, and A. lucayanum differed in domain architecture, we assumed that domain architecture deviation is more likely to reflect errors in gene prediction than true changes in gene structure [In cases where orthologous proteins of tructure . To disttructure ,34 to chTo decide whether the domain architecture deviation of the erroneous member(s) of the ortholog groups reflects error in gene prediction or true change in gene structure, we have re-annotated these genes using the FixPred protocol . The FixB. belcheri, 76% had orthologs in the human section of the UniProtKB/Swiss-Prot database , we assumed that this reflects an error of genome annotation rather than true loss of the gene. In such cases, we subjected genomic and/or transcriptomic databases to analysis by FixPred tools to find evidence for the presence of the ‘missing’ gene. Here, we illustrate this type of analysis with Ryk receptor tyrosine kinase.Out of the randomly selected proteins of database . In thisB. belcheri has a domain organization typical of Ryk receptor tyrosine kinases; they have a Wnt-inhinitory factor (WIF) domain that serves to bind Wnts [B. floridae was surprising  of the orthologs had full-length transcripts in the B. lanceolatum transcriptome. In the dataset of proteins involved in developmental regulation , all theB. belcheri, B. floridae, or A. lucayanum from those of their human orthologs reflect errors of gene prediction; in these cases, FixPred found genomic and/or transcriptomic evidence for the conservation of the domain architectures. In the case of B. lanceolatum, however, full-length versions of the protein fragments could not be reconstructed. In summary, our data suggest that the percentage of correct primary protein predictions tends to increase in the order B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, and that re-annotation with FixPred can verify the conservation of domain architectures, provided that genomic sequences are available. We can illustrate these conclusions with examples selected from We have shown that deviations in the domain organization of proteins of B. belcheri are orthologous with the C-terminal and N-terminal parts of XP_002607180.1 of B. floridae, respectively [B. floridae has the domain organization typical of WFIKKN proteins of vertebrates  of the WFIKKN gene and the genome assembly contained a single scaffold containing the 5′ part half of the WFIKKN gene (Sequence ID: LZCU01164105.1), permitting the reconstruction of the full-length sequence using the FixPred protocol. Analysis of the genomes and transcriptomes of B. belcheri, B. floridae, and A. lucayanum thus confirm that the domain architectures of WFIKKN proteins are conserved in lancelets.Proteins 328450_PRF0 and 328460 PRF0 of ectively . Comparitebrates . The ‘frKKN gene . The traB. belcheri has the domain architecture typical of calcium-binding mitochondrial carrier proteins, containing an EF-hand domain and three Mito_carr domains (B. floridae orthologue (XP_002602990.1) differs in domain architecture in that it has a C-terminal extension with a Peptidase_M2 domain. Because the XP_002602990.1 protein sequence violated basic dogmas that MisPred uses to detect gene prediction errors [B. floridae protein is mispredicted. Analyses of EST databases have confirmed this suspicion; ESTs originating from the putative ‘fusion’ region were found to align either with the N-terminal or the C-terminal fusion parts, but not both parts of the chimeric XP_002602990.1 protein, permitting the correction of the sequence [A. lucayanum; it contains full-length transcripts  of the calcium-binding mitochondrial carrier protein gene  supports the conclusion that the protein does not contain a Peptidase_M2 domain as it has a stop codon in the same region as its orthologs in the other lancelet species. Analysis of the genomes and transcriptomes of lancelets thus confirms that the domain organizations of calcium-binding mitochondrial carrier proteins are conserved in lancelets.The 330210_PRF0 protein of  domains . The pren errors , we suspein gene . The traB. belcheri and B. floridae proteins, however, differed from that of the human ortholog in as much as one of the B. belcheri orthologs fragments (256530_PRF0) lacks the JAB domain, another ortholog fragment (256520_PRF0) lacks the Myb_DNA-binding and SWIRM domains, whereas the B. floridae protein (B6MUN4.1) lacks the Myb_DNA-binding domain. Our analyses have confirmed that these deviations in domain architecture reflect errors in gene prediction that could be corrected with FixPred (MYSM1) genes is also supported by the analysis of the transcriptome of A. lucayanum; it contains full-length transcripts  of the MYSM1 gene. The transcriptome of B. lanceolatum had only one transcript fragment derived from its MYSM1 gene. Analysis of the genomes and transcriptomes of B. belcheri, B. floridae, and A. lucayanum thus confirm that the domain architectures of MYSM1 proteins are conserved in lancelets.Histone H2A deubiquitinases are conserved proteins involved in the regulation of nucleosome dissociation and transcription activation. The domain architecture of the human protein Q5VVJ2 (MYSM1_HUMAN) consists of a Myb_DNA-binding, a SWIRM, and a JAB domain. The architectures of the predicted  FixPred . The conB. floridae (Q0Q581_BRAFL), the domain architecture of the predicted sequence of the B. belcheri ortholog (Bb_056190F), however, is markedly different; it lacks two of the chordin (CHRD) domains, the C-terminal von Willebrand factor type C domain (VWC) domain is missing, the penultimate VWC domain is truncated, and the protein has a long C-terminal extension containing a polycystic kidney disease (PKD)_channel domain  of the CHRD gene. The transcriptome of B. lanceolatum had several transcript fragments derived from its CHRD gene. Although these fragments did not permit the reconstruction of the full-length protein, an EST (JT869941.1) matching the C-terminal part of the protein supports the conclusion that the protein does not contain a PKD_channel domain; it has a stop codon in the same region as its orthologs in the other lancelet species. Analysis of the genomes and transcriptomes of B. belcheri, B. floridae, and A. lucayanum thus confirm that the domain architectures of chordins are conserved in lancelets.Chordin is a key developmental protein that dorsalizes early chordate embryonic tissues by binding to ventralizing TGF-β family bone morphogenetic proteins and sequestering them in latent complexes. The domain architecture of the human protein is conserved in l domain . BecauseB. belcheri orthologs had domain architectures with no clear equivalents in the human section of the UniProtKB/Swiss-Prot database  domains [It must be emphasized, however, that the identity of domain architectures of lancelet orthologs, per se, does not guarantee that they are valid lancelet-specific innovations. Because of the similarity of lancelet genomes and the similarity of gene-prediction protocols, genome annotation may yield similarly erroneous predictions. This danger has been illustrated with the case of protein 018300_PFF0 of  domains . The ENCODE Genome Annotation Assessment Project  has showB. floridae and B. belcheri analyzed in the present work have been predicted by integrating the results obtained by de novo, homology-based, and transcriptome-based gene prediction methods [B. belcheri, prediction was aided by ~300 million ESTs [B. floridae gene, models were based on only about 480,000 ESTs [B. floridae transcriptome; whereas 83% of the predicted genes of the B. floridae complete genome sequence are represented in the B. lanceolatum transcriptome, only 41% of the genes were found to match the much smaller dataset from the B. floridae transcriptome [The original gene models of  methods ,18. In t000 ESTs . Oulion 000 ESTs  have alscriptome . B. floridae gene models were based on a significantly smaller dataset of ESTs than gene models of B. belcheri, misprediction may be more severe in the case of B. floridae genes. In harmony with this expectation, we have found that a higher proportion of B. belcheri proteins have the same domain architecture as their human Swiss-Prot orthologs than in the case B. floridae proteins  library, these authors have shown that more of the genome is intergenic than intronic, suggesting that the original genome annotation has erroneously fused or missed many genes. In a recent study, Francis and Wörheide have also pointed out major problems with the original genome assembly and genome annotation of the e genome . These ae models  had annoA. lucayanum [A. lucayanum, Yue et al. have identified 23,245 coding sequences (CDSs) (adult library) and 22,941 CDSs (20-h larvae library) of A. lucayanum, respectively [B. floridae, leading them to conclude that this lancelet genome contains 28,586 genes [The transcriptome of ucayanum  providedectively ,24. The 86 genes , a numbe86 genes .B. lanceolatum << B. floridae < B. belcheri < A. lucayanum, reflecting differences in the information-content of their transcriptomes. The results of our analyses confirm that the domain architecture differences of orthologous proteins of various lancelet species reflect errors of gene prediction; the proportion of correctly predicted proteins increases in the order


Ubiquitination plays a pivotal role in several cellular processes and is critical for protein degradation and signaling. E3 ubiquitin ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition. In order to achieve selectivity and specificity on their substrates, HECT E3 enzymes are tightly regulated and exert their function in a spatially and temporally controlled fashion in the cells. These characteristics made HECT E3s intriguing targets in drug discovery in the context of cancer biology. Post-translational modification of proteins by the addition of a ubiquitin (Ub) moiety can induce alteration in protein stability, function, activity, localization and interaction . This tiIn this review, we focus on the HECT-containing E3 ligases. Invariably at their C-terminus, all HECT E3s present the catalytic HECT domain, composed of a bulkier N-terminal lobe (N-lobe) that contains the E2 binding domain, and a C-terminal lobe (C-lobe) carrying the catalytic cysteine. The two lobes are connected by a flexible hinge region that allows the C-lobe to move around in order to facilitate the Ub transfer from the E2 to the E3 .According to the domain organization present in the N-terminal part of the proteins, the HECT E3s can be subdivided into three main families . The besThe activity of HECT E3s is tightly regulated in terms of chain specificity (mono- or poly-ubiquitination and Ub chain linkage), interaction with the E2 and recognition of the substrate.The ability to build linkage-specific poly-Ub chains appears to be an intrinsic feature of the HECT enzymes, as they are able to generate distinct Ub chains regardless of the paired E2 enzymes. NEDD4 family members primarily synthesize K63 chains , while EPrecise control of E3 ligase activity is needed to ensure that their functions are restricted until required. Several HECT E3s are kept in a catalytically inactive state by intramolecular interactions between the N-terminal region (either the C2 or the linker region between the WW domains) and the C-terminal HECT domain . For othA third level of regulation is represented by adaptor proteins that can modulate both the E2–E3 interaction and the interaction with the substrate. An example of the former is represented by SMAD7. SMURF1 and SMURF2 have low binding affinities for the E2-conjugating enzyme UbcH7, providing a point of control for regulating the Ub ligase activity through the action of auxiliary proteins. Indeed, SMAD7, functioning as a bridge between the E2 and E3, stabilizes an active complex and promotes, thus, the ligase activity . In otheNotably, HECTs themselves can function as adaptors for other conjugating enzymes as in the case of HERC2 whose binding to the N-terminal domain of E6AP increases the catalytic activity of E6AP .Finally, the catalytic activity of the HECT enzymes is often spatially and temporally regulated by post-translational modifications. Activating modifications can contribute to the release of auto-inhibiting conformational states of the E3s. For example, the phosphorylation of ITCH on the three residues of the proline-rich region releases the auto-inhibitory state generated by the binding of the C2 and the first WW domain to the HECT domain . LikewisUbiquitin ligases regulate a wide range of cellular processes and are involved in many human pathologies. Abnormal expression or dysfunction of HECT E3s have been shown in many different cancers. The current knowledge often suggests a dual role for these ligases in tumorigenesis, which might depend on the tissue context and/or additional events that affect their activity. Here, we will review the recent literature on E6AP, NEDD4, and HUWE1, and highlight excellent reviews for additional reading .The classical example of an HECT associated with cancer is E6AP. Since its discovery in 1993, it was evident that E6AP drives human papilloma virus (HPV)-induced cervical carcinogenesis, exerting its activity toward the tumor suppressor p53 through its association with the viral protein E6. E6 is an adaptor protein of the HPV and it is capable of binding to the N-terminal of EA6P and the DNA-binding domain of p53 , acting vs  as a more potent inhibitor. However, the binding mode and the specificity of this compound has not been experimentally validated.With the idea of identifying covalent modifiers of the catalytic cysteine of NEDD4, trations . ThroughA recent approach suggests the use of specific Ub mutants identified by phage display  to modulate HECT catalysis . The scrin silico screening of the hydrophobic pocket of the WW domains of SMURF1 led to the identification of compounds that possess features similar to the PPXY motif. These compounds bind the ligase and block SMAD1 ubiquitination, possibly disrupting the WW domain:SMAD1 interaction (A few molecules have been found to inhibit the HECTs, impairing substrate binding. An eraction . However50 values in the low micromolar range, leaving NEDD4 family members or the E1 and E2 enzymes unaffected. The compounds reduced the growth of colorectal cancer cells, but not that of HUWE1-depleted or normal epithelial cells of the colon (For many small molecules and inhibitors, the binding sites, the mechanism or the specificity have not been determined. By a high-throughput screening of small molecules, Eilers and co-workers identified two compounds that selectively inhibit the enzyme activity of HUWE1, as seen by a reduced substrate ubiquitination. Both compounds were found to inhibit the ligase activity with IChe colon . Howeverhe colon . With a he colon . The authe colon .Although tackling the ubiquitination system rather than the proteasome seems to be a promising avenue for therapeutic drug discovery, targeting HECT E3s to manipulate their activity is challenging for several reasons. First of all, we still lack the complete picture of their ubiquitome and their mechanism of action. Which substrates do HECT E3s ubiquitinate? What impact does ubiquitination have on their function proteolytic or non-proteolytic? How are these substrates recognized and how is their ubiquitination regulated in time and space and in different cellular conditions? What are the mechanisms the different HECT E3s apply to ubiquitinate their targets? So far, we only have a few answers for a small number of ligases and substrates due to the fact that ubiquitination is a dynamic and highly regulated process, and that the interaction with substrates is often transient with a low binding affinity. Besides the PPxY motif that is recognized by the WW domains of NEDD4 family members, no other substrate binding motif is known. An additional challenge is represented by redundancy. While E3s target multiple substrates, a specific substrate may be modulated by several E3s, depending also on the cell context. The high conservation of the HECT domain within the HECT family makes it a difficult target for which to develop specific inhibitory compounds. Finally, most of the HECTs act as both tumor suppressors and oncogenes, and more information is needed in order to find specific and effective compounds. Thus, acquiring more insights into the structural composition and the ubiquitination mechanism used by the different HECT E3s is of paramount importance in order to open new avenues for therapeutic interventions.JW, EM, and SP conceptualized and wrote this review. JW prepared the figure. All authors approved the final version of the manuscript and agreed to be accountable for the content of the work.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 toughening mechanism was found to be closely related with the morphology change from nanoscale droplet morphology to domain-in-domain morphology owing to the strong interfacial hydrogen bonding. The nanoscale morphology confinement and interfacial hydrogen bonding enhances the crystallization kinetics, while it lowers down the thermodynamic stability of the crystals. The toughening mechanisms were discussed based on these factors.Nanostructured polyamide 12(PA12)/polyketone (PK) blends were fabricated by melt compounding. The nanoscale droplet and domain-in-domain morphologies depending on PK content were observed. When the content of PK was 10 vol %, the impact strength of the blend jumped from 6.8 to 111.9 kJ/m Constructing nanostructures in polymer materials can lead to high performance because of the fascinating nanoeffect ,2,3. In b-poly(ethyl ethylene) (PEO-PEE) and poly(ethylene oxide)-b-poly(ethylene propylene) (PEO-PEP) diblock polymers with epoxy resins. They found that the nano-phase structures were generated through the self-assembly mechanism and that the nano morphology depended on the blend composition. Yang et al. 52] ratioFinally, the nanostructured morphology generates a high surface area, which in return promotes the interaction between PA12 and PK . The formed fine interfacial layers could enhance the interfacial adhesion and facilitate stress dissipation between the phases. In the impact test, the PK dispersed domain tended to deform or lead cavities to absorb the impact energy. Therefore, the nanostructured morphology and hydrogen bonding interaction are essential factors for the superior toughness of the blends.The morphology and crystallization of the PA12/PK blends showing greatly improved impact toughness were examined. The results demonstrated that PA12 and PK show intrinsic partial miscibility due to hydrogen bonding between the two polymers, which produces interesting phase morphologies depending on the PA12/PK ratio. When the content of PK in the blend was low, nano-sized isolated PK domains formed in PA12 matrix; the domain size increased as the PK content became higher. When the PK concentration exceeded 40 vol %, domain-in-domain morphologies were observed in the blends. The crystallization of both PA12 and PK components were accelerated, however, and resulted in less stable crystals according to the DSC and WAXD studies. The impact tests indicated that the PA12/PK blends showed significantly improved impact strength owing to the nanostructured morphology, interfacial hydrogen bonding and confined crystallization behaviors of the nanoscale domains. Our results bring new insights into the structure-property relationship of the nano-structured polymer blends.


A prominent challenge towards novel nanoelectronic technologies is to understand and control materials functionalities down to the smallest scale. Topological defects in ordered solid-state (multi-)ferroic materials, e.g., domain walls, are a promising gateway towards alternative sustainable technologies. In this article, we review advances in the field of domain walls in ferroic materials with a focus on ferroelectric and multiferroic systems and recent developments in prototype nanoelectronic devices. Materials research involving nanoscale properties of functional materials, especially in correlated electron systems, is faced with rich fundamental physics and a wide variety of phenomena that can potentially be exploited for new technology and applications. These systems present a great range of interesting aspects, such as superconductivity, magnetism, topological insulators, and ferroelectricity, to name just a few. Ferroic materials , possess a spontaneous polarization, spontaneous strain, and spontaneous magnetization or toroidal order. These materials can change their properties in a preconceived manner through the application of external fields. Thus, given their intrinsic structure, ferroic materials are ideally suited for functionality on the nanoscale. Ferroic phases typically arise in two or more distinct orientations of the order parameter; that is, they can form domains of uniform order which are separated by domain walls.Domain walls are one type of topological defect that can be found in ordered solid-state materials. The concept of using such topological defects as functional nanoscale elements  has giveIn this review, we confine ourselves to the latest advances in the field of domain walls in ferroic materials, with a focus on ferroelectric and multiferroic systems. Multiferroic materials correspond to systems exhibiting several ferroic orders. We start from a discussion of the basic properties of domain walls and later look into recent developments in prototype nanoelectronic devices.The transition of the order parameter in ferroic domain walls has been widely discussed from a theoretical perspective ,7,8,9. F3 x/[Ba(Ni0.5Nb0.5)O3−δ]1−x  interactions show a vast and interesting field of phenomena. Most of all they show that there is still a lot to be discovered and explained, which could lead to new prospects in future applications.So far, we have mainly discussed ferroelectric properties at (multi-)ferroic domain walls. In this section, we want to look at the magnetic properties in combination with electronic components in domain walls. Linking both orders can lead to new ways of manipulating electric and magnetic characteristics of multiferroic materials. The coexistence and coupling of magnetic and ferroelectric order are determined by the magnetoelectric effect. Depending on whether both ferroic orders appear independently or jointly, one talks about a type I or type II multiferroic, respectively. Extensive reviews on different types of magnetoelectric multiferroics can be found elsewhere ,136. Her3) revealed their highly complex domain structures. In h-RMnO3, each ferroelectric domain wall is accompanied by an antiferromagnetic wall with additional free antiferromagnetic domain walls [Magnetoelectric domain walls can occur in type I multiferroics, even though both ferroic orders appear independently. Experiments have shown that both multiferroic, as well as purely ferroic domain walls, exist in these materials. Extensive optical and SPM studies on hexagonal manganites  studies under the application of a small magnetic field have revealed a magnetic moment at ferroelectric/antiferromagnetic domain walls in h-ErMnO3 . Figure c moment ,150,151 c moment . Ferroelc moment ,154,155 omponent ,156.The appearance of a finite magnetic moment at different domain walls in these ferroelectric/antiferromagnetic multiferroics suggests an unexpected complexity of magnetic structures with domain walls that have the possibility to be manipulated by electric and magnetic fields.So far, we discussed materials where the magnetoelectric coupling at the domain walls is induced by their multiferroic domain properties. Unfortunately, magnetoelectric multiferroics mostly reside below room temperature, with rare exceptions like BFO. However, a wide range of ferroelectric and ferromagnetic materials do exist at room temperature. Recent experiments have shown that Neel-like domain walls in iron garnet films can possess a local polarization ,157. TheInhomogenous magnetic patterns are not restricted to domain walls; they should also lead to an electric polarization at magnetic vortices and Neel-like skyrmions . The feaNovel functional properties of ferroic domain walls, as outlined in previous sections, have fueled conversations of their potential in nanoelectronic devices and interconnects. However, the practical realization of ferroelectric solid-state domain wall device concepts has remained elusive until recently. This has been primarily due to issues associated with the injection of ferroelectric domain walls at precisely defined spatial locations and their controlled long-range displacement. Additionally, problems associated with precise control of electrical conduction at domain walls, conventional device configurations, and lack of experiments with suitable prototype device geometries and compatible ferroelectric film orientations have plagued rapid developments in the field.−4–10−1 Ωm) of the top electrode restricts the movement of the free charge in the electrode. As a result, the initial field  distribution under the top electrode is not uniform. This leads to nucleation of a reverse domain and domain walls at the tip location. With time, the field distribution evolves to a uniform state causing switching under the entire electrode. The time-dependent electric field evolution thereafter drives the propagation of the initially injected domain walls. Using this approach, domain walls can be deterministically injected at arbitrary spatial locations and displaced controllably over several micrometers. By controlling the geometry of the top electrode, simple domain wall multiplier circuits working either in series or parallel were fabricated [−1, which is still approximately three to four orders of magnitude smaller than that needed for nanosecond operation of submicron devices. Further, the applicability of the tip-top electrode approach requires the conductivity of metals to be high enough to screen the polarization, but at the same time low enough to limit the charge transport. These requirements can be fulfilled (to some degree) for metal electrodes fabricated using electron-beam-induced deposition [For a solid-state device comprising a ferroelectric film between two metal electrodes, the domain nucleation typically begins at one or more uncontrolled locations at the metal/ferroelectric interfaces. Therefore, the issue of the stochastic nature of domain nucleation and, as a result, the introduction of domain walls at random spatial locations must be addressed. Two strategies can be employed. The first one is based on tuning the strength of the applied field distribution in the ferroelectric film by introducing low permittivity structural defects  , such thbricated . Additiobricated . The fasposition .4, and devices were termed as ferroelectric domain wall diodes. This proof-of-concept experiment is an important milestone and potentially a precursor for realizing more complex ferroelectric domain wall devices such as wall shift register and domain wall logic. Here, although ferroelectric domain wall device research is still in its initial stages, it can be based on analogous research in the more mature field of ferromagnetism, which has already delivered demonstrator devices .3 are special because these films display only two out of the eight possible polarization directions with their in-plane components parallel to the film surface  with excellent endurance and retention characteristics. The encoding and nondestructive retrieval of information can be done at moderate biases, thus enabling low-energy operation. This study [In such a prototype domain wall device, both the interelectrode spacing and the lateral dimensions of one of the electrodes are restricted to a few hundred nanometers a to enabThe domain wall device we discussed above displays binary states . However, multilevel states can also be realized in these devices. There are several routes to achieve multilevel states including sequential injection of conductive domain walls as well as tuning the domain wall length and charge. The sequential domain wall injection has been shown ; howeverR~ρd; ρ and d are domain wall resistivity and length, respectively) of ferroelectric domain walls is directly proportional to the wall length. Therefore, domain walls of varying lengths can be thought of as the nanoscale analog of resistors of different resistances. As a result, one can potentially realize multilevel states in a two-terminal device by simply changing the wall length between metal electrodes. The wall length can, for instance, be influenced by the application of switching bias pulses of varying amplitude and duration. The feasibility of this idea was explored in an experiment in which domain wall devices with varying interelectrode spacings (from 400 to 90 nm), thus domain wall length, were prepared [I~e−d). Here, deep submicron interelectrode spacing (< 400 nm) is critical for the successful demonstration and operation of these domain wall devices. Further, the OFF:ON ratio (1 to ~103) increases with decreasing lateral spacing (400 to 90 nm) of metal contacts and points to high scalability  of these devices [The resistance  and stability of operation. For two-terminal device configurations, the OFF:ON ratio was around 100, while for the three-terminal devices it was ~3.In devices discussed thus far, the information is stored in domain walls. However, domain walls can also be employed to read information that is stored in the form of ferroelectric domains, as implemented in complementary metal–oxide–semiconductor (CMOS)-compatible commercialized ferroelectric random-access memories (FeRAM) ,177. In 0.7Sr0.3MnO3 (LSMO) electrodes separated by an ultrathin (4–5 nm) BaTiO3 layer. This head-to-head domain wall appears in the pristine state of the BaTiO3 and is caused by polarization pinning at the symmetric polar interfaces (TiO2/La1−xSrxO) with an effective ionic charge of +0.35 e−. The polar disruption at the head-to-head wall was argued to be sustained by electron donor oxygen vacancies, and it confines electrons to a wedge-shaped quantum potential well. This confinement drives energy quantization of electronic states in the well and affects the electron tunneling current of the device. Experiments below 100 K reveal conductance oscillations in the measured tunneling current and point to resonant transport through discrete unoccupied electronic states confined to the wall [Multiferroic tunnel junctions (MTJs) exploit the quantum phenomenon of tunneling electrons. In MTJs, the tunneling current between magnetic electrodes separated by an ultrathin ferroelectric barrier strongly depends upon the relative orientation of the magnetic moments of the electrodes and the polarization direction of the ferroelectric ,182,183.ltrathin –5 nm BaTthe wall . The meathe wall  further C [xSr1−xTiO3 (BST) films with a carefully chosen composition  were investigated. The domain wall phase diagram of BST exhibits a vertex at room temperature and, thus, a high variety of domain walls (domain size ~100 nm) in the as-prepared state. The experimental results showed gigahertz microwave tunability (1–10 GHz) and low dielectric loss. The device performance was comparable to that of bulk single crystals but was superior by a few orders of magnitude to state-of-art thin-film devices. Resonant domain wall fluctuations or oscillations in a domain wall rich sample, owing to proximity and accessibility of different domain wall variations, were determined as the main factors behind the observation of low dielectric losses and the exceptionally high-quality factors [Domain walls have recently also made an impact in the field of tunable dielectrics. Most telecommunication devices rely on the tunability of their frequency or the frequency agility in the radio spectrum. The usefulness of ferroelectrics in this area is well established . HoweverC , where tC  challeng factors .Another phenomenon that could potentially benefit from properties of ferroic domain walls is the negative capacitance in ferroelectrics. Ferroelectric materials with negative capacitance effects ,190,191 Domain walls and their unique properties continue to be widely explored. Over recent years researchers have gained significant new insight into fundamental properties and potential applications. Of special note is the utilization of the intrinsic structure of walls and the control of wall properties through order parameter coupling in multiferroic systems . FurtherLooking at the evolution of nanoelectronics devices based on domain walls, it is especially interesting to see how far the negative capacitance concept can be developed and if it can be applied in commercial devices ,190,191.


Prosopis laevigata) is a legume tree widely distributed in Aridoamerica. The mature fruit of this legume is a pod, which is currently underutilized and has high nutritional potential. In the present work, mesquite seed flour is described in terms of its nutritional value, as well as the effect of extrusion cooking on its bioactive components. Mesquite seed flour is rich in fiber (7.73 g/100 g) and protein (36.51 g/100 g), with valine as the only limiting amino acid. Total phenolic compound contents in raw and extruded seed flour were 6.68 and 6.46 mg of gallic acid equivalents/g (mg GAE/g), respectively. 2-2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity values in raw and extruded seed flour were 9.11 and 9.32 mg of ascorbic acid equivalent/g (mg AAE/g), respectively. The absorbance at 290 nm, as an indicator of generation of Maillard reaction product (MRP), was the same for raw and extruded samples. Apigenin was the only flavonoid found in mesquite seed flour (41.6 mg/kg) and was stable in the extrusion process. The water absorption index (WAI) and water solubility index (WSI) were changed significantly during extrusion. The expansion of mesquite seed flour extrudates was null due to the high protein and fiber content in the sample. Extrusion cooking of mesquite seed flour is a useful form of technology for the industrialization of this underutilized and nutritionally valuable legume.Mesquite ( Legumes have been an essential part of the human diet for centuries, with a major role in global food security, environmental challenges, and healthy diets . LegumesProsopis is comprised of a group of nitrogen-fixing trees belonging to the Fabaceae family distributed in arid and semiarid regions of Asia, Africa, and America. Prosopis species were a major staple food for indigenous peoples in arid regions of America before the arrival of Europeans [Prosopis is an indehiscent pod formed of an exocarp, a developed mesocarp, and a woody endocarp which protects the seed [Prosopis is a versatile ingredient with high potential for the food industry. It is rich in protein, sugars, and fiber, and is gluten-free [Prosopis species have been reported as a source of bioactive compounds with antioxidant, inflammatory, and antihypertensive activities [Prosopis alba showed high levels of proteins, minerals, fiber, and phenolic compounds, mainly flavones, with low content of total carbohydrates and fats [Prosopis laevigata is a good source of lysine, sulfur-containing amino acids, and total phenolic compounds, with higher radical scavenging capacity than soybeans and common beans [P. laevigata flours (for example in a baking process) increases the apparent total phenolic compound content and radical scavenging capacity, an effect associated with the generation of Maillard reaction products (MRPs) [Prosopis genera, and explore technological options for its processing, in the present work the seed flour of the specie P. laevigata, widely distributed in Aridoamerica, is described in its nutritional value, and the bioactive compounds content is analyzed in raw and extruded mesquite seed flour. The genus uropeans . The matthe seed . Pod floten-free ,9,10. Potivities ,11,12,13and fats . Pod floon beans . Thermals (MRPs) . In ordeGallic acid, 2-2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu’s phenol reagent, daidzein, genistein, myricetin, quercetina, kaempferol, and apigenin were from Sigma .P. laevigata at the experimental field of the Universidad Politécnica de Francisco I. Madero in the semiarid region of the Mezquital Valley in Hidalgo, Mexico. Collected yellow-brown mature pods were ground using a 900-W blender . Milled pods was sieved in a 30 mesh, retaining the intact endocarp. The intact endocarp was submitted to a second milling process, producing seed flour which passed through an 80 mesh and the retained brans.Dry mature pods were collected from trees of Crude fat, protein, moisture, fiber, and ash contents of seed flour were determined according to the procedures described in the Association of Official Analytical Chemists (AOAC), methods 920.39, 992.15, 925.09, 991.43, and 923.03, respectively .The amino acid content in raw flour was analyzed after hydrolysis with 6 N HCl, using a cation exchange separation column  with ninhydrin postcolumn derivatization, in an amino acid analyzer  . TryptopSeed flour was conditioned with purified water to obtain a moisture content of 16%. Seed flour was extruded in single screw extruder  equipped with a 19-mm diameter and 3:1 compression ratio screw, working at 170 rpm. Barrel temperatures were 80, 100, 120, and 150 °C for zones 1, 2, 3, and 4, respectively. Feeder rate was set at 30 rpm and the diameter of the exit die was 3 mm. Seed flour extrudate was dried and ground until it passed through 80 mesh. v/v) and centrifuged at 12,000 rpm/10 min. The extract was removed and the extraction process was done again with the residual pellet. Both extracts were mixed and diluted with 40% ethanol to obtain a final volume of 25 mL. These extracts were used for total phenolic compound content, radical scavenging capacity, and absorbance at 290 nm. For flavonoids, a replicate was prepared for each obtained extract and they were submitted to HCl hydrolysis with ethyl acetate aglycone recovery [Raw seed flour and extruded seed flour were extracted with aqueous ethanol . Samplesrecovery . The ethTotal phenolic compounds content was determined in raw and extruded seed flour extracts by the Folin–Ciocalteu reagent method . The absThe radical scavenging capacity of raw and extruded seed flour extracts was determined using the DPPH synthetic radical method . The absAnalysis of MRPs in extracts of raw and extruded flours was performed using the spectrophotometric method reported by Yu et al. . ApproprThe flavonoid content was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Dionex Ultimate 3000-DAD system (Thermo Scientific) supplied with an Acclaim 120 C-18 (4.6 × 100 mm) column . For sepThe water absorption index (WAI) and water solubility index (WSI) were assessed before and after the extrusion process . The expp ≤ 0.05). Assays were performed in triplicate, and expressed as means ± standard deviation. Data were submitted to analysis of variance (ANOVA) and means were compared by Tukey test  during thermal treatment have been associated with increases in the total phenolic compound content and radical scavenging capacity in pods flours of aevigata , flours aevigata , and in aevigata . The reaaevigata . These Maevigata ,20. In saevigata . Flours aevigata . In the aevigata . MesquitP. alba [Prosopis nigra [P. alba [The content of apigenin in raw and extruded mesquite seed flour is shown in P. alba ,13,14, ais nigra  without [P. alba ,30,31. D[P. alba ,33,34,35Prosopis seed flour extrusion. In the present work the extrusion cooking process of mesquite seed flour increases the WAI significantly. Water absorption indicates the amount of water immobilized by the material, as the dispersion by denaturalization of macromolecules such as proteins and starch during extrusion increase the WAI [Water absorption index (WAI) and water solubility index (WSI) of raw and extruded mesquite seed flour, together with the expansion index of extruded mesquite seed flour, is shown in  the WAI , an effe the WAI . In the  the WAI . Raw mes the WAI , an effe the WAI . Another the WAI . The extMesquite seed flour is a valuable plant food rich in good quality protein and active compounds. The extrusion cooking process of mesquite seed flour is an optional and versatile technology useful in the development of functional foods and industrialization of this underutilized legume.