Formins are proteins that assist in regulating cytoskeletal organization through interactions with actin filaments and microtubules. Metazoans encode nine distinct formin subtypes based on sequence similarity, potentially allowing for great functional diversity for these proteins. Through the evolution of the eukaryotes, formins are believed to have repeatedly undergone rounds of gene duplications, followed by diversification and domain shuffling, but previous phylogenetic analyses have shed only a little light on the specific origins of different formin subtypes. To improve our understanding of this in the case of the metazoan formins, phylogenetic comparisons were made here of a broad range of metazoan and non-metazoan formin sequences. This analysis suggests a model in which eight of the nine metazoan formin subtypes arose from two ancestral proteins that were present in an ancient unikont ancestor. Additionally, evidence is shown suggesting the common ancestor of unikonts and bikonts was likely to have encoded at least two formins, a canonical Drf-type protein and a formin bearing a PTEN-like domain. In vitiewed in ,6). The iewed in ,8. In adA detailed analysis of conserved motifs in diverse FH2 domains had suggested that the FH2 fold is likely to have arisen only once during the evolution of eukaryotes . Among vThe animal (metazoan) formins provide a group whose evolutionary origins are particularly interesting. Metazoan formins can be categorized into nine subtypes based on the degree of similarity of their FH2 domain sequences , designain vivo by RhoGTPases [The most common domain organization found among metazoan formins is that of the diaphanous-related formin (Drf), as found in DIAPH, DAAM and FMNL formins. Drfs have an extended N-terminus that includes a Rho-family GTPase-binding domain  followed by an armadillo repeat-rich diaphanous inhibitory domain (DID) and an α-helical dimerization domain (DD) ,15. For oGTPases ,15,23–25Several additional metazoan formin subtypes resemble Drfs with modest alterations . MWHFs lAmphimidon queenslandica, a member of the basal metazoan phylum Porifera, encodes formins from all nine subtypes. This suggests the nine subtypes were already in existence in the last common ancestor of the metazoans [Monosiga brevicollis, a close relative of metazoans, have suggested at least four metazoan subtypes  were already present in the common metazoan/choanoflagellate ancestor [Dictyostelium discoideum revealed a clear kinship between metazoan FMNs and several D. discoideum formins [We have relatively little insight as to how this diversity of metazoan formins arose. Members of all nine subtypes are widespread across many metazoan phyla. It may be particularly telling that the sponge etazoans . Compariancestor ,12. Comp formins ,11,12.Using a large set of sequenced non-metazoan genomes, this analysis attempts to revisit and expand on those earlier studies. Revealed are previously unappreciated relationships between specific metazoan formin subtypes and the formins of fungi, amoebozoans, and other non-metazoans. The results support a model in which eight of the nine metazoan formin subtypes originated from two formins that were present in an ancestral unikont.https://www.ncbi.nlm.nih.gov/) and the website of Ensembl Genomes , INF2, FMNL1, and FHOD1, Strongylocentrotus purpuratus LOC100890634 (a PHCF protein), and Caenorhabditis elegans FOZI-1 (a highly divergent FMNL-related protein). Non-exhaustive searches for formins in bikont species were performed by searching the relevant species dataset on the Ensembl Genomes website using the search term, "FH2". All identified formins are listed by species in Metazoan formin sequences considered here were identified previously . Unikontmes.org; ), and semes.org; . For uni2) . PredicAmino acid sequence alignments were performed in MegAlign (ver 13.0.0) of the Lasergene software suite  using Clustal W , with thEvolutionary histories were inferred from the sequence alignments by applying the Maximum Likelihood (ML) method using the LG model  + G  were aligned with those from eleven non-metazoan species (see below), and a ML tree was estimated ; and PH-PH-FH1-FH2-PH for the PHCFs -like domain, which is a composite domain that includes a protein tyrosine phosphatase (PTP)-like fold and a membrane-binding C2-like fold  formed a well-supported group  based on their FH2 domain sequences . PHYRE2 ike fold . The M. ld, only . Based oSummarizing these observations for the formins of Holozoa, distinct FMNL, MWHF, FMN, and INF proteins were present at least before the divergence of choanoflagellates and metazoans, while DAAM, PHCF, FHOD, and DIAPH-like proteins are likely to have existed in the common holozoan ancestor of filastereans, choanoflagellates, and metazoans . DistincFonticula alba, and from five representatives of four phyla from Fungi, Saccharomyces cerevisiae and Schizosaccharomyces pombe of Ascomycota, Ustiago maydis of Basidoimycota, Spizellomyces punctatus of Chytridiomycota, and Encephalitozoon intestinalis of Microsporidia  appeared among the holozoan DAAM proteins Figs , but its2 predicted no PH domains for Eint_101200, but instead predicted an N-terminal PTEN-like domain with PTP- and C2-like folds  clustered with the choanoflagellate PTEN-formins with moderate to strong support  domain at the extreme N-terminus of the fonticulid formin  had previously shown that it adopts an FHOD-like GTPase-binding (G2) fold [The N-terminal domain organizations of FHOD and FMN proteins differ dramatically . In thisproteins . Moreoveproteins . SupportG2) fold .One divergent feature of two apusozoan FHOD/FMN-related proteins was a predicted PH domain at their extreme N-terminus , but thiThese results suggest that the common unikont ancestor for Opisthokonta, Apusozoa, and Amoebozoa encoded an ancestral FHOD/FMN-related formin with a G2-DID-DD-FH1-FH2 domain organization . Among aMost remaining apusozoan and amoebozoan formins featured a Drf-type G-DID-DD-FH1-FH2 domain organization, often supplemented with additional domains and motifs unique to particular isoforms ,11. NoneArabidopsis thaliana and Physcomitrella patens, and two representatives of Heterokonta, Phaedactylum tricornutum and Phytophthora ramorum  and these non-Drfs (G2-DID-DD-FH1-FH2), arose from a duplication and divergence of an ancestral Drf. The presence of such non-Drfs among apusozoans, amoebozoans, and opisthokonts suggests this duplication and divergence occurred early in the unikont lineage, before the divergence of those three groups. Based on an absence of apparent further similarity of subgroups of the Drfs and non-Drfs of these organisms, this model suggests the ancestral Drfs and non-Drfs further multiplied and diverged independently within the apusozoans, amoebozoans, and opisthokonts.Within the opisthokonts, the ancestral Drf likely diversified early, explaining the observation that holomycotes (including the fungi) and holozoans (including metazoans and choanoflagellates) share several related groups of Drfs. One ancestral opisthokont Drf (Drf1) was likely similar in sequence to the metazoan DIAPH formins. Thus, DIAPH-like formins can be found broadly among the fungi, including the well-studied formins of the budding and fission yeasts. Within the metazoan lineage, the ancestral DIAPH-like formin appears to have duplicated very early, just after the divergence of metazoans from choanoflagellates, to give rise to the conserved DIAPH subtype and to the GRID2IP subtype which acquired a novel PDZ/HN domain-rich N-terminus.A second ancestral opisthokont Drf-type protein (Drf2) appears to have diversified independently in the holozoans and holomycotes, to give rise to DAAM, FMNL, and MWHF subtypes in metazoans and choanoflagellates, and to a group of holomycote formins that show intermediate relatedness to the DAAM/FMNL/MWHF subtype proteins. Additional ancestral opisthokont Drfs were also likely present to give rise to the formins of the holomycote Fungi-2 subtype, and possibly the INF subtype formins of metazoans and choanoflagellates. The model in Among the opisthokonts, the G2-DID-DD-FH1-FH2 non-Drf appears to have been lost from the holomycotes, but duplicated and diverged in the holozoan lineage. One duplicate lost its dimerization motif to become the FHOD subtype proteins with the domain organization G2-DID-FH1-FH2. The other duplicate lost all of its N-terminal domains and adopted a novel N-terminus, producing the FMN proteins with domain organization X-FH1-FH2. Thus, based on domain organization, the FMN proteins are unrecognizable as descendants of the ancestral non-Drf. However, the clear relatedness of the FMN FH2 domain sequences to those of FHOD proteins and ameobozoan and apusozoan non-Drfs strongly supports this origin. The presence of distinct FHOD and/or FMN subtype proteins in all the holozoans examined here suggests that the duplication and divergence of the ancestral non-Drf occurred in the holozoan lineage before the divergence of the metazoans, choanoflagellates and filastereans.Thus, this model suggests that eight of the nine metazoan formin subtypes ultimately arose from a G-DID-DD-FH1-FH2 Drf and a G2-DID-DD-FH1-FH2 non-Drf that were present in the last common unikont ancestor of the apusozoans, amoebozoans, and opisthokonts. The model suggests the PTEN-formin was also present in this ancestral unikont. Based on the single representatives of Apusozoa and Amoebozoa examined here, the PTEN-formin might have been lost from the lineages leading to those groups. Among the opisthokonts, PTEN-formins appear to have been lost among several lineages, including the metazoans, but were retained among others, including the fungi and choanoflagellates.The origin of the ninth metaozoan subtype, the PHCFs, is unclear. PHCF homologs were found in representatives from all the holozoan groups examined here, suggesting that the last common holozoan ancestor encoded a PHCF. However, among the formins sampled here, the PH-PH-FH1-FH2-PH domain structure is unique to PHCFs, and the FH2 domain sequences of PHCFs show no particularly strong relatedness to other eukaryotic formin subtypes. One possible explanation for this is that PHCFs might have arisen through horizontal transfer into the holozoan ancestor from some other lineage. The presence of formins with PH domains among heterokonts  points to a possible source. Alternatively, the ancestral PHCF might have arisen through a novel rearrangement that combined an FH2 domain with a series of three PH domains. This, coupled to a particularly high degree of sequence divergence for this FH2 domain might have obscured its relationship to other formins. Perhaps with identification of additional non-metazoan formins, the relationship between the PHCFs and the remaining members of the formin family tree will be discovered.While previous phylogenetic studies have delineated conserved subgroups of formins within particular kingdoms of organisms, they have generally failed at revealing relationships between formins from different kingdoms. With the availability of genomes from organisms more broadly distributed across the eukaryotic family tree, it is now becoming possible to trace some of these relationships. As shown here, the origins of the formin subtypes of the metazoans are deep. The evidence suggests a gradual process of formin duplication and divergence occurred over time during the evolution of metazoans from an ancient unikont ancestor.Formin diversity seems likely to contribute to the ability of metazoan cells to assemble a wide variety of cytoskeletal architectures, particularly actin-based ones. Formin diversity may also have contributed to the evolution and diversification of actin, itself, in metazoans. Actin plays essential roles in the cell, and interacts with bewildering number of proteins, placing strong constraints on its ability to evolve. It has been hypothesized that the ability of cells to assemble distinct populations of actin filaments, which provide alternative selective environments for actin, may have been a precondition to allow for the evolution of multiple actin isoforms in metazoans . ConsideS1 Table(XLSX)Click here for additional data file.S1 Text(TXT)Click here for additional data file.S1 FigEvolutionary histories for 180 FH2 domain sequences from the indicated species were inferred by the ML method using the LG + G model for 270 positions that were occupied ≥ 95% of FH2 sequences. Most previously identified major groups of unikont formins were reproduced here, as were the three plant formin subtypes , and a group of heterokont formins that was associated with the Class II plant formins. Formins that encode N-terminal PTEN-like domains are indicated with red arrows. All bootstrap values are indicated, and the scale bar indicates the number of substitutions per site for branch lengths.(TIF)Click here for additional data file.


As fermentation could reduce the negative effects of bran on final cereal products, the utilization of whole-cereal flour is recommended, such as brown rice flour as a functional food ingredient. Therefore, this study aimed to investigate the effect of fermented brown rice flour on white rice flour, white rice batter and its steamed bread qualities. Brown rice batter was fermented using commercial baker’s yeast (Eagle brand) according to the optimum conditions for moderate acidity (pH 5.5) to obtain fermented brown rice flour (FBRF). The FBRF was added to white rice flour at 0%, 10%, 20%, 30%, 40% and 50% levels to prepare steamed rice bread. Based on the sensory evaluation test, steamed rice bread containing 40% FBRF had the highest overall acceptability score. Thus, pasting properties of the composite rice flour, rheological properties of its batter, volume and texture properties of its steamed bread were determined. The results showed that peak viscosity of the rice flour containing 40% FBRF was significantly increased, whereas its breakdown, final viscosity and setback significantly decreased. Viscous, elastic and complex moduli of the batter having 40% FBRF were also significantly reduced. However, volume, specific volume, chewiness, resilience and cohesiveness of its steamed bread were significantly increased, while hardness and springiness significantly reduced in comparison to the control. These results established the effectiveness of yeast fermentation in reducing the detrimental effects of bran on the sensory properties of steamed white rice bread and encourage the usage of brown rice flour to enhance the quality of rice products. The past two decades have seen a rapid increase in consumer demand for healthy foods, which has prompted recent research to find methods for production of healthy and functional foods. The usage of whole grain cereal instead of milled cereals is one such trend for production of healthy and functional foods, as consumption of whole grain foods has shown a reduction in the risk of several diseases, such as cardiovascular diseases, obesity, diabetes and some types of cancers ,2. One oRice is a unique crop due to its colorless, soft taste, low sodium levels, easy digestible carbohydrates and hypoallergenic properties. Therefore, its flour is an attractive food material to be used for making gluten free foods . SteamedGluten free materials such as white rice flour do not have the required characteristics for production of leavened foods, since their proteins have no ability to develop a viscoelastic network such as gluten. In addition, lack of nutritional value has existed as a health problem of gluten free products specifically produced from white rice flour. Consequently, in recent years, there has been an increasing interest in using different food materials such as, gums, hydrocolloids and starches to enable developing a similar gluten network . MoreoveModification of whole-cereal flour prior to its usage by using simple food processing, such as fermentation, could eliminate the negative effects of the bran. Pre-fermentation of the whole-flour might increase fiber solubility due to enzyme reactions on the cell wall structure. The addition of pre-fermented wheat bran to wheat dough caused an increase in the bread volume of high fiber wheat bread . The addRecently, fermentation became a trend for production of healthy foods from whole grain cereals. Industrial application of the biotechnology of fermentation for the production of gluten free baked products is a promising innovation in the health foods industry. Thus, the objectives of the current study is to investigate the effect of fermented brown rice flour on white rice flour, white rice batter and its steamed bread (Apam) characteristics, which could encourage the usage of whole rice grains.Brown rice grains (MR219) and commercial baker’s yeast  were bought from a local supermarket in Selangor, Malaysia. Brown rice grains were ground using a Cyclotech™ (1093) grinder  and a 500 µm sieve to obtain brown rice flour (BRF), which was used for the fermentation process. Brown rice grains were milled using a rice miller  to obtain white rice grains that were ground to flour in a Cyclotech™ (1093) grinder  and sieved to produce white rice flour (WRF). Flour samples were packaged in polyethylene bags and stored at 4 °C. Fermentation conditions  were optimized to achieve moderate acidity (pH 5.5) of fermented brown rice batter using Minitab 14 software (data not shown). Fermented brown rice batter at the optimum conditions was dried in an air oven drier at 50 °C for 3 h, ground to flour sieved in a 500 µm sieve and verified as fermented brown rice flour (FBRF).The formula of 100 g of WRF which contains 2% sugar, 2% salt, 3% baker’s yeast and 93% volume water based on the flour weight was used to prepare steamed white rice bread (SWRB). The SWRB formula of 60% white rice flour substituted with 40% FBRF achieved the highest bread volume and the overall acceptability of the tested sensory properties among the other ratios that included 0%, 10%, 20%, 30%, and 50% FBRF (data not shown) . To prepPasting properties of WRF and WRF substituted with 40% FBRF was determined using a rapid viscos analyser  following the method of AACC 61-02 . Peak viThe dynamic rheological test was performed using a HAAKE Rheowin 600 rheometer,  at 32 °C using parallel plate geometry (35 mm diameter and 1 mm gap). Batter samples were prepared as mentioned earlier, but without adding the yeast to avoid interference of air cells formation. After placing the batter samples between plates; batter samples were allowed to rest for 2 min to relax the residual stress. The linear viscoelastic part was measured by performing stress sweep experiment at 1 Hz frequency prior to determination. Based on the linear viscoelastic region, frequency sweep test was carried out at 1% strain between 0.1 and 10 Hz. Storage modulus (Gʹ), loss modulus (Gʺ), complex modulus (G*) and tan delta (δ) values were obtained from the Rheowin software (3.3). All the rheological tests were done in five replicates and their average values were stated in the results.et al. [1) and filled with sago pearl. After that, the volume of sago pearl used was recorded (V2). Bread volume (V) was calculated based on the following formula:The volume of steamed breads was measured after 1 h of steaming according to the seed displacement method described by Hallén, et al.  using sa3/g).Specific volume was determined by dividing the volume of the bread by its weight  equipped with P/75 mm plate, 30 N load cell and trigger force of 5 g. After cooling at room temperature for 1 h, bread samples were prepared with approximately 2.5 × 2.5 × 2.5 cm dimensions taken from the crumb and compressed to 40% of its thickness , at a poOne way analysis of variance (ANOVA) and Tukey’s multiple range tests with a confidence interval of 95% were used to report the significant differences between the obtained results.et al. [et al.  who repoet al. [et al. [The viscoelastic properties of the WRB and WRB containing 40% FBRF were studied using a dynamic oscillatory test. The mechanical spectra of the WRB sample indicated that both elastic (G′) and viscos module (G′′) values were higher than that of WRB with FBRF at all the tested frequency ranges . In addiet al.  reported [et al.  also ind [et al. .3) and specific volume (2.46 cm3/g) in comparison to the reference sample . This finding supports previous research, where the addition of fermented barley flour to whole barley dough improved the overall quality of its bread. Its bread volume was significantly higher than that of the control [2 produced during fermentation, consequently increase the loaf volume [ control . Dough af volume . Howeverf volume . The modet al. [et al. [2 retention during dough proofing, and as a result increased the bread volume and the crumb softness [et al.  reportedet al. . Also, fet al. . Accordi [et al.  additionsoftness ,22.Addition of fermented brown rice flour to steamed white rice bread significantly improved rheological and textural properties as well as volume of the bread as indicated in this study. Currently, investigation of the effect of fermented brown rice flour on the nutritional value of steamed white rice bread is under way.


Persicobacter sp. CCB-QB2 is an alginate lyase with three domains — a carbohydrate-binding domain modestly resembling family 16 carbohydrate-binding module (CBM16), a family 32 CBM (CBM32) domain, and an alginate lyase domain belonging to polysaccharide lyase family 7 (PL7). Although AlyQ can also act on polyguluronate (poly-G) and polymannuronate (poly-M), it is most active on alginate. Studies with truncated AlyQ showed that the CBM32 domain did not contribute to enhancing AlyQ’s activity under the assayed conditions. Nevertheless, it could bind to cleaved but not intact alginate, indicating that the CBM32 domain recognises alginate termini. The crystal structure containing both CBM32 and catalytic domains show that they do not interact with one another. The CBM32 domain contains a conserved Arg that may bind to the carboxyl group of alginate. The catalytic domain, meanwhile, shares a conserved substrate-binding groove, and the presence of two negatively charged Asp residues may dictate substrate specificity especially at subsite +1. As Persicobacter sp. CCB-QB2 was unable to utilise alginate, AlyQ may function to help the bacterium degrade cell walls more efficiently.AlyQ from  Alginate oligosaccharides composed of 3–25 monomers, meanwhile, have multiple biological activities such as stimulating the growth of human keratinocytes2 and endothelial cells3, as well as promoting growth and root elongation in rice and barley5.Alginate is a major component in the cell walls of brown algae, composed of mannuronate (M) and guluronate (G) arranged as 1,4-linked polysaccharides. It is commercially extracted from seaweed and widely used in the food and pharmaceutical industries. Due to its biocompatibility and ease of gelation, alginate has proved a favourable biomaterial as hydrogels for medical applications such as tissue engineering and drug deliveryβ-elimination mechanism to yield 4,5-unsaturated sugar at the non-reducing end. Alginate lyases are classified as either endolytic, which cleave the polysaccharide internally, or exolytic, which cleave the polysaccharide from the end to produce monomers, and both types can exhibit poly-MG, poly-G or poly-M specificity. Among the presently classified 23 polysaccharide lyase (PL) families in the CAZy (Carbohydrate Active enZyme) database (http://www.cazy.org/), alginate lyases are found in families PL5, PL6, PL7, PL14, PL15, PL17 and PL18. These families are structurally diverse, with PL7, PL14 and PL18 alginate lyases, for example, sharing a β-jelly roll topology6. Alginate lyases can play an important role in producing biologically active alginate oligosaccharides for potential therapeutical and biotechnological applications, as well as monosaccharides for biofuel production8.Alginate is degraded by alginate lyases via a Persicobacter sp. CCB-QB2, an agarolytic bacterium isolated from seaweed (genus Ulva) in a coastal area of Malaysia9, was found to encode a PL7 alginate lyase, AlyQ, in its genome. Like many multidomain alginate lyases, AlyQ contains two additional carbohydrate-binding modules (CBMs) at its N terminus. CBMs bind to a wide variety of carbohydrates, and are commonly found appended to polysaccharide degrading enzymes such as xylanases and chitinases to help target complex substrates like insoluble polysaccharides10. AlyQ’s first domain moderately resembles family 16 CBM (CBM16), while the second domain belongs to family 32 CBM (CBM32). In order to understand the relationship between these CBMs and the catalytic domain, we have characterized AlyQ’s activity and substrate binding, as well as solved the structure of a truncated version containing the CBM32 and catalytic domains.Persicobacter sp. CCB-QB2, but the expressed enzyme was insoluble. A BLAST search found an almost identical protein from Persicobacter sp. JZB09 (WP_060686070), which however contains an extra N-terminal 48 residues. Comparison of both genes revealed a missing cytosine in the alyQ gene, whose presence was verified with resequencing. The final gene encodes a protein of 572 residues that also includes the additional 48 residues, sharing 93% identity with the Persicobacter sp. JZB09 protein. In addition to the three domains, domains A–C .AlyQ was cloned initially according to its annotated gene (NCBI WP_053404615) from the genome of AlyQ had the highest activity at 50 °C while it became largely inactivated at 30 °C, retaining only 30% of activity, and below Fig. . It was Km and kcat, determined from the Lineweaver–Burke plot, were 2.42 mM and 32.21 s−1 respectively  from Zobellia galactanivorans12.Among the different substrates, AlyQ showed the highest preference for alginate Fig. . Its actPseudoalteromonas PL18 alginate lyases such as AlyPEEC13, AlyA14 and aly-SJ0215, this CBM16-like domain is cleaved during enzyme maturation to yield only a single catalytic domain. In aly-SJ02, it has further been shown to bind to the catalytic domain when both separate domains were co-expressed in E. coli cells, suggesting a role in facilitating the folding of the catalytic domain16. In contrast, CBM16 domains are reported to facilitate substrate degradation, as deleting the two CBM16 domains of a mannanase from Caldanaerobius polysaccharolyticus, Man5A, greatly reduced its activity17. The crystal structures of these two CBM16 domains further revealed a binding site that could accommodate a chain of five sugar residues18.Domain A, a putative CBM sharing modest similarity with CBM16, is found in most PL18 alginate lyases. In several BC, was constructed. The catalytic efficiency (kcat/Km) of AlyQBC was not significantly different from that of AlyQ  which instead contains two CBM32 domains and shares 61% identity with AlyQBC, the characterized AlyPI from Pseudoalteromonas sp. CY24 which contains only a single CBM3219, as well as the single-domain alginate lyase from Vibrio variabilis (GenBank GAL28632) which is 70% identical to domain C, suggesting that domain A may not necessarily be required for the proper folding of the catalytic domain.The CBM16-like domain of aly-SJ02 which shares an identity of 34% with AlyQ’s domain A, in comparison, did not seem to form large oligomerslyQ Fig. , which wBC (discussed below) that domain A may be placed in close proximity to the catalytic domain C, hence interactions between the two may not be impossible.As at present the ability of these CBM16-like domains to bind any carbohydrate has not been reported, it is thus unclear how domain A may facilitate AlyQ’s function. This domain probably may not be cleaved in AlyQ, at least not in the same manner as the PL18 alginate lyases due to the absence of the Gly-containing cleavage sites. It is noted from the crystal structure of AlyQ20. The single-domain YeCBM32 from Yersinia enterolitica, 36% identical to domain B, has been shown to bind to polygalacturonic acid21, whereas the two CBM32 domains from a Paenibacillus chitosanase, DD1 and DD2 of 29–32% identity with domain B, bind specifically to chitosan22. To study domain B’s function, a truncated version containing only the catalytic domain, AlyQC, was further constructed. However, its Km and kcat values were very similar to those of AlyQBC , and both itsN-terminal and C-terminal ends converged to hydrogen bond each other, potentially placing domain A close to the catalytic domain.In order to study AlyQ’s mechanism, both full-length AlyQ and the truncated AlyQher Fig. . Residue2+ ion is found coordinated by the side chains of the strictly conserved D209 and T214, and the main chains of N206, N211, T214 and T307 27. Residues making up the sugar binding groove are highly conserved in both enzymes. In order to predict the AlyQ’s mechanism of action, a structure of Sphingomonas sp. A1 alginate lyase A1-II′ complexed to the GGMG ligand (PDB 2ZAA)28, which is also similar to AlyQ , was superposed onto domain C  shares the same conformation of subsite −1 with A1-II′, while the rest contain either an Asp27 or Glu12 similar to AlyQ’s D447 . This binding site of AlyQ is also not well conserved, and therefore the possibility that the EDTA effect on AlyQ may be due to other factors, for example Ca2+-assisted substrate binding to neutralise the carboxyl group as observed in certain PL families6, cannot be ruled out. In the absence of strong evidence, this ion was modelled as a K+ ion derived from the crystallisation buffer. Intriguingly, C355 from this loop and C338 form a disulphide bond which, again, is not conserved in the other alginate lyase structures.The catalytic domain of AlyQ is also found to bind an ion coordinated by the side chains of D351, T353 and E357 Fig. , which iDTA Fig. , it coulPersicobacter sp. CCB-QB2 is an agarolytic bacterium that possibly feed on seaweed. Although it can degrade alginate and agar, the bacterium does not seem to be able to utilise them as it could not grow when supplemented with only either one. In the bacterial alginate metabolic pathway, alginate is first depolymerized into oligosaccharides by endolytic alginate lyases, then further degraded into unsaturated monosaccharides by exolytic alginate lyases. These monosaccharides are spontaneously converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), which is then converted into 2-keto-3-deoxy-D-gluconic acid (KDG) by KDG reductase30. Persicobater sp. CCB-QB2’s genome, however, does not encode an exolytic alginate lyase that plays the critical role in producing DEH.Persicobacter sp. CCB-QB2 does contain many key enzymes for agarose metabolism, which may explain its agarolytic activity, and displayed a diauxic growth behaviour when supplemented with tryptone and agar as nitrogen and carbon sources9, the cells could have prioritized tryptone metabolism over agar metabolism during the diauxic growth. These polysaccharide-degrading enzymes including AlyQ, therefore, may likely function to help the bacterium degrade the structurally complex cell walls of red and brown algae for nutrient acquisition. By anchoring the extracellular enzyme around cleaved alginate, AlyQ’s CBM32 domain may help concentrate the enzyme on enlarging a single cleaved site instead of the enzyme spreading over a large area. Alternatively, AlyQ may function to degrade other polysaccharides that contain certain uronic acids, allowing the bacterium to utilise them as a carbon source.While Nde I and Hind III sites, with a six-His tag inserted before the stop codon. The ligated vector was then transformed into Escherichia coli BL21(DE3) cells and verified by DNA sequencing. The truncated enzymes, AlyQBC, AlyQA, AlyQB and AlyQC, were similarly constructed.The full length of AlyQ, excluding its N-terminal signal peptide (C29–Q572), was cloned into the pCold III vector (TaKaRa) between the 600 reached 0.6, and isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. After 16-hour incubation, the cells were harvested by centrifugation, resuspended in 50 mM Tris–HCl, 100 mM NaCl, pH 8.0, sonicated and centrifuged again to remove cell debris.Cells were grown in 1 L of LB broth supplemented with 100 µg/ml ampicillin at 37 °C. The culture was cooled to 15 °C when the OD2+-charged HiTrap IMAC HP column , and eluted with a gradient of 0–1 M imidazole. The protein was then further purified by size-exclusion chromatography using a Superdex 200 column . Protein purify was assessed on 10% SDS-PAGE gels. All other truncated enzymes were similarly purified.The supernatant was first applied to a Ni31. Briefly, 50 g of sodium alginate (Sigma) was partially hydrolysed in 1 L of 0.3 M HCl at 100 °C for 20 minutes. After centrifugation, the supernatant containing poly-MG was neutralised with NaOH and precipitated with ethanol. The insoluble precipitate was further hydrolysed with 1 L of 0.3 M HCl for 24 hours, centrifuged and dissolved with 1 M NaOH. After adjusting the pH to 2.85 and centrifugation, the precipitate containing poly-G was dissolved in 10 mM NaOH while the supernatant containing poly-M was neutralized with 0.1 M NaOH, and both were precipitated with ethanol.Poly-M, poly-G and poly-MG alginate blocks were prepared by partial acid hydrolysis of alginatePurity of the synthesized alginate blocks were verified with circular dichroism analysis, with the poly-M’s mannuronate content estimated to be 90% and poly-G’s guluronate content 80%. The mannuronate content of sodium alginate was about 60%.All measurements were performed in triplicate and the mean values were calculated. AlyQ activity was determined by measuring continuously the increase in absorbance at 235 nm, which is due to the formation of the 4,5-unsaturated bond at the non-reducing end. Reactions were carried out at room temperature with 1 mg/ml alginate in 20 mM Tris–HCl, 500 mM NaCl, pH 7. The optimal pH was determined by replacing the buffer with acetate for pH 4–5, MES for pH 6, and Tris–HCl for pH 7–9, whereas the optimal temperature was determined at 10–60 °C with the substrate incubated accordingly for five minutes prior to measurements. Substrate specificity studies were carried out by replacing alginate with either poly-G or poly-M, while EDTA effects were evaluated in the presence of 1 and 5 mM EDTA.2O loss during polymerisation. The Km and kcat values were determined from the Lineweaver–Burk plots, using an extinction coefficient of 6,150 M−1 cm−1 for measuring the unsaturated product concentration32.The kinetic parameters were measured by incubating 0.05–3 mg/ml alginate at 30 °C before adding the enzyme to a final concentration of 70 nM. As alginate consists of mannuronic acid and guluronic acid of the same molecular weight, 176 g/mol was assumed for each residue after subtracting 18 g/mol for HB, were performed using size-exclusion chromatography. Equal volumes of 10 mg/ml substrate , which was either intact or treated beforehand with minute amounts of the catalytic domain, and 3 mg/ml AlyQB were mixed, incubated for 20 minutes, and then applied to the Superdex 200 column.Substrate binding studies of AlyQ’s CBM32 domain, AlyQBC was crystallised at 10 mg/ml using the sitting-drop vapour diffusion technique at 20 °C. Crystals were formed in 30% PEG 3,350, 0.1 M sodium acetate pH 4.0, and 0.2 M potassium phosphate. Diffraction data for a flashed-cooled crystal at 100 K were collected on a Rigaku MicroMax-007 HF X-ray generator equipped with an R-AXIS IV++ area detector, and processed with the Rigaku CrystalClear software in space group P41212.AlyQ21 (PDB 2JDA) and Aly-127 (PDB 1UAI) were used for solving the structures of both domains B and C by molecular replacement with Molrep33. The combined structure was then built and refined using REFMAC34 and Coot35. Data collection and refinement statistics are summarised in Table 36, manually edited and presented with ESPript37. Figures were generated using PyMol (http://www.pymol.org).The structures of YeCBM32Supplementary Information


A H2O2-responsive and plaque-penetrating S2P–CeO2–ASOs nanoplatform was developed for the effective silencing of mTOR and treatment of atherosclerosis. 2 NW)-based RNA interference (RNAi) oligonucleotide delivery nanoplatform for the effective silencing of mTOR and treatment of atherosclerosis. This nanoplatform is composed of the following three key components: (i) a stabilin-2-specific peptide ligand (S2P) to improve plaque targeting and penetration; (ii) polyethylene glycosylation (PEGylation) to extend in vivo circulation time; and (iii) a high aspect ratio CeO2 core to facilitate endosome escape and ensure “on-demand” release of the RNAi payloads through competitive coordination of cytosolic hydrogen peroxide (H2O2). Systemic administration of the nanoplatforms efficiently targeted stabilin-2-expressing plaque and suppressed mTOR expression, which significantly rescued the impaired autophagy and inhibited the atherosclerotic lesion progression in apolipoprotein E-deficient (ApoE–/–) mice fed with a high-fat diet. These results demonstrated that this H2O2-responsive and plaque-penetrating nanoplatform can be a potent and safe tool for gene therapy of atherosclerosis.The mammalian target of rapamycin (mTOR) that controls autophagy and lipid metabolism is pivotal for atherosclerosis initiation and progression. Although blocking the mTOR function with rapamycin and its analogs may stimulate autophagy and consequently attenuate lipid storage and atherosclerotic lesions, only limited success has been achieved in clinical applications due to the unsatisfactory efficacy and safety profiles. In this study, we engineered a cerium oxide nanowire (CeO For atherosclerosis therapy, the barriers to effective in vivo siRNA delivery mainly include serum nuclease degradation, atherosclerotic plaque targeting, plaque and cell membrane penetration, escape from the endosome, and release of ASOs or siRNAs in the cytoplasm.in vivo, the nanoparticle delivery platform should be tailored to exhibit the following features: (1) good biocompatibility, (2) a long blood circulation time and high EPR effect, (3) plaque-penetrating ability and VSMC/Mφ-targeting specificity, (4) an efficient endosomal escape ability and the “on-demand” release of active ASOs or siRNAs into the cytoplasm, and (5) an imaging function for monitoring the release profile and therapeutic efficacy.RNA interference (RNAi) using an antisense oligonucleotide (ASO) or short-interfering RNA (siRNA) is an attractive alternative to inhibiting otherwise intractable therapeutic targets.2 NW)-based RNAi oligonucleotide delivery nanoplatform capable of satisfying all of the above-mentioned criteria and demonstrated its high efficiency in suppressing mTOR expression and atherosclerosis progression. This nanoplatform consisted of a high aspect ratio CeO2 NW functionalized with PEG  mice. Once inside the cell, the high aspect ratio CeO2 core was able to pierce the endosomal membrane, thus facilitating the loaded ASOs escape from the endosome prior to lysosomal degradation. As demonstrated in previous atherosclerosis studies,2O2). In the presence of H2O2, the absorbed ASOs are liberated from the CeO2 surface due to the stronger binding ability between peroxo and Ce4+ compared to that between phosphate oxygen and Ce4+.2O2-triggered release. Targeted delivery and “on-demand” release of ASOs to high-fat diet-fed ApoE–/– mice efficiently depleted mTOR expression and rescued the impaired autophagy, which led to a dramatic reduction in foam cell formation and atherosclerotic lesion progression 3 as the Ce source.2 NW with a dimension of 100–150 nm was selected as the core unit, which is the optimal size for EPR-mediated targeting and endosomal escape and allows for maximal RNAi oligonucleotide loading. Transmission electron microscopy (TEM) showed that the obtained CeO2 NWs presented a smooth surface with the length being 130 nm and width being 9 nm .2 NWs in PBS (pH 7.4) was determined to be –14.2 ± 0.4 mV, which allowed for electrostatic interactions with positively charged maleimide–PEG–amine (MW 2000). The reversed zeta potential of 2.2 ± 0.5 mV indicated that PEG was successfully coated onto the CeO2 core surface. The plaque-penetrating peptide S2P, Cys-Arg-Leu-Thr-Leu-Thr-Val-Arg-Lys-Cys, was further grafted to the PEG-capped CeO2 by thioether bonds between the thiol groups on Cys and maleimide groups on PEG, resulting in a much higher positive zeta potential (4.3 ± 0.3 mV). The S2P-PEGylated CeO2 NW was then wrapped with mTOR-specific ASO1 and ASO2  in DNA and Ce ions (Ce4+) at the particle surfaces. The loading efficiency of the ASOs was calculated using a NanoDrop spectrophotometer. The average number of ASOs loaded in S2P-PEGylated CeO2 NWs was estimated to be 435 DNA/NW , hydroxyl radicals (˙OH), cysteine (Cys), glutathione (GSH), ascorbic acid (AA), and adenosine triphosphate (ATP), possibly coexisting with H2O2, revealed that these species did not cause a remarkable release of ASOs  was employed to evaluate its in vitro plaque-targeting ability. The uptake of the S2P–CeO2–ASOs nanoplatform was facilitated through a stabilin-2-mediated endocytosis pathway. Stabilin-2 is a transmembrane protein expressed predominantly in VSMCs of atherosclerotic plaques  of S2P peptide  of CeO2 NW–DNA coordination complexes,2–ASOs nanoplatforms were localized inside the endosomes/lysosomes at the early time point (2 h). When the nanoplatforms entered the cytoplasm (6 h), the presence of H2O2 , a marked decrease in the red fluorescence intensity was observed, further confirming that the nanoplatform allowed for “on-demand” release of ASOs in response to cytosolic H2O2.Next, the endosomal escape ability of S2P–CeO2 Fig. S9 induced 2–ASOs nanoplatform toward enhanced mTOR gene silencing, various controls and time-silencing dependence studies were carried out to investigate and optimize the in vitro intracellular mRNA silencing mechanism involved in the mTOR knockdown. 2–ASOs at an 80 ng ASO concentration, as compared to 18% achieved with CeO2–ASOs (vs. bar iv), which was ∼4 times lower than S2P-conjugated silencing abilities. It has also been demonstrated that S2P–CeO2–ASOs better facilitate the ASO internalization process, as the nanoplatform offers an 18-fold enhanced silencing when compared to free ASOs in solution at the same concentration (vs. bar ii). Furthermore, to ensure that the silencing efficiency was a result of the H2O2-triggered release of ASOs, cells treated with catalase and S2P–CeO2–ASOs and cells treated with S2P–CeO2 cores lacking the ASOs (2–ASOs showed a fast silencing response (52% after a 6 h treatment), reaching a silencing percentage plateau 24 h post-treatment -induced VSMC foam cell formation, which was manifested by a decreased lipid droplet accumulation . As shown in t1/2) of S2P–CeO2–ASOs was around 9.33 h, which was comparable to the t1/2 of CeO2–ASOs (∼9.16 h), but far longer than that of free ASOs (t1/2 < 20 min). This prolonged half-life in the blood circulation was mainly due to the protection of the PEG outer layer and stable nature of the Ce4+–phosphate oxygen coordination conjunction. The in vivo plaque-targeting ability of S2P–CeO2–ASOs was assessed by intravenously injecting Cy5-tagged ASOs loaded nanoplatforms into plaque-bearing ApoE–/– mice . 2O2  was performed 1 h before S2P–CeO2–ASOs injection, stabilin-2 blocking significantly reduced the aorta accumulation of S2P–CeO2–ASOs. In comparison, CeO2–ASOs or free ASOs presented a high accumulation in the liver and kidneys and an extremely low accumulation in the aorta. 2–ASOs with H2O2 scavenger catalase (1 mg kg–1), further confirming the ability of the nanoplatform to release ASOs “on-demand” in response to H2O2.Having established that S2P–CeO2–ASOs accumulated in the aorta, the leaky vasculature allows them to penetrate and be retained in the plaques. To evaluate the plaque-penetrating ability of the S2P–CeO2–ASOs, aortic arches were isolated from mice and their localization was determined by an immunohistochemistry study. Immunofluorescence micrographs of aortic arch cross-sections (2–ASOs can (shown with the red fluorescence of Cy5) easily pass through the arterial wall and become trapped by VSMC in atherosclerotic plaques (shown with the green fluorescence of α-SMA) via S2P mediated stabilin-2 targeting. As expected, non-coated CeO2–ASOs and free ASOs did not show plaque-specific targeting. These results strongly demonstrated the deep plaque-penetrating and H2O2-triggered release characteristics of S2P–CeO2–ASOs.It has been identified that the blood vessels surrounding atherosclerotic plaques are defective and immature.sections  clearly 2–ASOs nanoplatforms were intravenously administered into ApoE–/– mice fed with a high-fat diet . Aortas were harvested three days post-injection and the in vivo mTOR expression was evaluated by western blot analysis. By virtue of plaque-penetration, S2P–CeO2–ASOs induced >75% knockdown of the mTOR expression in the aortas. In contrast, there was <26% knockdown for the CeO2–ASOs and free ASOs controls . After 12 weeks, the S2P–CeO2–ASOs treatment had the best therapeutic results in limiting atherosclerotic lesion progression in the ApoE–/– mice on a high-fat diet compared to the controls. Examination of the aortic arch with a stereo dissecting microscope (en face preparations (2–ASOs treatment. Notably, S2P–CeO2–ASOs showed good biocompatibility, as evidenced by the lack of histological changes in tissues of the heart, liver, spleen, lung, or kidneys and no obvious influence on the mouse body weight (Fig. S17 and S18To examine the inhibition of the mTOR expression in aortic atherosclerotic lesions, S2P–CeOFig. S16A. As a reFig. S16B. To evalcroscope  showed aarations  revealed2O2-responsive and plaque-penetrating S2P–CeO2–ASOs nanoplatform for targeted mTOR ASOs delivery. In vitro and in vivo results demonstrated that this nanoplatform has a long blood circulation time, and can efficiently pass through the arterial wall, penetrating into the plaque through specific recognition between S2P ligands and overexpressed stabilin-2 on VSMCs. With the high aspect ratio CeO2 core and H2O2-based competitive coordination, this nanoplatform can effectively escape from endosomes and release therapeutic ASOs “on-demand” into the cytoplasm, leading to a significant suppression of the mTOR expression and atherosclerotic plaque formation in ApoE–/– mice fed with a high-fat diet. After 12 weeks of treatment, the nanoplatform showed a more effective therapeutic outcome compared to mTOR inhibitor rapamycin. Accordingly, this targeted gene-silencing nanoplatform opens up new opportunities for developing CeO2-based nanotheranostics and possibly a robust atherosclerosis treatment strategy for clinical applications.In summary, we have successfully developed a HThere are no conflicts to declare.Supplementary informationClick here for additional data file.


This aggregation supposedly involves a weighting procedure because individuals care about different parts of their lives to varying degrees. Thus, composite measures of well-being should be more accurate if domain satisfaction scores are weighted by the importance that respondents assign to the respective domains. Previous studies have arrived at mixed conclusions about whether such a procedure actually works. In the present study, importance weighting was investigated in the Panel Study of Income Dynamics . Both weighted composite scores and moderated regression analyses converged in producing the conclusion that individual importance weights did not result in higher correlations with the outcome variable, a global measure of life satisfaction. By contrast, using weights that vary normatively across domains  significantly increased the correlation with global life satisfaction . These results converge with findings from other fields such as self-concept research, where evidence for individual importance weighting seems elusive as best.Bottom-up models of life satisfaction are based on the assumption that individuals judge the  What happens in people's heads when they are asked how satisfied they are with their lives? So-called bottom-up models are based on the assumption that people assess the conditions of their lives across various life domains and then aggregate these pieces of information to arrive at their judgments e.g., . BeginniSuch a weighted bottom-up model has a strong intuitive appeal: Surely, it makes sense to expect that somebody who cares a lot about his or her family will be less satisfied with his or her life in general when family life is unsatisfactory, whereas somebody who does not care about relatives would probably not be affected too much. A similar importance-weighted model has been popular in self-esteem research and can be traced back as far as William James' “The principles of psychology” see , illustrIn theory, such an idiographic approach is fairly straightforward. Respondents report (a) how satisfied they are with the central domains of their lives and (b) how important these domains are, either by ranking them or by assigning ratings. Researchers can then weight the satisfaction ratings by the importance ratings to arrive at a personalized composite score of overall life satisfaction. Such a measure should be a more valid reflection of how respondents' lives are going than a simple sum score, which represents the implicit assumption that each domain is equally important—in order words, a sum score is equal to applying uniform weights to all domains. The weighted composite score can then be used to predict other variables. Most studies on this topic have relied on correlations between the weighted composite score and a global measure of life satisfaction to gauge the validity of the weighted score. If the weighted composite score shows a higher correlation than an unweighted sum score, this is interpreted as evidence that the importance weighting hypothesis has merit.empirically speaking, studies have frequently failed to find evidence that importance weighting results in improved validity—including Campbell et al., themselves , followed by others. For example, However, 1 Their second point  addresses the idea of weighted models in general: Domains incorporated into questionnaires have already been selected to be universally relevant, and thus, they should be important to almost all respondents. Importance weighting should therefore be unnecessary.Moderated regression analyses provide another way to assess importance weighting that is built on less strict assumptions and provides a test that is insensitive to the scaling of the importance ratings . In moderated regression analyses, the outcome  is predicted from satisfaction ratings, importance ratings, and their interactions. A positive interaction between a person's satisfaction in a specific domain and the respective importance rating is interpreted as evidence for the merit of importance weighting. In comparison with composite scores, moderated regression analyses distinguish between the main effects of satisfaction and importance ratings and their interactions, whereas weighted composite scores result in one estimate in which these sources of variation are intertwined.N = 2,164; note that this is the same sample as used in very indirect test of importance weighting.Lastly, Taken together, the current state of the literature does not allow for a clear and straightforward judgment to be made about importance weighting. Results are difficult to integrate across studies because different authors used different methodological approaches , then there would actually be no need to collect importance ratings. In addition, we tested the relationship between domain importance and domain satisfaction. Many researchers conducting studies investigating importance weighting have implicitly assumed that these two types of ratings are orthogonal, which would admittedly render the weighting procedure somewhat more conceptually “elegant.” Thus, the researchers who conducted such studies did not report whether they found a correlation between the importance and satisfaction ratings. However, if the importance of a specific domain was strongly correlated with satisfaction with that domain, the information provided by the two items would become redundant.To test the central hypothesis of importance weighting, we applied weighting procedures and moderated regression analyses, and we discuss the methodological properties of both approaches. We elaborate on the crucial distinction between normatively as compared to individually weighted scores, an issue that has caused lots of confusion and false claims in the literature. At last, we report additional exploratory analyses to test whether importance weighting is relevant in only some domains.Data came from the well-being supplement of the Panel Study of Income Dynamics (PSID) 2016. The PSID is a nationally representative panel survey of families in the United States, produced and distributed by the Survey Research Center at the University of Michigan. In 2016, a questionnaire on well-being PSID-WB;  was addeM = 50.55, SD = 14.37. Women comprised 56.35% of the sample.Heads of household and their partners who were at least 30 years old by December 31, 2015 were eligible for the PSID-WB in 2016. Eligible panel members were mailed an invitation letter including the web address of the survey and login credentials and received a $20 check upon completing the survey. Of the 10,689 eligible cases, 8,341 responded (78% response rate). Age ranged from 30 to 97, strongly disagree, 2 = somewhat disagree, 3 = neither agree nor disagree,4 = somewhat agree, 5 = strongly agree). The scale had a satisfactory reliability of α = .89. Due to missing responses to SWLS items, 160 respondents had to be excluded.Respondents answered the Satisfaction With Life Scale SWLS; , one of not at all important, 1 = a little important, 2 = somewhat important, 3 = very important, 4 = extremely important).2 The phrasings of the items as well as domain abbreviations that will be used throughout the manuscript can be found in Respondents reported the importance of 10 different life domains on a 5-point response scale . These 10 items corresponded to the 10 importance items  applied to all respondents. We restricted analyses to all respondents who answered all 10 domain satisfaction items. Thus, the final sample consisted of N = 5,049 respondents (54.03% women) with a mean age of 46.82 years (SD = 12.13). Notice that because of this considerable sample size, even small effects can reach the conventional threshold for statistical significance. In the results section, we will consider changes in R2 to evaluate whether importance weighting is able to account for meaningful amounts of variance in the outcome variable.Respondents reported their satisfaction with 10 different life domains on a 5-point response scale . The domain that showed the highest variability in importance, faith, still showed considerable concentration at the high end of the scale: 49.97% reported that having a strong religious faith was extremely important to them, and 25.27% rated it very important, whereas 9.88% of respondents chose one of the options at the lower end of the scale .Given the high mean scores, it was no surprise that variability in the importance ratings was generally low. For example, 71.52% of respondents reported that their family was completely satisfied with their family life, and 41.59% were very satisfied, whereas only 5.17% used one of the two response options at the lower end of the scale .Domain satisfaction scores consistently showed somewhat higher variability for all domains except for faith, although the responses were still concentrated in the upper half of the scale. For example, 35.75% of respondents reported that they were Taken together, both the domain importance and domain satisfaction ratings tended to be located in the upper half of the response scales. In particular, domain importance items tended to be answered with the two highest response options and accordingly, variability in those items was limited.Are respondents more or less satisfied with domains that are more important to them? This question could be approached from two perspectives.compared with other respondents more/less satisfied with their health compared with other respondents? This question can be answered by investigating bivariate correlations between domain importance and domain satisfaction, and this analysis would result in one estimate per domain .From a variable-centered perspective, it would make sense to ask: Are respondents who rate health as more important compared with other domains more/less satisfied with their health compared with other domains? This question could be answered by investigating profile correlations between domain importance ratings and domain satisfaction ratings, and this analysis would result in one estimate per respondent . Note that the two approaches are intrinsically related on a statistical level  to .45 (faith), all ps < .001, with the exception of the domain finances that showed quite a distinct pattern with virtually no correlation between the importance of finances and satisfaction with finances : Respondents who gave high ratings to the importance of financial security were not more or less likely to report being satisfied with their financial situation.For all domains except for finances, there was a positive correlation between domain importance and domain satisfaction. Respondents who rated domains as more important also said they were more satisfied with these domains. This can be seen in could reflect a general tendency to report high importance and high satisfaction—respondents might have a certain tendency to reply with high or low values on the rating scales, and this might result in the appearance that satisfaction and importance are correlated for each of the domains. However, these correlations were specific to the domains to some extent, ruling out the possibility that this alternative explanation could completely account for the pattern. Although there were some substantial correlations across domains  = 29.89, p < .001. As a robustness check, we standardized each variable separately and then recalculated the profile correlations. This procedure ensured that the individual profile correlations were not confounded by normative patterns . Again, the average profile correlation coefficient (r = .30) was positive and significantly different from zero, t = 45.77, p < .001.3For each respondent, a profile correlation coefficient was calculated by correlating the 10 importance items with the 10 corresponding satisfaction items. Correlations were Fisher Taken together, importance and satisfaction were mildly correlated both across and within respondents. Respondents who considered a domain more important were also more satisfied with that domain. Furthermore, on average, if a respondent considered a specific domain more important than other domains, he or she was also more satisfied with that domain than with other domains. However, all these correlations were moderate in magnitude, so that there is no reason to assume that importance and satisfaction ratings are essentially redundant.Does weighting by importance increase the amount of variance that domain satisfaction ratings can explain in a measure of global life satisfaction? Previous studies have frequently calculated importance-weighted composite scores and have subsequently compared the correlation between these weighted scores and simple (unweighted) sum scores.i indexing the different life domains.To arrive at an individually weighted composite score, each domain satisfaction rating for each respondent is multiplied by the respective domain importance rating, all these products are summed, and the sum is divided by the number of all importance ratings made by the respective respondent, that is: r = .993, p < .001, see r = .695 (R2 = 48.37%), the correlation between the individually weighted composite and the SWLS score was only slightly higher, r = .699 (R2 = 48.86%).This individually weighted composite was highly correlated with the simple sum score of all domain satisfaction ratings  explanations for the incremental validity of the individually weighted composite.normative importance effect. For example, respondents might collectively assign higher weight to a domain that is indeed more strongly related to global life satisfaction across respondents . In a regression model predicting global life satisfaction from only the domain satisfaction scores, this would show up as different regression coefficients for the various domain satisfaction ratings. Such a normative importance effect would make it superfluous to collect individual importance ratings if the researcher knew which domains the average respondent considered more important.First, the added validity might stem from a idiosyncratic/individual importance effects. For example, a respondent who assigns a higher weight to a specific domain might be more strongly influenced by this specific domain, regardless of the importance assigned to this domain by other respondents. In a regression model predicting global life satisfaction from domain satisfaction, domain importance, and their interaction, this would be indicated by a significant interaction effect. In order to account for such individual importance effects, one would have to collect individual importance ratings.Second, the added validity might stem from individual applies importance weights when forming summative judgments.This distinction between normative and individual weighting has played a crucial role in discussions about importance weighting in self-esteem research  and is eacross respondents. This normatively weighted composite, To assess the evidence for the two different types of weighting within the multiplicative weighting framework, we additionally generated a weighted composite score in which the individual domain satisfaction ratings were multiplied by the average importance rating r = .9997, p < .001) and the individually weighted composite . Its correlation with the SWLS score was comparable to the other two composites , and it significantly outperformed the unweighted composite according to Steiger's test .In the initial model (Model 0), the SWLS score was predicted from the 10 satisfaction ratings, and all coefficients were constrained to equality. This model is based on the assumption that each of the 10 domains is equally important for life satisfaction. Estimating this model yielded exactly the same results as correlating the unweighted composite  with the SWLS score. Thus, it was no surprise that in this model, R2 = 51.63% of the variance in the SWLS score was explained by the predictors , indicating that it might be preferable to estimate the weights assigned to the domains from the data instead of based on the importance ratings, although the difference was rather small.In the next step, the SWLS score was predicted from the 10 domain satisfaction ratings, but the coefficients of the predictors were allowed to vary as in regular linear regression analyses. This conceptually corresponds to the normatively weighted score above. All in all, main effects of the 10 domain importance ratings. In this model, again, slightly more variance in the outcome could be explained  = 11.178, p < .001. Thus, the domain satisfaction importance ratings themselves were seemingly able to explain some variance in the global life satisfaction measure, but the gain was negligible.The next model additionally incorporated the R2 = 52.20% or 0.26% more than the model without interaction terms, coefficients in 2(10) = 9.08, p = .003. However, results from the cross-validation procedure indicated that this model was not really preferable to the previous model, R2cross-validated = 51.23%. When comparing the models without cross-validation, the interaction terms seemed to contribute to the prediction of the outcome—but the performance of the two models was virtually identical when applied to new data points from the same population , suggesting that the in-sample performance of the more complex model might be an overestimation due to an overfitting of the data at hand. Thus, results from the moderated regression analyses did not support the idea of individual importance weighting.4The key regression model that was computed to test the importance weighting hypothesis additionally incorporated the interaction between the domain satisfaction ratings and the corresponding domain importance ratings. Thus, the outcome (SWLS score) was predicted from the 10 domain satisfaction ratings, 10 domain importance ratings, and 10 interactions. In this model, again, slightly more variance was explained  was virtually identical to the performance of the moderated regression model without within-subject centering described above  and thus also failed to support the idea that the importance * satisfaction ratings play a large role.We modified the moderated regression approach to be closer aligned with a person-centered perspective. For that purpose, we centered the importance ratings N = 7,439, 56.27% female, ageM = 49.79, ageSD = 13.92). In Model 0 , 48.41% of variance could be explained. Allowing the coefficients of the domain satisfaction ratings to vary (Model 1) again resulted in an improved prediction of the SWLS . Incorporating the main effects of the domain importance ratings (Model 2) again lead to a statistically significant (p < .001) change in R2, with a small gain of 0.23% . Including the importance * satisfaction interactions in the last step (Model 3) again lead to a statistically significant increase in R2 (p < .001), but once again cross-validation led to the conclusion that the difference was completely negligible .Seven of the domains  are arguably relevant to almost all respondents, regardless of their living circumstances. We thus repeated the central moderated regression analyses only including those seven domains, which allows for a considerably larger sample size  from the ten domain satisfaction ratings, the ten domain importance ratings, and the ten satisfaction * importance interactions. This approach also allows us to include respondents with missing values on any of the included items using the full-information maximum likelihood (FIML) estimator, resulting in a sample size of R2 = 2.98%, ΔAIC = −275, ΔBIC = −216. When additionally estimating the coefficients of the importance ratings, Model 2, the conventional fit measures CFI, RMSEA and SRMR remain virtually unchanged, and an additional 0.34% or variance in global life satisfaction can be explained. According to the AIC, this model is to be preferred over Model 1, ΔAIC = −14. However, at this point, the BIC already indicates that the more parsimonious Model 1 should be preferred, ΔBIC = 52, as it puts a heftier penalty on the increased complexity given the large sample size.The largest increase in model fit as well as explanation of variance can once again be seen moving from Model 0 to Model 1, when allowing the coefficients of the satisfaction ratings to vary by domain, ΔR2 = 0.27%), which parallels the observation that including the interaction terms in the moderated regression increased the (unadjusted) R2. Again, according to the AIC, this more complex model is somewhat preferable (ΔAIC = −8), but the BIC again imposes a larger penalty on the added complexity, resulting in a preference for the more parsimonious previous model (ΔBIC = 58). The disagreement between the two information criteria can be explained by the fact that they are trying to answer different questions  to .57  as can be seen in the effect of being more or less satisfied with one's friends than would be predicted on the basis of both satisfaction with and the importance of the other life domains. When holding everything else constant, the interpretation of the interaction term becomes even more complex.Inspection of the model coefficients of the moderated regression model including all domains  0.04 point increase in global life satisfaction, p = .001. A significant positive interaction indicated that for jobs that were rated as more important, the effect of job satisfaction on life satisfaction was more pronounced, p < .001. For example, for a person who rated his or her job as 1 point more important than the average, a 1 point increase in job satisfaction was associated with a 0.37 + 0.05 = 0.42 increase in global life satisfaction. The same pattern emerged for satisfaction with marriage (pinteraction < .001) and satisfaction with faith (pinteraction < .001). The domains finances and friends also showed this pattern in weaker form and met the conventional threshold for significance, whereas the domains hobbies (pinteraction = .074) and family (pinteraction = .091) displayed trends in the same direction that could be labeled “marginally significant” at best. It is noticeable that only one domain—housing— showed a negative interaction , but it was still very close to zero. Overall, the pattern of results thus suggests that there is some evidence for the expected moderating effect of importance ratings although the strength of this evidence varies across domains. However, as we will show below, it is questionable whether these coefficients should be interpreted as support for a bottom-up weighted model of life satisfaction.Some domains clearly showed the expected interaction pattern. For example, at the average level of job importance, a 1-point increase in job satisfaction predicted a 0.37 point increase in global life satisfaction, The high intercorrelation within the importance ratings and within the satisfaction ratings raised the question of whether these interactions are sufficiently specific. For example, it is possible that there is a general satisfaction factor , and a general importance factor . Both might reflect either substantial factors, such as personality predispositions, or, in a more mundane interpretation, response biases. The two factors might interact such that individuals who think that life domains per se are important tend to be more strongly affected by their domain satisfaction when judging their overall life satisfaction. Such a general explanation would not go well with a bottom-up weighting approach that implies that the importance that a person ascribes to family  moderates the effect of family satisfaction on general life satisfaction.p = .005) interaction between the average satisfaction and average importance, albeit with a negative coefficient, which does not support this alternative account. Furthermore, model performance (R2 = 48.55%) was worse than in the domain-specific moderated regression analyses reported above.As a first (crude) test of such an alternative explanation, we ran a moderated regression model in which we predicted global life satisfaction from the average satisfaction rating across all domains, the average importance rating across all domains, and the interaction of these two factors. Indeed, in this model, we found a statistically significant . However, the interaction between job importance and hobby satisfaction had a similar magnitude . Furthermore, the effect of job satisfaction was affected by the importance of finances to a similar degree (Panel D).Second, the coefficient for the interaction between the importance of marriage and marriage satisfaction was the largest of the marriage importance interactions . However, to a lesser degree, the importance of marriage also potentially moderated the effects of satisfaction in a couple of other life domains . Furthermore, the interaction between marriage satisfaction and importance of finances was comparable in magnitude (Panel D).Third, the coefficient for the interaction between the importance of faith and satisfaction with faith was the largest of the faith importance interactions . Importance of friends also moderated the association between the SWLS score and satisfaction with faith (Panel H) but to a lesser extent.https://osf.io/m3ezs/).Taken together, only one out of 10 life domains yielded clear support for domain-specific, distinct importance weighting. Note that the results remained almost unchanged when all involved variables were standardized prior to analysis , we provide the corresponding figure on the OSF (that life domain (and not the importance of a different life domain).All things considered, our results suggest that importance weighting of domain satisfaction ratings does not improve the prediction of global life satisfaction. Moderated regression analyses including all ten life domains simultaneously indicated that the inclusion of the interactions between importance and satisfaction did not increase the amount of variance explained when taking into account the increased model complexity. Investigating only one domain at a time revealed somewhat more promising results with significant interactions for multiple domains. However, cross-domain analyses suggested that most importance-satisfaction interactions were not domain specific, which is why these significant interactions can hardly be interpreted as evidence for a bottom-up model in which the effect of satisfaction in a specific life domain is modulated by the importance of It is worth noting that this lack of support for importance weighting on the individual level aligns with similar findings from another substantive field, the research of self-esteem. In self-esteem research, the idea that one's overall self-evaluation is the importance-weighted average of self-evaluations in specific domains has been popular for more than 100 years , but empWhy does importance weighting, despite its intuitive appeal, fail to find support in empirical data? In the following, we will outline possible explanations, which are not mutually exclusive, and their implications for further research.The chance of detecting individual importance weightings is zero if all respondents consider a certain domain to be equally important see also . For exaany other life domain and global life satisfaction.This explanation is somewhat supported by our findings regarding the domain of faith. For this domain, importance ratings showed the highest variability, and they were also only weakly correlated with importance of other life domains. In line with a weighted bottom-up model, the importance of faith moderated the association between satisfaction with faith and global life satisfaction but not the association between satisfaction with Researchers could try to purposefully include life domains that are likely rather unimportant to part of the respondents. For example, one could draw inspiration from the study by Campbell et al.  already noted that the stability of their importance ratings was lower than those of other measures, raising questions about the reliability of single-item importance measures. Of course, a lack of reliable variance in single item importance measures would also limit their validity and hence undermine chances to detect the effects of importance weighting. This could be solved by the inclusion of more reliable multi-item measures. For example, the SEM approach suggested by Apart for low reliability, there are other potential reasons for a lack of validity of domain importance ratings. In addition, one could argue that the weights assigned to various life domains when forming a global life satisfaction judgment are not weighted according to importance, but rather according to some other variable. For example, values have been suggested as one moderator . Schimmawithin subjects. In such a study, it should be possible to detect interindividual differences in domain weighting, assuming that such differences are somewhat stable  assessment— people might assign exaggerated weights to certain life domains (e.g., income; So far, we have assumed that it is sensible to evaluate the validity of individual importance weighting by using a global life satisfaction measure as criterion. The validity of global life satisfaction measures has been extensively illustrated by countless plausible associations with, for example, non-self-report measures and life circumstances . However


Increasing Ca content results in decreasing domain size and enhancement of the polar disorder. For the composition with x = 0.32 the characteristic domain size is similar to that reported for relaxor Sr0.61Ba0.39Nb2O6 (SBN61). However, decay of an artificial macroscopic domain in CBN32 takes place below the macroscopic transition temperature, contrary to SBN61, where random fields stabilize it above the transition temperature. We can conclude that CBN with 0.26 ≤ x ≤ 0.32 does not display classical relaxor behavior and might be considered as a disordered ferroelectric.Polar structures of Ca The properties of CBN are still a matter of discussion. Based on the results of thermal expansion measurements and resonant ultrasound spectroscopy, CBN28 was classified as a relaxor [Relaxor ferroelectrics with the tetragonal tungsten bronze (TTB) structure have attracted significant attention as the lead-free alternative to well-known PbMgoperties . In thisx = 0.61 ,6. Recent of SBN . Contrar relaxor . BrillouNb2O6 CBN0x system relaxor . HoweverNb2O6 CBN0x system relaxor . It was  relaxor .k-space spectroscopy [Important information about relaxor or ferroelectric behavior of CBN crystals can be obtained by investigation of their polar structures. Scanning and transmission electron microscopy studies of the (001) cut of CBN28 crystals revealed fine 180° domain patterns with domain sizes in the range of 30–200 nm ,13. Thestroscopy . A nonuntroscopy .ac voltage applied between a sample and a nanometer sharp tip. The phase of the first harmonic of this deformation provides information about the local polarization orientation, while its amplitude is proportional to the local piezoelectric coefficient. PFM has been successfully applied to the investigation of SBN single crystals, where a cross-over from ferroelectric to relaxor behavior on increasing of Sr concentration was accompanied by an enhanced polar disorder [Non-destructive direct studies of the evolution of polar structures are possible using piezoresponse force microscopy (PFM) . PFM is disorder . Moreovedisorder .In this article, we report on piezoresponse force microscopy (PFM) studies of CBN single crystals. As in the case of SBN, a diminishment of domain size was found in CBN as the Ca/Ba ratio increases. The characteristic size of nanodomains in CBN32 is comparable with that in SBN61. However, contrary to relaxor SBN61, the depolarization of a macroscopic domain in CBN31 occurs below the transition temperature, but not above it.C,  is the value of the piezoresponse signal at a point with coordinates . From the shape of the autocorrelation function, information about the symmetry and regularity of the domain structure can be obtained. In particular, the width of the central peak in the C map is a measure of the polarization correlation radius, i.e., domain size. To evaluate it, the autocorrelation function was averaged over all in-plane directions , b were obrections c and ther is the distance from the central peak, <ξ> is the average correlation radius, and the exponent h (0 < h < 1) is a measure of the roughness of the “polarization interface” [Here, terface” . We obse2 by applying a dc voltage of −40 V. As a result, a uniformly poled area appeared  and that presumably is a relaxor. We prepared a “macroscopic” domain by scanning an area of 5 × 5 µmappeared a. On heaThe investigation of the non-poled area shows that nanodomains can be still observed at this critical temperature . While t0.61Ba0.39Nb2O6 single crystals [2+ ions are restricted to the square A1 sites, whereas the pentagonal A2 sites are nearly fully occupied by Ba2+ exclusively [2+ is equally distributed between A1 and A2; additionally, A2 is less than 50% occupied by barium. Therefore, less structural disorder and, correspondingly, weaker random fields are expected for CBN as compared to SBN [Thus, we observed that the induced “macrodomain” disappears on approaching the transition temperature, while the nanodomains still exist. This behavior is in contrast to one reported for Srcrystals . In the crystals . Seeminglusively . In contd to SBN . We alsod to SBN , we can d to SBN . We suggxBax1−Nb2O6 single crystals, where x = 0.26−0.32, were grown by the Czochralski method [k = 2 N/m were used. The measurements were performed by applying an ac voltage with an amplitude acU = 5–10 V and a frequency f = 50 kHz. A heating stage was used to conduct PFM measurements between room temperature and 185 °C.The studied Cai method . ExperimIn CBN single crystals, increasing Ca content results in both decreasing domain size and local polarization. In general, in compositions with more Ca content, the polar disorder becomes more pronounced. The characteristic nanodomain size of the CBN32 single crystal is similar to that of relaxor SBN61. However, the decay of an artificial macroscopic domain (depolarization) in CBN32 takes place below the temperature of the maximum of the dielectric permittivity, but not above it like in the case of SBN61. Based upon the obtained data and earlier dielectric permittivity data , we cons


Bordetella pertussis reversibly folds into a β-roll secondary structure upon calcium binding. In this review, we discuss how the conformationally dynamic nature of the peptide is being engineered and employed as a switching mechanism to mediate different protein functions and protein-protein interactions. The peptide has been used as a scaffold for diverse applications including: a precipitation tag for bioseparations, a cross-linking domain for protein hydrogel formation and as an alternative scaffold for biomolecular recognition applications. Proteins and peptides such as the RTX domains that exhibit natural stimulus-responsive behavior are valuable building blocks for emerging synthetic biology applications.The isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from  Bordetella pertussis is a Gram-negative bacteria, first isolated in 1906 and is the causative agent of whooping cough . I. I71]. ISerratia marcescens serralysin into a 2-D protein mesh network. The RTX domain served as a spacer between two other proteins ) and the spacing of the proteins in the mesh could be controlled by varying calcium concentrations . . 69]. The amino acids on the faces of the Block V RTX domain can be mutated without affecting the calcium-dependent β-roll structure formation. Thus the RTX domain offers a unique capability among repeat proteins in that a binding face can be engineered while leaving the amino acids involved in the calcium-dependent conformational change unchanged. We explored whether the techniques used to engineer other repeat proteins, like the DARPins , could bE. coli proteins [RTX peptide libraries were created where the eight amino acids on one face of the peptide were randomized to express all 20 possible amino acids . This liproteins .Nature has evolved the RTX domains as necessary participants in the type 1 secretion system, which enables protein structure and function to be directed extracellularly. Characterization of these novel domains can shed light on disease pathology, as well as to create new therapeutic opportunities. Protein engineering approaches have been used to elucidate the biophysical behavior of these domains providing new insights into the stability and folding mechanisms of these proteins. Since the RTX domains exhibit highly specific stimulus-responsive switching behavior leading to a well-characterized disordered-to-ordered structural transition, several groups have explored these domains as building blocks for synthetic biology applications. Native RTX domains have been used to control spacing between proteins, as well as to subtly alter the conformation of an enzyme. Designed RTX repeats have been utilized to construct new peptide materials, and to create a desirable phase change that can be exploited for bioseparations. Rationally-mutated β-roll domains have been created with hydrophobic faces that can be used to drive self-assembly leading to hydrogel formation. Most recently, directed evolution approaches have been used to select β-roll domains with affinity to a target protein (lysozyme) leading to a novel stimulus-responsive capture and release capability that has been demonstrated through protein affinity chromatography. It is becoming clear that conformationally-dynamic and/or intrinsically-disordered peptide motifs are playing important roles in biological processes and as these peptides are further explored and characterized, they will lead to further biophysical insights as well as additional biotechnological uses. In summary, the Block V RTX domain of CyaA, with its remarkable conformational behavior, demonstrates how fundamental insights into biomolecular mechanisms underpinning disease pathology can inspire new technologies. It is likely that further advances will soon be made in applications including nanomedicine, theranostics, artificial viruses, and other biotechnological areas and these efforts can be supported by fundamental studies in toxinology.


Ferroelectric macro-porous BaTiO3 ceramics for piezoelectric applications are manufactured for a range of relative densities, α = 0.30–0.95, using the burned out polymer spheres method. The piezoelectric activity and relevant parameters for specific applications are interpreted by developing two models: a model of a 3–0 composite and a ‘composite in composite’ model. The appropriate ranges of relative density for the application of these models to accurately predict piezoelectric properties are examined. The two models are extended to take into account the effect of 90° domain-wall mobility within ceramic grains on the piezoelectric coefficients α ≤ 0.45 and achieve a high squared figure of merit This work demonstrates the potential of porous BaTiO