Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues.Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic . In orde N-acetyl galactosamine  residues in a calcium-dependent manner . ASGR is a C-type lectin that is abundantly expressed on the cell surface of hepatocytes in the liver . The funt manner . The ASGt manner  and the t manner . Mice lat manner . In contt manner .The interaction of ASGR with its ligands is well studied and shown to occur in clathrin-coated pits on the basolateral membrane of hepatocytes . Upon biAntisense oligonucleotides (ASOs)—short, chemically modified oligonucleotides—are attractive candidates for targeted therapeutic delivery via ASGR ,18. ASOsWe recently showed that conjugation of tri-antennary GalNAc ligands to ASOs enhances potency 10–60-fold for suppressing gene targets expressed in hepatocytes ,23. Furt125I-α1-acid glycoprotein (AGP)—a reported high affinity ligand for the ASGR—to determine the receptor binding properties of GalNAc-conjugated ASOs  on a reverse phase column. Purity and mass of ASOs were determined using ion-pair liquid chromatography–mass spectrometry (LC–MS) analysis . The residue was desalted by HPLC on reversed phase column to yield the 5΄-GN1, GN2 and GN3 conjugated ASOs. The ASOs were characterized by ion-pair-HPLC–MS analysis  at 55°C for 4 h and then cooled and added 10% (v/v) of 40% methylamine in water. Heating at 55°C was continued for additional 12–14 h to cleave GalNAc-conjugated ASOs from support, remove protecting groups, and hydrolyze the UnyLinker™ moiety. Oligonucleotides were purified by ion-exchange chromatography using a gradient of NaBr across a column packed with Source 30Q resin as described before. Pure fractions were desalted using HPLC on a reversed phase column. Purity and mass of oligonucleotides were determined using ion-pair LC–MS analysis (ASOs containing 1–3 hydroxy-proline (HP) GN modifications were synthesized according to the procedure reported . In brieanalysis .Microsome fractions containing ASGR were enriched from fresh or frozen Balb/C mouse livers as previously described ,26. BrieN-Boc-1,3-diaminopropane  in dichloromethane (50 μl), and triethylamine  were added. After stirring for 3 h the reaction mixture was diluted with dichloromethane (50 ml) and washed with 1 M aqueous NaHSO4 solution (3 × 50 ml), aqueous saturated sodium bicarbonate (2 × 25 ml) and brine (50 ml). Organic phase separated, dried (Na2SO4) and concentrated. The residue obtained was dissolved in dichlormethane (3 ml). To this trifluoroacetic acid (1 ml) was added and the reaction mixture was stirred at room temperature for 1 h. Reaction mixture was diluted with toluene (10 ml) and evaporated under reduced pressure. The residue thus obtained was dissolved in acetonitrile (2 ml) and co-evaporated with toluene (2 ml) under reduced pressure to yield GalNAc cluster amine S2 (Scheme S1). LRMS  m/z calculated for C81H134N9O35 [M+H]+: 1794.0, found 1793.8. GalNAc cluster amine S2  was dissolved in DMF (250 μl) and Cy5-NHS-Ester  was added. To this triethylamine  was added and allowed to shake for 18 h. The reaction mixture was concentrated under reduced pressure. To the residue obtained aqueous ammonia  was added and vortexed to get a solution and resulting solution was allowed to shake at room temperature for 2 h. The reaction mixture was diluted with water (20 ml) and purified by HPLC on a reverse phase column to yield GN3-SulfoCy5 Tracer S3 . 1H NMR  δ 1.17 , 1.26–1.64 , 1.66-1.78 , 1.82 , 1.88–1.96 , 2.05–2.18 , 2.33 , 2.95–3.10 , 3.39–3.47 , 3.50–3.84 , 4.02 , 4.31 , 6.22 , 6.49–6.57  7.27 , 7.72–7.80 , 8.01 ; LRMS  m/z calculated for C95H153N11O33S22 [M+H]+: 2039.4, found 2039.5.Synthesis of Tracer was accomplished as described in 2, 5 mg/ml bovine serum albumin (BSA), and 0.1% Triton X-100. 10 μl of GalNAc ASO of various concentrations was added to the mixture and pre-incubated for 15 min. Then, 12.5 μl GN3-sulfoCy5 tracer S3 was added to the mixture for a final concentration of 1.25 nM . Using polarized excitation and emission filters, the instrument measures fluorescence perpendicular to the excitation plane (the ‘P-channel’) and fluorescence that is parallel to the excitation plane (the ‘S-channel’), and then it calculates FP in millipolarization units (mP) as follows:We adapted assay conditions reported by Kornilova with a few changes outlined . Instead* G) / (S + P * G)] * 1000. The ‘G-factor’ is measured by the instrument as a correction for any bias toward the P channel. Specific binding was calculated by subtraction of the mP value of the highest competitor concentration utilized after addition of galactose to a final concentration of 1 mM.mP = [, 5΄-ATGCGACTTGTCAGGCTGG-3΄ (reverse primer) and 5΄-CGTGGAGAACCGCAGCCTCCATT-3΄ (probe). RNA was normalized to total RNA using RiboGreen, and all experiments were performed in triplicate.Freshly isolated mouse hepatocytes were placed in wells with growth medium containing 10% FBS, antibiotic-antimycotic, HEPES, glutamine and varying amounts of ASO or PBS control. Cells were maintained at 37°C and 5% COad libitum. Tissues were collected, weighed, flash frozen on liquid nitrogen, and stored at –60°C.Animal experiments were conducted in accordance with the American Association for the Accreditation of Laboratory Animal Care guidelines and were approved by the Animal Welfare Committee . The animals were housed in microisolator cages on a constant 12-h light–dark cycle with controlled temperature and humidity, and were given access to food and water ASGR1 –/– (JAX#009105) and their respective controls, C57BL/6NJ (JAX#005304), were obtain from The Jackson Laboratory. 5–6 mice/group were dosed by subcutaneous injection of saline or ASO 38 at 0.3, 1, 3 and 10 mg/kg or ASO 39 at 3, 8 and 24 mg/kg once and sacrificed 72 h after dosing. A 6 mm biopsy punch was used to remove an ∼50–100 mg piece of liver tissue, the tissue was placed in 1.5 ml of 1% beta-mercaptoethanol in buffer RLT (Qiagen) and homogenized for 10 s with a TH-01 tissue homogenizer  at 35 000 rpm. The homogenized liver tissue was placed on dry ice to freeze the homogenate and stored at –80°C until the RNA purification was performed. GCGR mRNA expression was analyzed by qRT-PCR using the OneStep RT-PCR system . The sequences of primers and probe used for mouse glucagon receptor (GCGR) are 5΄-ATTTCCTGCCCCTGGTACCT-3΄ for the forward primer, 5΄-CGGGCCCACACCTCTTG-3΄ for the reverse primer, and 5΄-CCACAAAGTGCAGCACCGCCTAGTGT-3΄ for the probe.Six- to eight-week-old C57BL/6 mice (Charles River Laboratories) were treated according to the indicated treatment schedules. A 6 mm biopsy punch was used to remove an ∼50–100 mg piece of liver tissue, the tissue was placed in 2 ml of 8% beta-merccaptoethanol in UltraPure™ Guanidine Isothiocyanate Solution (Thermo Fisher) and homogenized for 10 s with a TH-01 tissue homogenizer  at 35 000 rpm. The homogenized liver tissue was placed on dry ice to freeze the homogenate and stored at –80°C until the RNA purification was performed. The sequences of primers and probe used for mouse scavenger receptor B1 (SRB1) are 5΄-TGACAACGACACCGTGTCCT-3΄ for the forward primer, 5΄-ATGCGACTTGTCAGGCTGG-3΄ for the reverse primer, and 5΄-CGTGGAGAACCGCAGCCTCCATT-3΄ for the probe.® Pro 96 total RNA Purification Kit (Thermo Fisher) with on column DNase I (Thermo Fisher) treatment and target mRNA was measured by RT-qPCR using the Express StepOne SuperMix Kit (Thermo Fisher). Part of the initial total RNA eluates were diluted to a concentration of approximately 10 ng/μl and the actual concentration was determined using the Quant-IT Ribogreen RNA assay (Thermo Fisher). Approximately 50 ng of total RNA was combined with 10 μl Express PCR Supermix, target specific forward and reverse primers and hydrolysis probe , 0.4 μl ROX Reference Dye and 2 μl of Express Superscript Mix in a total reaction volume of 20 μl in a 96-well PCR plate. Samples were run on StepOnePlus thermocyclers with the following cycling protocol: 15 min at 50°C, 2 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. Cq values were derived using the StepOne software v2.3 and relative levels of mRNA expression for each sample was determined by comparing the sample Cq to the Cq values of a dilution series  of concentrated RNA, from a pool of the initial RNA eluates. Relative target expression was then normalized to total input RNA levels based on the Ribogreen assay values. The normalized sample values were then divided by the average value of all samples from the appropriate control group to generate the reported relative expression of the target mRNA. Data are mean values ± standard deviations.Reduction of target expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the StepOnePlus System (Thermo Fisher). Briefly, RNA was extracted from ∼50–100 mg of liver tissue from each mouse using a 96-well format RNeasy Kit (Qiagen) or the PureLinkKi values) were determined with GraphPad Prism 5 software using non-linear regression for curve fit assuming one binding site with one concentration of ligand and multiple concentrations of competitor. Concentration and dissociation constants (KD) of tracer were constrained to 1.25 and 9 nM, respectively. Effective doses to cause 50% inhibition (ED50) values were determined by plotting log dose versus mRNA relative to untreated controls using a four-parameter fit with variable slope and constraining bottom = 0 and top = 1. Statistical significance of in vivo knockdown experiment in ASGR knockout mice were calculated using a two-way ANOVA analysis.Inhibition constants  mRNA using chemistries described in Figure A widely accepted model for ASGR binding proposes a tri-antennary scaffold with precise distances between the sugar moieties and the linker scaffold as the optimal ligand ,29 Figu. To dete1–4, Figure 5–8, Figure 9–12, Figure 13–16, Figure First, we examined the effect of ASO single-strandedness, backbone charge and chemical composition on ASGR binding in the FP competition binding assay. To evaluate the effect of backbone composition, ASOs containing full phosphorothioate  of the single stranded SRB1 ASO 4, gave a several thousand-fold enhancement in binding affinity relative to the unconjugated ASO or the monovalent GalNAc sugar itself (Ki ∼ 860 μM)  . The imp31) or one sugar to the 3΄- and the 5΄-end (ASO 32) of the ASO resulted in further enhancement in ASGR binding affinity relative to the one GalNAc designs above (ASOs 29–30). Interestingly, ASOs 31 and 32 showed similar binding affinity and activity as ASO 28 in which the three GalNAc sugars were displayed in a linear arrangement. The improved ASGR binding and activity of ASO 32 with a GalNAc sugar on either end of the oligonucleotide is especially interesting as this arrangement does not conform to the previous model for ASGR binding which proposed the importance of precise orientations and distances between the GalNAc sugars for optimal binding ..31) or o29:33 and 4:34) which is functionally active in cells as the complementary phosphodiester strand is cleaved within endosomes to liberate the parent ASO . Once more, we observed that GalNAc valency had a greater impact on ASGR binding and activity as compared to the spatial orientation of the GalNAc sugars. Interestingly, the ASO duplex with a GalNAc sugar attached to either end of each strand , showed the strongest ASGR binding and activity comparable to the single stranded three GalNAc ASO 28  mRNA in ASGR knockout mice Figure . GCGR wae Figure . The ASGe Figure .ASGR is primarily expressed on hepatocytes and therefore is well-suited for receptor-mediated delivery of drugs . Target in vitro and in vivo, characterized by ∼10-fold increase in activity compared with unconjugated ASOs. ASGR binding of ASOs conjugated to two or three GalNAc sugars was not affected by PS content, length, charge, or spatial arrangement of GalNAc sugars. In contrast, single strandedness, PS content, length and charge affected the ASGR binding and in vitro and in vivo activity of one GalNAc ASOs.In the model for binding to ASGR mediated by GalNAc sugars, single stranded or even duplexed ASOs, conjugated to 3 GalNAc sugars bind the ASGR with the highest affinity, and can undergo ASGR-mediated endocytosis Figure . ASOs coin vivo activity was reduced by only 2-fold. These results suggest that the ASGR binding affinity of one GalNAc ASOs in the competition binding assay is not directly proportional to their in vivo activity which may be a reflection of the higher stoichiometry of one GalNAc ASO ligands required to displace the three GalNAc tracer from ASGR membranes (Figure It is important to note, that although ASOs conjugated to one GalNAc showed 10-fold reduced binding affinity compared to ASOs conjugated to two or three GalNAc sugars, their s Figure .An unexpected outcome from our work was the realization that the ASGR is potentially involved in the uptake of unconjugated PS ASOs into hepatocytes. The importance of the PS modification for enhancing the protein binding properties of oligonucleotides has been long recognized ,36. We rOur binding data suggests that the unconjugated ASO does not interact with the receptor within the carbohydrate binding pocket given the high concentrations of unconjugated ASO required to displace the ASGR membrane bound GN3-SulfoCy5 tracer. It is more likely that the ASO interacts with basic amino acids on the exposed surface of the receptor and this interferes with binding of GalNAc within the shallow carbohydrate binding pocket in ASGR1 . This hyThe X-ray crystal structure of the ASGR1 ectodomain shows that the carbohydrate binding pocket is shallow and solvent exposed . As a reThe generally accepted stoichiometry for ASGR1 and ASGR2 is 2:1. However, others have reported stoichiometries ranging from 2:1 to 5:1 for ASGR1 and ASGR2 based on immunoprecipitation assays . Thus, oThe contribution of ASGR towards uptake of PS ASOs was further confirmed in hepatocytes and in ASGR knockout mice where loss of ASGR expression coincided with loss of activity for both, GalNAc-conjugated and unconjugated ASOs. However, ASO activity was not completely ablated in ASGR knockout mice indicating that other pathways for ASO uptake are operative in these cells. Presumably, the ability of the PS backbone to enhance ASO protein binding promotes interactions with other cell-surface proteins which allows the ASO to be internalized into cells via multiple pathways.Conjugation with GalNAc sugars can improve the potency of second-generation ASOs in animals, as a direct result of enhanced delivery of the modified ASOs to hepatocytes via binding to ASGR. The optimal structure for efficient delivery is a single stranded ASO conjugated to two or three GalNAc sugars, as its potency is not affected by the composition of the ASO backbone. However, single stranded ASOs conjugated to a single GalNAc sugar also possess therapeutic potential for enhancing ASO potency via targeted delivery to the liver. The insights gained from this study support other efforts to further characterize properties of GalNAc-conjugated and unconjugated ASOs using the validated FP assay for high-throughput screening of potential therapeutics. GalNAc-conjugated ASOs represent an advanced therapeutic platform for liver-targeted delivery and are progressing into human clinical trials . Such enSupplementary DataClick here for additional data file.


The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials.Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA N-acetylglucosaminidase, glucocerebrosidase, exo-1,4-β-glucosidase, and exo-1,3/1,4-β-glucanase, that have been characterized tosidase , N-acetynosidase , and β-gnosidase . They ard-glucan exohydrolase Exo1 from Hordeum vulgare (barley) was reported 6-sandwich domain 2, containing the acid/base catalyst. The contribution of different domains in supplying crucial catalytic residues was a highly unusual feature of GH3 enzymes. Furthermore, in the last few years many new structural studies have shown a great variety in domain composition and arrangement of typical GH3 β-glycosidases, having up to four separate domains (8N-acetylglucosaminidases (NagZ) members showing that, despite a few having two domains, most Gram-negative bacteria encode single domain enzymes, and all of them have the acid/base catalyst in an unusual histidine/aspartate dyad located in a flexible loop of the (α/β)8 barrel  analysis. The structural and biochemical analysis of the GlyA1 hydrolase presented in this study shed new light on comparative catalysis and evolutionary model studies as well as phylogenetic relationships.Here, activity screening of a metagenomic library created from rumen fluid led us to the isolation of a novel β-glycosidase, GlyAp-nitrophenyl-β-d-glucoside (pNPβGlc) and p-nitrophenyl-β-d-cellobioside (pNPβCel). We identified a positive clone (designated SRF4) that is highly active against both substrates. The fosmid with insert SRF4  was fully sequenced. A gene herein designated as glyA1 encoding a potential GH3 β-glycosidase (GlyA1) was identified out of the 38 distinct genes on the hit fosmid. The deduced molecular mass and estimated pI value were 101,849 Da and 4.86, respectively. This 921-amino acid-long putative protein exhibited a maximum amino acid sequence identity of 59% to a similar protein in public databases (with a top hit EDO57841.1 from Clostridium sp.). A search of oligonucleotide patterns against the GOHTAM database  showed similarity to glycosidases of unknown Clostridia (phylum Firmicutes). BLASTP search with identified protein sequences showed good matches for many of them against corresponding proteins in Eubacterium and Prevotella and members of Lachnospiraceae, Clostridium, Ruminococcus, and Bacteroides. Most likely, GlyA1 has thus its origin in the phylum Firmicutes, and the presence of a phage gene may, however, indicate a horizontal gene transfer of the carbohydrate metabolism genes from Firmicutes to Bacteroidetes. Those microbes are known to be abundant in the ruminal environment and are thought to play key roles in the breakdown of proteins and carbohydrate polymers  collected from rumen-fistulated, non-lactating Holstein cows  was scredatabase  and TBLApolymers , 31.1) was cloned, expressed in E. coli BL21 (DE3), and purified. The hydrolytic activity was analyzed using 18 synthetic model p-nitrophenyl (pNP) derivatives with different sugars as well as a series of 11 additional oligosaccharides. Their specific activities (units/g protein) (mK), the catalytic rate constant (kcat), and the catalytic efficiency (kcat/mK) values  and pNP-N-acetyl-β-d-galactosaminide  was below detection limits, and thus the enzyme does not have β-N-acetylglucosaminidase nor β-N-acetylgalactosaminidase activity. As shown in pNPβCel was the preferred substrate, mainly due to the higher affinity for this substrate as compared with other pNP sugars. The purified recombinant hydrolase was also assayed for their activities toward different polymeric substrates. Using specific activity determination, GlyA1 hydrolyzed all short cello- and xylo-oligosaccharides tested (degree of polymerization (DP) from 2 to 5), with longer substrates being slightly preferred (kcat/mK) while using the non-activated substrates cellobiose and xylobiose were lower than those found for pNPβCel and pNP-β-xylobiose (pNPβXylb), respectively, mainly due to a significant decrease of kcat values for the natural disaccharides  found for other substrates is from low to very low mainly due to lower catalytic rates. The enzyme also exhibited activity against lichenan, suggesting that is able to hydrolyze substrates with mixed β-1,3/1,4 linkages. No activity was detected using avicel or filter paper, as well as toward substrates without β-1,4 linkages such as β-1,3 glucan or mixed β-1,3/1,6 linkages such as laminarin. Accordingly, the enzyme showed a clear preference for short cello-oligosaccharide substrates, which may likely be produced in natural settings from the cellulose components of plant cell walls due to the action of glucanases in the ruminal fluid. Other substrates such as gentibiose  and sophorose (or 2-O-β-d-glucopyranosyl-α-d-glucose) were also hydrolyzed to a similar extent as cellobiose and xylobiose. The optimum activity for GlyA1 was observed within a mesophilic range (45–65 °C) and within a neutral or slightly acid pH (6.0–7.0), being most active at 55 °C and a pH close to 6.5 (The gene encoding putative GH3 β-glycosidase (GlyAprotein)  and the ) values  were detis order . The actreferred . The catcharides . A compae to 6.5 .1-ΔCt, was created in the vector pQE80L. After purification, activity was determined for the 18 sugars being hydrolyzed by the wild-type enzyme, so the effect of the C-terminal region was tested. As shown in A mutant containing a missing C-terminal region, herein referred to as GlyA1 were obtained after more than 3 months with PEG3350 as the precipitant, and they were cryoprotected into 25% d-glucose to obtain the complex with this sugar. The structure was solved by molecular replacement using the domains from T. neapolitana β-glucosidase as independent search models. Refinement and analysis of electron density maps allowed modeling of the chain containing residues 3–798 but did not show any density to build the C-terminal segment 800–921, suggesting a putative cleavage of this region in the slow crystallization step. The low numbers of crystals impeded analysis of the intact protein by mass spectrometry, but SDS-PAGE analysis of protein solution samples revealed the presence of two bands after incubation at room temperature or treatment with proteases. Therefore, the sample was incubated with subtilisin previously to the crystallization step, which accelerated formation of many good quality crystals, under similar conditions and with the same space group. These crystals were cryoprotected into 20% glycerol, and this molecule was found bound at the active site. Furthermore, crystals from a truncated construct containing residues 2–799 (GlyA1-ΔCt) also grew in a week with ammonium sulfate as the precipitant and, despite having different shape, yielded the same cell and space group, which is consistent with the hypothesis that the wild-type sample was cleaved. These crystals were used to obtain the complexes with d-xylose and d-galactose. Many attempts done to crystallize the complete enzyme were unsuccessful. Also, a construct with residues 800–921, containing the isolated C-terminal region (GlyA1-Ct), failed to crystallize. Crystallographic data and refinement statistics for the four structures here presented are given in Preliminary crystals from the wild-type GlyAd-glucan glucohydrolase (8 barrel domain 1 linked to an (α/β)6-sandwich domain 2 (A); both of them provided residues that make up the active site. The later reported structures from T. neapolitana , although its function is unknown 6 sandwich (previous domain 2) is located at the N terminus and the FnIII domain is sequentially inserted between this and the (α/β)8 barrel (A). Additionally, a 120-residue segment attached to the C terminus most surely folded into an additional domain.The first solved structure from barley β-ydrolase  showed tdomain 2 A; both oa reesei , Aspergiergillus , 14, and innocua  β-glucos innocua , showed arxianus  and Strenezuelae  that als unknown . To expa8 barrel A. AdditiB displays the 3D structure of the solved 3–798 region of GlyA1, which present overall dimensions of 85 × 65 × 45 Å. The N-terminal (α/β)6-sandwich domain  is followed by the FnIII domain  and the (α/β)8 barrel domain . Two long segments connect the three domains . Linker 1 (residues 220–277) and half of linker 2 (residues 411–443) are tightly wrapped over the core structure, whereas the rest of linker 2 (444–467) forms an extended arm that clasps the (α/β)8 barrel. Finally, the regions at the beginning and the end of the chain are making a two-stranded β-sheet that laces the core structure at the top.Fig. 21 (α/β)6-sandwich domain, containing the catalytic acid/base residue Glu-143, superimposes onto the corresponding domain from the T. neapolitana β-glucosidase, with a root-mean-square deviation (r.m.s.d.) of 1.6 Å for 202 eq Cα positions (39% sequence identity). The same comparison with the other structurally known GH3 gives deviations in the range 2–2.5 Å (20–25% sequence identity). The FnIII domain seems more structurally conserved along the GH3 family; GlyA1 is most similar to those in the β-glucosidases from T. neapolitana, with r.m.s.d. = 1.5 Å , and K. marxianus, with r.m.s.d. = 1.6 Å , but the same analysis gives values in the range 1.8–1.9 Å (21–28% sequence identity) against the other GH3 enzymes containing this domain. Finally, the (α/β)8 barrel, which contains the nucleophile Asp-709, is most similar to the corresponding domain in the β-glucosidases from T. neapolitana , K. marxianus, , S. venezuelae , and T. reesei . Equally to GlyA1, all these domains present a deviation from the canonical (α/β)8 barrel topology, which was first observed in the T. neapolitana β-glucosidase. Thus, their first α-helix of the eight β-α motifs is missing, which has the consequence of making strand β2 reversed and antiparallel with the other seven strands. The different deviation from the canonical topology found at this domain is consistent with the higher deviations found in the structural comparison of GlyA1 with other GH3 enzymes, in the range 2.5–3 Å (16–20% identity).Comparative analysis using the Dali  server r1 core is structurally rather conserved with known β-glucosidases with equivalent domain architecture (C). The superposition of the T. neapolitana β-glucosidase onto the structure of GlyA1 reported here shows small differences in the orientation of some of the helices (D). The main difference is the long arm that links the FnIII to the (α/β)8 domain in GlyA1, which is missing in T. neapolitana β-glucosidase. There are also significant differences in the loops surrounding the active site both in length and orientation, which must be related to the different substrate specificity, as commented below.Interestingly, the GlyAitecture C. The su helices D. The ma1 is located at the molecular surface, at the interface between the (α/β)8 barrel domain, which provides the nucleophile Asp-709 and the (α/β)6-sandwich domain, contributing to the Glu-143 acid/base catalyst (A). The participation of Asp-709 in substrate hydrolysis was confirmed by site-directed mutagenesis (D709A) in GlyA1 and GlyA1-ΔCt, as mK and kcat values could not be determined from the data obtained due to the activity value being below the detection limit. It is a pocket of 12 Å deep with a narrow entrance 4–6 Å wide. A detailed structural comparison with the T. neapolitana β-glucosidase (A) reveals the main differences in loop conformation observed around the active site that are responsible for making a deeper catalytic pocket in GlyA1. First, loop β7-α7 of the (α/β)8 barrel, following the nucleophile Asp-709 (residues 711–726), has an 11-residue insertion that extends away from the pocket and interacts with the long segment linking the FnIII domain to the barrel, which is missing in the T. neapolitana β-glucosidase. Here, Arg-717 makes an ion pair with Glu-447 at the small helix located in the middle of the extended linker, which helps in stabilizing this region. An important feature of this β7-α7 loop is the presence of Trp-711, close to the nucleophile Asp-709, that protrudes from the surface and delineates a narrow catalytic pocket. Moreover, and despite loop β3-α3 (residues 536–550) being shorter in GlyA1, Arg-538 clearly bulges into the pocket contributing to constrict it even more.The active site of GlyAcatalyst A. The pacosidase A reveals6-sandwich, similarly to that observed in T. neapolitana β-glucosidase, this domain is shaping the active site by means of two loops, residues 139–152 containing the acid/base catalyst Glu-143 and residues 100–113 enclosing Trp-106 that clearly projects into the catalytic pocket. Interestingly, the last loop is markedly flexible as it is deduced from the fact that it could only be fully traced in the ligand-free crystal, containing only glycerol in the active site, and in the galactose-soaked crystal of the truncated form. In contrast, the crystals of the full-length and truncated forms, soaked into glucose and xylose, respectively, showed poor density that precluded tracing residues 104–107. Furthermore, the traced loops showed significant conformational changes in the different crystals at Trp-111, coupled to a change in Phe-147 from the adjacent 139–152 loop (A), reinforcing its intrinsic mobility. The loop equivalent to 100–113, which is highly variable within GH3 enzymes, was proposed to be involved in recognition of large substrates from the crystal structure of T. neapolitana β-glucosidase, which showed some disorder that precluded tracing of a segment equivalent to that not observed in some GlyA1 crystals. Noteworthy, the non-visible region of T. neapolitana β-glucosidase includes Trp-420 that, consequently, may be defining additional binding subsites, similarly to Trp-106. However, the remaining sequence is not conserved, with both Phe-147 and Trp-111 being unique to GlyA1, and therefore, the substrate recognition mode presented by the two enzymes to accommodate the substrate may be different.With respect to the (α/β)152 loop A, reinfoB). This subsite is well conserved among known GH3 β-glucosidases and, with the exception of the acid base catalyst, is made up entirely by residues from the (α/β)8 barrel domain. Thus, residues from the loops emerging from the central β-strands are making a tight net of hydrogen bonds that accommodate the glycon with all its OH groups making at least two polar interactions. The glycon moiety is located by stacking to Trp-710, and the acid base catalyst Glu-143 and the nucleophile Asp-709 interact with the O1 and O2 hydroxyls, as is expected in GH enzymes. The other residues making subsite −1 are Asp-532, Arg-597, Lys-630, His-631, Arg-641, and Tyr-677. Xylose and glucose are bound in an identical position, and the glycerol molecules observed in the ligand-free crystals are mimicking the positions occupied by C2, C3, C4, and C5 from both sugars. The additional polar interaction made by the glucose O6 hydroxyl appears consistent with the higher affinity observed in GlyA1 toward glucosides as compared with xylosides. Thus, as shown in mK = 2.4 ± 0.3 mm) was ∼2-fold higher than that for xylobiose (mK = 4.7 ± 0.2 mm). Interestingly, soaking of crystals with galactose showed that this sugar displays a semi-chair conformation at subsite −1 by flattening of the C4 atom that has the axial hydroxyl substituent . In this way, galactose is accommodated by essentially the same polar interactions observed in the glucose complex, thereby explaining the activity of the enzyme on β-galactosides. However, the energy cost of getting the substrate ring distortion is reflected by the lower β-galactosidase activity, as given in 1 to accept different sugars (from high to low and very low specificity).Soaking with xylose and glucose showed a clear density indicating that both sugars occupy the catalytic pocket subsite −1 in a relaxed chair conformation B. This sstituent B, inset.T. neapolitana β-glucosidase that presents an active site opened to the solvent with only subsite −1 being defined, more subsites are apparent in GlyA1. To delineate a putative +1 subsite, we modeled the position of the non-hydrolysable substrate analogs thiocellobiose and thiogentibiose by structural superimposition on the previously reported experimental barley complexes  is manifested by the lower mK value with pNPβCel compared with pNPβGlc and by the lower mK value with pNPβXylb compared with pNPβXyl , a Glc-4Glc-3Glc-4Glc chain (purple), and a Glc-4Glc-4Glc-3Glc (yellow). These sugars have been docked by superimposition of their non-reducing units onto the observed glucose at the GlyA1 complex. The hydrophobic patch defined by Trp-106 and Trp-711 may fit the oligosaccharides at subsites +1 and +2, and the long side chain of Lys-723 seems available to make polar interactions with the hydroxyl groups defining a possible subsite +3. The putative existence of at least three subsites in the GlyA1 active site would be in agreement with the tendency of an increased activity against longer cello- and xylo-oligosaccharides  8 barrel domain. At the opposite face, GlyA1 Trp-106 is structurally equivalent to Tyr-443 and Trp-434 from the barley and T. reesei enzymes, although all of them come from different loops within the (α/β)6-sandwich domain. Interestingly, other enzymes present an aromatic residue in a position identical to Trp-106, but they are provided by the PA14 domain, Phe-508 in the case of the K. marxianus β-glucosidase, or by a long loop coming from the other subunit, Tyr-583 in the case of the L. innocua β-glucosidase dimer (data not shown) (Comparison of the GlyAcosidase  and barlllobiose E 7) dis dis1-Glct shown) . This fe1, we explored its overall flexibility and putative shape in solution by SAXS experiments. Thus, we compared the molecular descriptors of the complete construct with respect to the truncated construct GlyA1-ΔCt, lacking the C-terminal domain. For this purpose, several solutions with varying concentrations were measured for each sample, and their scattering curves were merged to extrapolate idealized data. Analysis of the scattering curves shows a good fit to the Guinier approximation, which indicates that the samples are not aggregated. Also, the calculated radii of gyration (gR) are consistent across the range of measured concentrations. Then, the overall size descriptors can be properly determined for each construct.Because of the unfeasibility in crystallizing the full-length GlyAgR and the maximum distance (Dmax) for the complete protein are only slightly higher than the truncated protein, which may be indicate that the extra C-terminal domain is not too extended from the core structure. In support of this hypothesis, the pairwise distance distribution function P(r) calculated for both constructs shows a similar unimodal pattern consistent with a single domain protein in both cases. Furthermore, the analysis of the scattering function by the Kratki plots is consistent with the expected profile for a folded protein with a clear peak, in contrast what is observed in multidomain proteins with flexible linkers that present several peaks or smoother profiles. Consequently, we do not observe in the data calculated from the complete protein any of the signs that may be indicative of molecular flexibility, i.e. large gR and Dmax, absence of correlation in the P(r) function, or smooth Kratky plots. Therefore, SAXS analysis appears consistent with a compact overall shape of the complete GlyA1, in which the extra C-terminal region would not define a marked separate or flexible domain but rather it could be folded over the core three-domain structure.First of all, the calculated molecular masses from both samples are close to the expected values , indicatab initio models were generated for complete GlyA1 from SAXS data. First, two models of the last 120 residues (GlyA1-Ct) were obtained, as explained under “Experimental Procedures,” with both showing an overall β-sandwich topology. This topology is related to carbohydrate-binding domains within families CBM6 and CBM35, to which GlyA1-Ct presents 15–20% sequence identity, although the equivalent carbohydrate-binding motifs, typically clusters of conserved aromatic residues, are not evident in its surface. Then, three runs of CORAL were computed by considering the experimental structure of the truncated protein and each of the two models. The six models obtained are shown in 1-Ct may be somewhat packed between the two domains making the core structure and, interestingly, with a putative linker somehow exposed to solvent. This feature might possibly explain the proteolysis observed in the complete protein.To test the feasibility of this hypothesis, 1 keeps the location of the active site at the interface between the (α/β)8 barrel and the (α/β)6-sandwich domains. As mentioned above, N-acetylglucosaminidases are built by a single domain, with its (α/β)8 barrel holding both the nucleophile and acid/base catalyst. Interestingly, the Bacillus subtilis NagZ shows the two-domain composition but still keeps the catalytic residues at the (α/β)8 barrel (1) within the GH3 family, we have carried out a phylogenetic analysis based on the sequence of its (α/β)8 barrel domain . Sequences representative for each of the domain architectures found in the GH3 domain were selected . The five topologies selected for this study are ABB, ABB-ABS, ABB-ABS-FLD, ABS-FLD-ABB, and ABB-ABS(PA14)-FLD (ABS (α/β)6-sandwich; FLD is fibronectin-like type III domain). The resulting phylogenetic tree given in N-acetylglucosaminidases . Insertion of the ABS module is associated with three different nodes  were appended after ABS. At node b, fusion of C-terminal FLD seems to occur close to ABS addition because most sequences contain both modules. GlyA1 and the other GH3 enzymes with inverted topology arose within this cluster. The phylogenetic analysis shows that the inverted topology is predominantly found in Firmicutes, although it is also present in at least another phylum (Actinobacteria) and even Archaea. Furthermore, it appears clearly associated to enzymes belonging to bacteria dwelling in the digestive tract of animals.Our structural analysis illustrated that the permuted domain architecture of GlyA8 barrel . TherefoIn this work, a functional metagenome library analysis was used to identify a β-glycosidase from a plant polymer-degrading microorganism populating the rumen of a dairy cow. The enzyme most likely originated from the genome of a representative of Firmicutes phylum known to be abundant in the ruminal environment , 31.1 hydrolase presented in this study sheds new light on the mechanisms of the catalysis and evolutionary patterns of the GH3 family. Our data demonstrated that GlyA1 has a permuted domain topology. It is well documented that the formation of new domain combinations is an important mechanism in protein evolution. The major molecular mechanism that leads to multidomain proteins and novel combinations is non-homologous recombination, sometimes referred to as “domain shuffling.” This may cause recombination of domains to form different domain architectures. Proteins with the same series of domains or domain architecture are related by descent (i.e. evolved from one common ancestor) and tend to have the same function . It is well documented that these wide ranging gene transfer events take place at high frequency in the rumen  strongly suggest that GlyAhe rumen , 38. Pro1 that is composed of an N-terminal (α/β)6-sandwich domain, followed by the FnIII domain, and the (α/β)8 barrel domain. Based on sequence data, a C-terminal domain was expected after the (α/β)8 barrel domain. However, attempts to crystallize the C-terminal region of the protein were unsuccessful, and its functional role was unclear. Biochemical characterization of the GlyA1 and GlyA1-ΔCt proteins revealed that the C-terminal domain does not affect the overall substrate profile of the protein, but rather it affects the catalytic performance, which is significantly lower in the truncated GlyA1-ΔCt protein. This suggests that most likely the C-terminal domain may not have a direct role in substrate binding, but still it might disturb the dynamics of the proximate mobile loop (residues 100–113), which seems directly involved in catalysis.Structural analysis illustrates the permuted domain composition of GlyA1-Ct homologous sequences shows that these domains are attached to GH3 β-glucosidases from a ruminal environment, and this feature points to a possible function related to this ecosystem. However, its presence is not related to the permuted domain topology, as only half of the sequences included in the GlyA1 cluster . Nevertheless, although its involvement in binding small substrates does not seem apparent, this domain might be playing a role in positioning or locating the enzyme to distal positions of a yet unknown polymeric substrate by recognizing specific but still unidentified substitutions. Alternatively, it could play a role in keeping the enzyme attached to the cell surface, facilitating the intake of its products and conferring the bacteria an advantage over competing organisms. Interestingly, the analysis of the GlyA cluster  contain 1 here presented uncovers new features of GH3 enzymes and provides a template for a novel subfamily, including members with permuted domain topology. It also allows picturing the GlyA1 active site architecture and the molecular basis of its substrate specificity. More work is needed to have a complete picture of the intricate molecular mechanisms that these highly diverse enzymes have evolved to tailor specificity. It will contribute to improve our knowledge about enzymatic carbohydrate degradation and open up new avenues for biocatalysis.In conclusion, the analysis of GlyAE. coli Rosetta2  for cloning and expression of wild-type protein and the genetic constructs in pQE80L vector were cultured and maintained according to the recommendations of the suppliers.Chemicals and biochemicals were purchased from Sigma and Megazyme  and were of pro-analysis (p.a.) quality. The oligonucleotides used for DNA amplification were synthesized by Sigma Genosys Ltd. . The pNPβGlc and pNPβCel. For screens, the plates  were covered with an agar-buffered substrate solution . Positive clones were detected by the formation of a yellow color. One positive clone, herein designated as SRF4, was selected, and its DNA insert was fully sequenced with a Roche 454 GS FLX Ti sequencer  at Life Sequencing S.L. , and the predicted genes were identified as described previously  and a deleted version (residues 2–799) lacking the C-terminal domain (GlyA1-ΔCt) were amplified by PCR with 4GF (CACGAGCTCAATATTGAAAAAGTGATACTTGATTGG) as forward oligonucleotide and 4GR1 (AGCCGTCGACTTACTGCTGCTTTTTAAACTCTATTCG) or 4GR2 (AGCCGTCGACTTACACTCTTCCTGCTATCTCAACC) as reverse oligonucleotides, respectively. The SRF4 fosmid was used as the template. The PCR conditions were as follows: 95 °C for 120 s, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 120 s, with a final annealing at 72 °C for 500 s. The PCR products were analyzed and agarose gel-purified using the Mini Elute gel purification kit . The PCR products were digested with SacI/SalI and cloned in vector pQE80L to generate plasmids GlyA1-pQE and GlyA1ΔCt-pQE, respectively. The coding sequence of the C-terminal domain  was amplified with oligonucleotides CT1F (CACGAGCTCATAGAAGAGGATGCATTCGATATAG) and 4GR1 and cloned in the SacI/SalI sites of pQE80L (plasmids Ct-pQE). GlyA1-pQE was used as a template to introduce the mutation D709A by PCR with primers M1 (TGGTGGGCTCAGGTTAATGACC) and M2 (GGCAGTCATCACAATACCCTTAAAGCC), as described previously  was transformed with the selected plasmids; the clones were selected on LB agar supplemented with ampicillin (100 μg/ml) and chloramphenicol (68 μg/ml) and stored with 20% (v/v) glycerol at −80 °C.The full coding sequence of GlyAeviously . The cod1 and GlyA1-ΔCt, using the QuikChange II XL mutagenesis kit from Agilent Technologies, Inc. , with TGGTGGGCTCAGGTTAATGACC and GGCAGTCATCACAATACCCTTAAAGCC as forward and reverse oligonucleotides, respectively. The resulting variant plasmids were transferred into E. coli strain Rosetta2  and selected on the LB agar supplemented with the same antibiotics as parental plasmids.Mutation D709A was introduced into the corresponding pQE80L plasmids containing genes encoding GlyA1 and GlyA1-ΔCt variants, as well as GlyA1-Ct in the pQE80L vector, a single colony (E. coli Rosetta2) was grown overnight at 37 °C with shaking at 200 rpm in 100 ml of 2× TY medium  containing ampicillin (100 μg/ml) and chloramphenicol (68 μg/ml), in a 1-liter flask. Afterward, 25 ml of this culture was used to inoculate 1 liter of 2× TY medium, which was then incubated to an A600 nm of ∼0.6 (range from 0.55 to 0.75) at 37 °C. Protein expression was induced by 0.9 mm isopropyl β-d-galactopyranoside followed by incubation for 16 h at 16 °C. The cells were harvested by centrifugation at 5000 × g for 15 min to yield 2–3 g/liter pellet (wet weight). The cell pellet was frozen at −80 °C overnight, thawed, and resuspended in 3 ml of 20 mm phosphate buffer, pH 7.4, 500 mm NaCl/g of wet cells. Lysonase bioprocessing reagent  was then added (4 μl/g wet cells) and incubated for 30 min on ice with rotated mixing. The cell suspension was then sonicated for a total of 1.2 min and centrifuged at 15,000 × g for 15 min at 4 °C; the supernatant was retained. The His6-tagged enzyme was purified at 4 °C after binding to a nickel-nitrilotriacetic acid His·Bind resin . The columns were pre-washed with 20 mm phosphate buffer, pH 7.4, 500 mm NaCl, and 50 mm imidazole, and the enzyme was eluted with the same buffer but containing 500 mm imidazole. The monitoring of the enzyme elution was performed by SDS-PAGE and/or activity measurements, using standard assays (see below). After elution, protein solution was extensively dialyzed with 20 mm Tris, pH 7.5, 50 mm NaCl by ultrafiltration through low adsorption hydrophilic 10,000 nominal molecular weight limit cutoff membranes , after which the protein was maintained at a concentration of 10 mg/ml; the protein stock solution was stored at −20 °C until used in assays. The purity was assessed as >95% using SDS-PAGE, which was performed with 12% (v/v) polyacrylamide gels, using a Bio-Rad Mini Protean system. Prior to crystallization assays, 2 mm dithiothreitol (DTT) was added.For enzyme expression and purification of wild-type and mutant GlyAmK and kcat) were first determined using pNP sugars (read at 405 nm) in 96-well plates, as described previously (pNP substrates tested included those containing α-glucose (pNPαGlc), α-maltose , β-glucose (pNPβGlc), β-cellobiose (pNPβCel), α-arabinofuranose (pNPαAraf), β-arabinopyranose (pNPβArap), α-xylose (pNPαXyl), β-xylose (pNPβXyl), β-xylobiose (pNPβXylb), α-fucose (pNPαFuc), α-rhamnose (pNPαRha), α-mannose (pNPαMan), β-mannose (pNPβMan), α-galactose , β-galactose , β-lactose (pNPβLac), N-acetyl-β-d-glucosaminide (pNPGlcNAc), and N-acetyl-β-d-galactosaminide . For cello-oligosaccharides (DP from 2 to 5), gentibiose and sophorose, the level of released glucose was determined using a glucose oxidase kit (Sigma). The level of released xylose from xylo-oligosaccharides (DP from 2 to 5) was determined using the d-xylose assay kit from Megazyme . Substrate specificity was investigated also using carboxymethylcellulose, lichenan, barley glucan, laminarin, and avicel . Specific activity for all these sugars was quantified by measuring release of reducing sugars according to Miller , a protein concentration of 0.23 μm and 10 mg/ml of the sugar or polysaccharide were used in 50 mm sodium acetate buffer, pH 5.6, T = 40 °C. The pH and temperature optima were determined in the range of pH 4.0–8.5  and 20–65 °C in assays containing a protein concentration of 0.23 μm and 10 mg/ml pNPβGlc, which was used as standard substrate. BR buffer is a “universal” pH buffer used for the range pH 2–12. It consists of a mixture of 0.04 m H3BO3, 0.04 m H3PO4, and 0.04 m CH3COOH that has been titrated to the desired pH with 0.2 m NaOH. Optimal pH was measured at 40 °C, and the optimal temperature was determined in the same buffer used in the kinetic assays. In all cases, absorbance was determined immediately after reagent and enzyme were mixed using a microplate reader every 1 min for a total time of 15 min . All reactions were performed in triplicate. One unit of enzyme activity was defined as the amount of enzyme required to transform 1 μmol of substrate in 1 min under the assay conditions, with extinction coefficients as in Ref. m−1 cm−1) corresponding to its amino acid sequence.Specific activity (units/g) and kinetic parameters  were explored by high-throughput techniques with a NanoDrop robot (Innovadyne Technologies Inc.), using different commercial screens as follows: PACT and JCSG+ Suites from Qiagen; JBScreen Classic 1–4 from Jena Bioscience; and Index, Crystal Screen, and SaltRx packages from Hampton Research. These assays were carried out using the sitting drop vapor-diffusion method in MRC 96-well crystallization plates (Molecular Dimensions).Initial crystallization conditions for the complete GlyAm ammonium sulfate, BisTris, pH 5.5. For data collection, crystals were cryoprotected in mother liquor supplemented with 25% (w/v) d-glucose before being cooled in liquid nitrogen. Diffraction data were collected at the German Electron Synchrotron . Diffraction images were processed with XDS (12121. The structure was solved by molecular replacement using MOLREP (T. neapolitana (PDB code 2X42), but the search was made in two steps. First, the region containing residues 2–315 was used for finding a partial solution. Then, another round of molecular replacement, with the region 321–721, was computed. Preliminary rigid-body refinement was carried out using REFMAC (R-factor of 15.7 (Rfree 17.8). The free R-factor was calculated using a subset of 5% randomly selected structure-factor amplitudes that were excluded from automated refinement. Many attempts to reproduce and improve these crystals were unsuccessful, until in situ proteolysis of the sample with subtilisin was tried. Resulting crystals grew after 15 days in the same conditions, but at pH 7.0, they were cryoprotected in 20% (v/v) glycerol and showed the same space group and cell content. Then, the truncated GlyA1-ΔCt construct (residues 1–798) was tested. Initial crystallization assays were accomplished as described above, and several hits were obtained. Best crystals were grown in 2.0 m ammonium sulfate, 0.1 m BisTris, pH 5.5, and belonged to the same space group. The asymmetric unit contains a single molecule, with a Matthews's coefficient of 2.73 and a 54% solvent content within the cell.Elongated bars grew after 3 months in 20% polyethyleneglycol (PEG) 3350, 0.2 with XDS  and scalwith XDS  leading g MOLREP  with refg REFMAC . Subsequg REFMAC  combinedd-xylose or d-galactose were performed with the truncated construct in mother liquor solution implemented with 5–50 mm ligand. Then, the crystals were flash-frozen into liquid nitrogen using mother liquor plus 20% (v/v) glycerol or ethylene glycol as cryoprotectants. The ligands were manually modeled into the electron density maps and were refined similarly to that described above. Although a mixture of α- and β-anomers may exist in solution, only the β-form of the monosaccharides was observed at the active site of the different complexes. For the docked glucotetraose coordinates, not present in the Protein Data Bank, a model was built by the on-line carbohydrate-building program GLYCAM  were dialyzed against the same buffer  for 18 h. SAXS measurements were performed at ESRF on beamline BM29, equipped with a Pilatus 1M detector. Each sample concentration, prepared by dilution of these stock solutions, was measured in 10 frames, 1-s exposure time per frame, at 4 °C, at a sample-to-detector distance of 2.867 m, using an x-ray wavelength of 0.991 Å. No radiation damage was observed during the measurements. The SAXS curves for buffer solutions were subtracted from the protein solution curves before analysis.GlyAI(q) function using the ATSAS software package (gR) for each protein was calculated by Guinier plot using the program PRIMUS, and the pair distribution function P(r) and the maximum particle size Dmax were obtained by the program GNOM. Then, POROD was used to calculate the excluded volume of the particle, as well as the molecular weight of each sample.The scattering curves from six gradual concentrations, from 0.3 to 5 mg/ml, were scaled and averaged to obtain the  package . The rad1. All of them predicted a topology corresponding to carbohydrate-binding domains of families CBM6/CBM35, but they differed in the length of the linker attaching this domain to the core protein. Finally, models obtained from Swiss-Model (2W46 and 1UYX), each predicting a loop of 32 or 5 residues, respectively. Both entries share less than 20% identity with the C-terminal region of GlyA1.Several homology and threading modeling programs were tried to obtain a model of the last 123 residues of GlyAss-Model  and CPHmss-Model  servers 1-ΔCt coordinates and both templates, alternately; linkers were built as dummy atoms. The fit of the CORAL models to the SAXS experimental data were evaluated by the χ2 value calculated from the program CRYSOL 8 barrel domain was analyzed in a phylogenetic tree. The predicted protein sequences were aligned against the National Center for Biotechnology Information non-redundant (NCBI nr) database using BLASTP algorithm. We downloaded all 27,499 GH3 sequences deposited in public databases. They were grouped within five different domain architectures as follows: ABB , ABB_ABS , ABB_ABS_FLD , ABB_ABS_PA14_FLD , and ABS_FLD_ABB (328), where ABB, ABS, FLD, and PA14 refer to (α/β)8 barrel domain, (α/β)6-sandwich, fibronectin-like type III domain, and protective antigen PA14 domain, respectively. We discarded those sequences (848) from the ABB_ABS group longer than 700 amino acids, as they represent enzymes with unidentified domains downstream from the ABS module. Subsequently, the sequence corresponding to the ABB domain was extracted from all of the five sub-groups. An additional filter was applied to remove ABB sequences with coverage lower than 60% of the consensus domain defined by Interpro or Pfam databases (i.e. with less than 200 amino acids). The final number of sequences was the following: ABB , ABB_ABS , ABB_ABS_FLD , ABB_ABS_PA14_FLD , and ABS_FLD_ABB (289). For each of the five sub-groups, redundant sequences (those sharing more than 50% identity) were eliminated to select sequences that belong to different taxonomic groups. Following this procedure, the final selected sequences were as follows: ABB (132), ABB_ABS (54), ABB_ABS_FLD (The positioning of the sequence of the GlyA_ABS_FLD , ABB_ABS_ABS_FLD . MultiplJ. S. A., M. F., and J. P. conceived and coordinated the study. M. V. P. and M. F. contributed to screening, gene cloning, and enzyme production and characterization. P. N. G. contributed to metagenomics clone resources. J. S. A., B. G. P., and M. R. E. designed the crystallographic work and the SAXS experiments and interpreted the results. M. R. E. performed all the crystallography and SAXS experiments. J. M. N. and J. P. performed the phylogenetic analysis. J. S. A. and M. F. wrote the paper, and all authors read and commented on the manuscript.


Extrusion process of the most acceptable porridge flour was optimized by response surface methodology (RSM). Barrel temperature (130–170°C) and feed moisture content (14%–20%) were the independent variables which significantly (p < .05) affected in vitro protein digestibility, vitamin A retention, total polyphenol, phytic content, and iron and zinc extractabilities. Optimization of the extrusion process improved the nutritional quality of the instant flour.Malnutrition is one of the foremost causes of death among children below 5 years in developing countries. Development of nutrient‐dense food formulations using locally available crops has been proposed as a means to combat this menace. This study optimized the extrusion process for the production of a nutritious amaranth‐based porridge flour. Least cost formulations containing grain amaranth, groundnut, iron‐rich beans, pumpkin, orange‐fleshed sweet potato, carrot, and maize were developed and evaluated by a sensory panel ( Protein–energy malnutrition is associated with more than 50% of childhood mortality in developing countries  is a fast‐growing, high‐yielding, stress‐resistant, nutritious crop with potential to contribute to the alleviation of malnutrition and nutritional deficiencies , chapatti (a thin pancake of unleavened bread cooked on a griddle), kabalagala , porridges, and sauces were developed from grain amaranth composite flours in Uganda to address the dietary inadequacies  that complement the nutritional profile of grain amaranth were selected on the basis that they are rich sources of the target nutrients .2.1Grain amaranth, common bean (ROBA 1 variety), and maize flours were obtained from Nutreal Ltd., Kampala, Uganda. Raw orange‐fleshed sweet potato tubers (NASPOT‐10 variety) were obtained from a farmer in Bombo, Uganda. Fresh carrots, pumpkin, and groundnut were purchased from Kalerwe market, Kampala, Uganda.2.2Groundnuts were sorted and milled into flour using a local cast iron mill. The method of Dauthy  was adop2.32.3.1Concept 4 creative software  was used to generate six formulations Table  whose co2.4The formulations were prepared and conditioned to a moisture content of 20% and thoroughly mixed using a mixer for 15 min and extruded using a DP70‐III double screw inflating food machine . The extruder conditions were feeding frequency of 30 Hz, cutting frequency of 50 Hz, and barrel temperature of 60°C, 130°C, and 150°C in first, second, and third zones, respectively. After extrusion, the samples were cooled to room temperature under natural convection conditions. The samples were then milled into flour using a 30 B‐C milling machine , packed in polythene bags, and stored at 4°C.n = 30) mainly comprised of students in the School of Food Technology, Nutrition, and Bio‐engineering, Makerere University. A 9‐point hedonic scale was used to score the acceptability of the different porridges.Six different porridges were prepared by mixing 200 g of each of the composite flours in 800 ml of boiling water with constant stirring for about 4 min. Thirty grams of sugar was added per liter of the ready porridges. Sensory acceptability was determined by a semitrained panel  was varied as shown in Table After sensory acceptability screening, the extrusion process of the most acceptable formulation was optimized. Each independent variable  was used to simultaneously optimize the amaranth‐based flour's in vitro protein digestibility, total polyphenol, phytate, protein content, vitamin A retention, and iron and zinc extractability after carrying out the analyses below.2.5.1Total phytate content was determined by the titrimetric method described by Sarkiyayi and Agar , with thwhere A is % protein in the sample before digestion and B is % protein after enzyme digestion.2.5.2After optimization, consumer acceptability tests were carried out by 48 semitrained panelists using a 9‐point hedonic scale (1 = dislike extremely to 9 = like extremely). The instant porridge was prepared as described earlier , Porridges were prepared with different concentrations of flour  and their viscosities measured using a Brookfield DV II + Pro Viscometer at 55°C. Flour rates that could produce porridges of drinkable viscosities  were suitable for child feeding . The pasting temperature (PT), peak viscosity , holding strength or trough viscosity (the trough at the minimum hot paste viscosity), final viscosity, breakdown , and setback  were obtained with Thermocline for Windows software. The viscosities were presented in centipoise (cP).2.7Data for optimizing amaranth‐based flour extrusion conditions was analyzed by response surface methodology (RSM) procedures using design‐expert statistical software . Data for consumer acceptability and pasting properties were analyzed using the Statistical Package for Social Science (SPSS) software (version 17). Mean and standard deviations were computed. T test and analysis of variance (ANOVA) were used to calculate significant differences in treatment means and least significant difference technique was used for separation of means .33.1p ≤ .05) affected the consumer acceptability scores of porridge (Table p ≤ .05) different and more acceptable than other formulations. Formulation with grain amaranth, beans, groundnut, maize, and fresh potato flour (Formulation 1) had the lowest score. Therefore, formulation 6 was selected for optimization. Acceptability of this formulation could be attributed to the inclusion of pumpkin flour which improved the textural quality and flavor characteristics of the porridge thus masking the taste of grain amaranth. Kulkarni and Joshi (bhajjiya (an Indian savory).Formulations of the composite flours significantly  affected the nutritional composition of the instant amaranth‐based flours Table . The relR2 (coefficient of determination) ≥ 0.80, a significance level of p < .05, and coefficients of variance and (CV) values ≤ 10%; all these parameters could be used to decide the satisfaction of the modeling.The predictive models were tested for adequacy and fitness by analyses of variance (ANOVA). According to Myers and Montgomery , a good 3.2.1Equation X1) and feed moisture content (X2) had a negative linear and interactive effect on protein content of the instant amaranth‐based flour (Equation Barrel temperature (Equation . IncreasEquation . High teEquation .3.2.2X2) on in vitro protein digestibility resulted in an increase in in vitro protein digestibility  had negative linear effect on vitamin A content implying that it decreases with increasing barrel temperature (Equation X2) had positive linear and quadratic effects. Loss in vitamin A may be attributed to thermal degradation which appears to be the major factor contributing to β‐carotene loss during extrusion .Barrel temperature (Equation . Feed mo3.2.4X1) and feed moisture content (X2) had negative linear effects on the total polyphenol content of the composite flour (Equation Barrel temperature (Equation .(7)TotaTotal polyphenol content of the instant flours reduced with increasing feed moisture content and barrel temperature. Polyphenols are naturally occurring substances in plants which inhibit non‐heme‐iron absorption and interfere with protein digestibility Bravo,  by inhib3.2.5Equation X1) and feed moisture content (X2) had significant negative linear effects on the phytic acid content of the flours. Phytic acid is present in foods in varying concentrations of 0.1%–6.0% . Barrel temperature (X1) and feed moisture content (X2) had positive linear effects on zinc extractability (Equation The predictive model describing the effects of barrel temperature and feed moisture content on zinc extractability of the amaranth‐based instant flour was significant (Equation . ExtractEquation . ExtractEquation . ExtrusiEquation . IncreasEquation . Indeed Equation .(9)Zinc3.2.7X1) and feed moisture content (X2) on the iron extractability of the extruded flours.Equation Feed moisture content had a positive quadratic effect Equation  on iron 3.3A barrel temperature of 169°C and feed moisture content of 14% were chosen as the optimum conditions since they had the highest desirability (0.756). These processing conditions resulted in instant amaranth‐based flour with 18.32% protein content, 82.82% protein digestibility, 217.63 mg/100 g total polyphenol content, 54.86 mg/100 g total phytates, 553. 80 μg/RAE 100 g vitamin A content, 49.06% extractable zinc, and 17.32% extractable iron.3.4p < .05) higher than for the control porridge. The mean acceptability score for aftertaste of the control porridge was significantly (p < .05) higher than that for the amaranth‐based porridge. Inclusion of pumpkin flour in the amaranth‐based instant porridge resulted in a product with a very smooth consistency which is most likely responsible for higher texture and thickness scores. From the panelists' point of view, amaranth‐based porridge leaves an undesirable distinct aftertaste when consumed unlike the maize‐based porridge.The consumer acceptability of the amaranth‐based composite instant porridge was compared with a common commercial maize‐based instant porridge Table . The mea3.53.5.1The amaranth‐based and control instant porridges attained the drinking viscosity  at 20% 20 g/100 ml) and 18% (18 g/100 ml) flour rates, respectively  higher breakdown viscosity, peak time, and pasting temperature, and a significantly (p < .05) lower peak viscosity, trough I, and final viscosity when compared to the commercial maize‐based instant porridge.The pasting properties of amaranth‐based instant porridge are presented in Table Pasting properties are important indices for determining the cooking quality of flours PBIP, . Peak vi4This study demonstrated that extrusion cooking can be used to develop acceptable and nutritious instant composite flours from locally available foods. It further revealed that ingredient complementation can be strategically applied in the development of extruded foods to boost sensory acceptability. Barrel temperature and feed moisture content do have varying effects on nutritional quality of extruded foods thus necessitating process optimization for products. Increasing barrel temperature and feed moisture content leads to a decrease in polyphenol, phytic acid contents, and iron extractability, but increases zinc extractability. Increasing feed moisture content favors retention of vitamin A. Optimal processing conditions for amaranth‐based porridge flours developed in this study were 14% feed moisture content and a barrel temperature of 169°C.None declared.


Quantifying global tissue deformation patterns is essential for understanding how organ-specific morphology is generated during development and regeneration. However, due to imaging difficulties and complex morphology, little is known about deformation dynamics for most vertebrate organs such as the brain and heart. To better understand these dynamics, we propose a method to precisely reconstruct global deformation patterns for three-dimensional morphogenesis of curved epithelial sheets using positional data from labeled cells representing only 1–10% of the entire tissue with limited resolution. By combining differential-geometrical and Bayesian frameworks, the method is applicable to any morphology described with arbitrary coordinates, and ensures the feasibility of analyzing many vertebrate organs. Application to data from chick forebrain morphogenesis demonstrates that our method provides not only a quantitative description of tissue deformation dynamics but also predictions of the mechanisms that determine organ-specific morphology, which could form the basis for the multi-scale understanding of organ morphogenesis. Quantifying deformation patterns of curved epithelial sheets is challenging owing to imaging difficulties. Here the authors develop a method to obtain a quantitative description of 3D tissue deformation dynamics from a small set of cell positional data and applied it to chick forebrain morphogenesis. While the former have been studied for several decades, the latter—macroscopic geometrical information about physical tissue deformation—has been lacking. Recent advancements in imaging techniques and fluorescent probes have made total cell recordings possible, especially in flat, small, and relatively transparent tissues such as Drosophila germband and wing, and Zebrafish skin6. Based on tracking data, collective cellular behaviors and tissue deformation dynamics at single-cell resolution during development have been studied using velocimetric methods.Understanding how three-dimensional (3D) organ morphology is determined during development and regeneration is one of the ultimate goals in biology. This is important not only for pure scientific interests but also for potential medical applications for controlling and designing functional organs. To achieve these goals, it is essential to clarify the quantitative relationships between microscopic molecular/cellular activities and organ-level tissue deformation dynamics10. One reason for this lack of information is the difficulty in measurement; in general, tissue morphologies are achieved through complex 3D deformation of highly curved sheet-like structures, either cystic or tubular. Furthermore, high resolution deep imaging is often quite difficult. In addition, to achieve high temporal resolution, methods of embryo culture which maintain normal function over an appropriate period are required. From the analytical perspective, image processing that can automatically distinguish individual cell trajectories from a dense cell population is often difficult, which itself is an important issue in this field12. Furthermore, although sheet deformation occurs in 3D space, its actual structure is two-dimensional (2D) even with curvature. Thus, in order to analyze deformation dynamics, it is necessary to introduce a 2D curvilinear coordinate system onto the sheet with the involvement of a non-Euclidean metric. As will be described below, the 2D coordinate system and metric is, in general, different at each developmental time point with differing morphology, making it difficult to perform ordinary velocimetric analysis.Aside from these notable exceptions, we have little knowledge of tissue deformation dynamics during the morphogenesis of many vertebrate organs including brain, heart, and even artificially synthesized organoids derived from ES and iPS cells13. By combining differential-geometrical and Bayesian frameworks, the difficulties listed above are overcome. In particular, with this method, manifold- and tensor-based descriptions are adopted, allowing it to be applied to any tissue described with arbitrary coordinate systems. This is critically important for analyzing the deformation of curved structures because orthonormal coordinate systems cannot be applied to them and because curvilinear coordinate systems defining the surface itself can differ with changes in morphology. With our method, positional information from just 1–10% of the total cells within a tissue is adequate for reconstructing the global deformation pattern with sufficient accuracy, which ensures the feasibility of analyzing many vertebrate organs with complex morphologies. Moreover, the sparse cell labeling makes it easier to distinguish individual cells even if the microscopic resolution is not high.Against this background, we propose a method to reconstruct tissue deformation dynamics for 3D morphogenesis of curved epithelial sheets from a small set of positional cellular data with limited resolution. This method is a generalization of that proposed in our previous study which focused on flat tissuesThe performance of the proposed method is validated using both simulated and in vivo data. In particular, we focus on the process of tissue evagination and confirm with simulated data that the spatial patterns of deformation characteristics calculated from reconstructed tissue deformation maps show clear signatures for distinguishing different mechanisms that generate similar morphologies. Then, as an actual biological target, we apply this method to morphogenesis during early development of the chick forebrain from somite stage (SS) 5 to SS13; SS5 corresponds to the very beginning of optic vesicle (OV) formation from a simple neural tube and at SS13 a fully evaginated OV and overall more complex morphology is present , the deformation of a sheet can essentially be described as a 2D map that relates the positions before and after deformation for each cell. Thus, in order to analyze sheet deformation dynamics, we need to start by allotting 2D coordinates to points on a curved sheet. Epithelial tissues often have sac like or tubular morphologies that can be approximated by closed surfaces, for which the spherical harmonics expansion (SHE)  would be the best choice for obtaining 2D coordinate charts  differs depending on the position in the given 2D coordinate system Fig. . This islas Fig. . Since tmentclass2pt{minimThis metric 18. As is the case in many vertebrate organs, the scale of the whole organ (102−104 μm) is generally much larger than that of a single cell (100−101 μm), and thus it is useful in tissue-level deformation analysis to regard them as continuums by averaging the variability in the shape and size of individual cells. Continuum deformation can be described as a map, and as with positional coordinates of cells, the deformation map also has 3D (denoted by x = ϕ(X)) and 2D , the posterior probability for the parameter has the form P(θ|u) ∝ P(u|θ)π1(θ|η1)π2(η1|η2). P(θ|u) is the probability of observing the data point u for a fixed parameter set θ. Conversely, as a function of θ with u fixed, this acts as a likelihood function. In regards to this data distribution, we assumed additive noise obeying isotropic Gaussian distribution on the tangent plane at each point in the 3D representation , and the transformed distribution is no longer isotropic but anisotropic due to the anisotropy of the metric i-th point), the variance-covariance matrix σ2 is the magnitude of the noise. In the computation, ui) as shown in the rightmost term in Eq. (π1(θ|η1) and π2(η1|η2) are the prior distributions for the parameter θ and for the hyper-parameter η1. η2 is also the hyper-parameter necessary for determining the shape of the distribution π2. As biologically plausible prior information, we assumed the smoothness of deformation both inside the tissue as well as at its boundary. Specifically, the first spatial derivative of the deformation gradient tensor For a given data point ier Fig. . For eacP(u|θ)π(θ|η)dθ; then, using these values, the discretized deformation map θ is obtained by maximizing the posterior probability21. Finally, from the relationship Using these conditions, in the computation the values of the hyper-parameters are first determined by maximizing the marginal likelihood function ∫We evaluated the performance of our method by using artificially generated data Figs.  and 5. Cz = f , and another case in which noise anisotropy was adjusted based on the metric tensor at each position .Next, in order to generate data reflecting more realistic situations, we performed mechanical simulations for evagination of an elastic membrane As with the second example of in silico validation described in the previous section, the 2D coordinates on the neuroepithelial sheet were given by the SHE. Figures We then examined the spatio-temporal patterns of the deformation characteristics. As for the area growth rate of the apical surface, no clear bias was observed throughout the entire period we analyzed SS5-SS13), although some variations were present depending on position. In contrast, deformation anisotropy showed a clear pattern, with high values and globally oriented direction along the medio-lateral axis throughout the entire period of forebrain analysis although, at later stages , the definition of the M-L axis became ambiguous due to the morphological complexity and the differences in the degrees of deformation anisotropy for the OV and non-OV regions became clearer. These results clearly show that anisotropic deformation  along the medio-lateral axis, and not local area growth, is the predominant mechanism underlying the dynamic change in forebrain morphology during the period of our focus, which we think could not be readily predicted by simply comparing the morphologies shown in Fig. , althougFurthermore, we examined the changes in tissue thickness during morphogenesis. We first confirmed that, consistent with previous histological studies, tissue thickness and cell height itself gradually decreased over time . At each stage, the distribution of thickness was narrow  than in microscopes, a larger range of tissues (1–100 mm) can be scanned using magnetic resonance imaging, computed tomography, or ultrasound imagingOur investigation has also shown that the quantitative analysis of tissue deformation dynamics is useful for predicting mechanisms that determine organ-specific morphology. As demonstrated in the example of tissue evagination, spatial patterns of deformation characteristics, such as directional distribution of deformation anisotropy and surface expansion, show clear signatures for distinguishing background mechanisms that generate similar morphologies. In particular, during chick forebrain development, strong and globally oriented deformation anisotropy along the medio-lateral axis was observed throughout the entire region as well as relatively non-biased area growth patterns. This strongly suggests that the evagination is driven mainly by cell intercalation along a specific axis, not by local growth, which was confirmed by additional experiments involving the inhibition of cell proliferation and quantification of cellular characteristics.37 and PCP proteins40, and mechanical constraints such as external forces and boundary conditions44. Quantitatively linking such force generation mechanisms with tissue deformation dynamics would enable the development of models for explaining morphological anomalies and diversity among species, and would aid the morphological design of artificially constructed organoids.The anisotropic deformation through dynamic cellular rearrangement implies the existence of anisotropic stress distribution within the tissue. Previous studies on epithelial tissue development of different organs/species have identified some factors for generating mechanical anisotropy, these include the localization of phosphorylated Myosin45. Another example is Drosophila germband extension, evident in mutants in which posterior midgut invagination does not occur 43. In that case, without posterior midgut invagination, the extrinsic or boundary force is diminished, while cell rearrangement and tissue elongation still occur, although to a lesser degree than in wild types. Does this tendency toward a directional relationship between cell shape and cell rearrangement share a common mechanism among different systems? This would also be an interesting issue to study from a mechanical viewpoint.Furthermore, during the analysis of major cell shape axis orientation, we found that in the evaginated region, the axes were perpendicular to the direction of anisotropic tissue deformation Fig. . This teAs previously described, the teleost OV is composed of two types of cells, epithelial-like marginal cells and mesenchyme-like core cells, and during OV evagination the core cells vertically intercalate into the layer of marginal cells, which is considered to contribute to OV formation. In contrast, in amniotes such as chick, the eye field is composed of a single layer of neuroepithelial cells. Our current analysis suggested that the intercalation of epithelial cells within this layer is the predominant cellular mechanism that induces anisotropic tissue deformation. Despite differences in the initial cell arrangements and the direction of cell intercalation between chick and zebrafish, both embryonic tissues elongate along a common medio-lateral axis. Comparative analysis of the molecular mechanisms that induce the evagination process in both embryos will be an interesting future topic from an evolutionary perspective.46.Fertilized chicken eggs (Shiroyama Farm) were incubated in a humidified incubator at 38 °C to obtain Hamburger and Hamilton (HH) stage 4 embryos47. We amplified the myrVenus gene  by PCR, using following primers: myrVenus forward 5′-AAGCTTATGGGAAGCAGCAAGAGCAAGCCAAAGATGGTGAGCAAGGGCGAGG-3′ and myrVenus reverse 5′-TCTAGA TTACTTGTACAGCTCGTCC-3′. Using these fragments, we replaced the EGFP gene in the pN2-EGFP47 with the myrVenus gene resulting in pN2-myrVenus.To label the rostral portion of the neural tube, we used fluorescent protein expressing plasmids containing the N2 enhancer of Sox248 with some modifications. For electroporation and 3D imaging, the vitelline membrane was removed in 123 mM NaCl solution. For neural epithelial cell labeling, we electroporated pN2-myrVenus plasmids into the future neural tube region. We used a CUY21EX electroporator (BEX) at the following settings: poration pulse 7 V, 1 pulse of 50 ms. Afterward, 3 V driving pulses, five pulses of 50 ms with a 50 ms interval.The embryos were explanted using the EC culture method33, then incubated the embryo in a CO2 incubator  until the desired stage. The addition of a drop of glutathione-coated Q-rods was done prior to neural tube closure. At that stage, the apical surfaces of the neuroepithelial cells are exposed on the outside of the embryo, thus we could label the apical, not basal, surface. In the other method, we first incubated the electroporated embryo in a CO2 incubator  until the desired stage. Then, we introduced Q-rods inside the neural tube using a glass capillary tube (tissue was pierced from a posterior approach so it did not affect the morphogenesis of the forebrain region). Note that, at target stages, the neural tube was already closed. The latter method was more efficient for labeling tissues, that is more Q-rods  were attached to the apical surfaces. In particular, the latter method was necessary for introducing sufficient numbers of small Q-rod aggregations at later stages (after SS10).For labeling the apical surface, we used two approaches. In one method, we positioned the electroporated embryo with the dorsal side up, and added a drop of Glutathione-coated Q-rodsThe size of each Q-rod used was <100 nanometers, but objects chosen for analysis in our study only included small aggregations of Q-rods with a diameter of 1–20 micrometers  that were clearly detected by our microscopic set-up and distinguishable from one another in the manual tracking process. The small aggregations tended to remain on the apical surface.Live 3D imaging was performed using an upright microscope  equipped with an Olympus 25×/NA1.05 XLPLN25XWMP objective and a multi-photon femtosecond laser . For the live imaging, an electroporated embryo was immersed in 1× PBS. The 3D morphology of the neural tube was determined by stacking the optical section images along the Z-axis . For each Z-level, four X-Y images (512 × 512 pixels each) were tiled so that the entire region of the prospective brain was included. The embryo was then returned to culture until the next imaging time point. All images were taken at room temperature. Imaging intervals (incubation time) were 2 h. Within each interval, embryo development progressed to the next somite stage. Although this developmental speed is slightly slower than that in normal development (1.5 h/somite), the morphology itself appeared normal.Initial image processing was done with Imaris 7.6 (Bitplane). In order to obtain the 3D morphology, we first manually traced the apical and basal surfaces of the neural tube for each section along different axes. By stacking the traced data, the 3D morphology of the rostral neural tube lumen was reconstructed. The result of manual tracing could be obtained as a set of dots, as shown in Fig. From the positional data of the Q rods, the 2D deformation map was estimated using the proposed method. The analytical procedure was similar to that used for the validation with artificially generated data  were identified by DAPI staining. The cell division orientation was quantified as the angle between the medio-lateral axis  and the line through the centers of the two daughter cells. In all Figures of In Fig. The data that support the finding of this study are available from the corresponding author upon reasonable request.Supplementary InformationSupplementary Figures, Supplementary Notes and Supplementary References


P < 0.05) the lipid, protein and ash contents, and decreased (P < 0.05) the levels of carbohydrates and total dietary fiber. BTF0%, BTF2.5% and BTF5% received the highest (P < 0.05) scores for acceptance and preference. Despite this apparent consumer preference for low or no levels, TF can be added to bread at levels below 12.17% (P < 0.05) without triggering consumer rejection. TF changed (P < 0.05) the sensory characterization of bread because of a disagreeable flavor, aroma, and texture; however, airy appearance, sticky in the teeth and cream color did not influence the overall liking. TF at 5% enhanced the nutritional value while maintaining acceptable sensory scores for bread, constituting a potential strategy to satisfy consumer and industry requirements.Six bread formulations with different levels of tilapia-waste flour  were analyzed for nutritional composition and sensory characterization. Tilapia-waste flour (TF) increased ( Brycon cephalus) [Consumer demand for healthier foods has become increasingly widespread in recent years . Bread mephalus) , and shrephalus) . Howeverephalus) . To the Oreochromis niloticus) is one of the most commercially important freshwater fish species, contributing approximately 9% of the total amount of fish produced globally [Although Nile tilapia  characterized formulations of bread fortified with different levels of tilapia flour, from the point of view of nutritional value and sensory attributes; (2) determined the overall liking for all bread formulations; and (3) determined the cutoff point (COP) for wheat flour replacement by tilapia-flour in wheat bread, using a consumer-based approach.A total of 9.0 ± 0.3 kg of mechanically separated meat (MSM) of tilapia, packed in polyethylene bags, was purchased from a commercial fish farm . The MSM was dried for 12 h at 65°C in a forced-air convection oven  in the bakery pilot plant, to obtain the tilapia flour.®, ACH Food Companies, Inc., Pederneiras, São Paulo, Brazil), 50 g sugar , 20 g salt , 50 g dough improver , 33 g vegetable fat , and 640 mL water , lipid (AOAC method 991.36), protein , ash (AOAC method 920.153), and total dietary fiber (AOAC 991.43) were determined for the six bread formulations described in One hundred consumers  were invited to participate in the study. They were recruited among workers and students at Embrapa Food Technology , according to their interest in and availability to participate in the study. All participants consumed bread regularly. Their socio-demographic characteristics are shown in Each bread formulation described in The study comprised affective tests  and a descriptive evaluation using check-all-that-apply (CATA) questions. The socio-demographic characteristics of consumers were also noted.Participants evaluated the acceptability of the bread formulations using a 9-point structured hedonic scale, ranging from 1: dislike extremely to 9: like extremely. Next, they were asked to try the sample and to answer “yes” or “no” to the following questions, to establish the sensory COP of wheat flour replacement by tilapia-flour in wheat bread by the survival-analysis method :a) “Suppose that you bought this product to eat or that it was served to you in your home. Would you consume it?”b) “Suppose that this product is new on the market. Would you buy it?”After that, the sensory characteristics were evaluated using CATA questions . The CATFinally, the participants answered the last question “Would you be interested in eating bread with a higher amount of proteins and minerals?” Prior to the analysis, no information about the aim of the study was provided to participants. Water at room temperature and unsalted crackers were served to participants to refresh their sense of taste between samples.P < 0.05) were performed on the data for proximate composition, energy value, total dietary fiber, and hedonic scores, to determine differences among all the bread formulations. In addition, Internal Preference Mapping was carried out to detect consumer preferences among the different formulations. For the questions, the answers were categorized as 0 (no) and 1 (yes), and survival-analysis statistics  with a 50% rejection probability [P < 0.05) among bread formulations related to each CATA term included in the questionnaire were identified by Cochran’s Q test. All data were submitted to Multifactorial Analysis (MFA) to determine the parameters that were influenced by replacement of wheat flour with tilapia-flour. The demographic data and the final question were evaluated by the frequency of each response. All statistical analyses were carried out using R language [Analysis of variance (ANOVA) and Tukey’s test (bability  were applanguage  with a 9P < 0.05) the lipid, protein, and ash contents, and decreased (P < 0.05) the levels of carbohydrate and total dietary fiber (P < 0.05) moisture content and the lowest (P < 0.05) energy value. No differences (P > 0.05) were observed in moisture contents and energy values among other tilapia-bread formulations at 0%, 2.5%, 5%, and 10%. These results are strongly related to the wheat and tilapia flour compositions. Tilapia flour has a low carbohydrate level (< 1.5%) and high levels of protein (> 45%), lipid (> 25%), and ash (> 3%) [The substitution of wheat flour by tilapia flour increased (ry fiber . BTF15% h (> 3%) , while th (> 3%) ,24. Althh (> 3%) ,24, the h (> 3%) ,25–27. Oh (> 3%) . Similarh (> 3%) , Bastos h (> 3%) , and Jeyh (> 3%) .P < 0.05) liking scores among samples, BTF10% and BTF15% received intermediate acceptance (P < 0.05), while BTF20% received the lowest (P < 0.05) overall liking score. No difference (P > 0.05) was observed among BTF0%, BTF2.5%, and BTF5%, and between BTF10% and BTF15% for hedonic scores related to overall liking. Similarly to our study, other authors also reported successful acceptance after addition of 5% fish-protein concentrate in biscuits [BTF0%, BTF2.5%, and BTF5% received the highest  were less preferred by participants, especially BTF20% . The incn powder , and a sn powder . On the n powder  reportedcream color, moist, crumbly, salty taste, hard, raw, acid taste, sticky in the teeth, cheese flavor, oily, and cheese aroma  was observed in overall liking among BTF0%, BTF2.5%, and BTF5%. BTF10% and BTF15% were described as moist, salty taste, acid taste, raw, cheese aroma, cheese flavor, odd flavor, and crumbly. BTF20% was perceived by consumers as having strong aroma, oily, compact texture, compact appearance, odd aroma, dark color, and hard. To the best of our knowledge, no study has evaluated the sensory characterization of fish-enriched bread by CATA questions. Previous studies have reported sensory changes, mainly in appearance, texture, and flavor (strong taste or flavor), and decreased overall liking in bread formulations fortified with high amounts of seafood flour [odd flavor. On the other hand, breads are commonly known as airy and sticky foods [BTF0% and BTF2.5% were characterized mainly by the attributes od flour ,32. Jeyaod flour  concludeky foods ,34. Thersoft, airy appearance, spongy, bread aroma, and light color, which were descriptive terms perceived for BTF0%, BTF2.5%, and BTF5%  were enough to contribute to nutritional enrichment of bread. Tilapia-waste flour is easy to prepare, has good chemical stability at room temperature, and has a high nutritional value containing essential amino acids and fatty acids that are beneficial to human health ,42. AlsoWould you be interested in eating bread with a higher amount of proteins and minerals?), the majority of the consumers (72%) stated that they were interested in eating bread with higher amounts of proteins and minerals. Today’s consumers require foods that are functional, convenient, and healthy [In response to the final question  could be used without detrimental effects on overall liking. In addition, our results indicated that the intention to consume and to purchase enriched bread can be maintained with levels < 12.17% and < 6.86% tilapia-waste flour, respectively. Finally, the results suggest that bread fortified with tilapia-waste flour at 5% (wheat flour replacement by 10% of TF) can be a potential alternative for the food industry to satisfy the current nutritional and sustainability requirements of consumers.S1 Table(XLSX)Click here for additional data file.