Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1–16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1–16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen,  Candida albicans is an important opportunistic human pathogen that persists in the microbiome of healthy individuals causing slight candidiasis with hardly any serious symptoms under normal conditions ATP , Mg2+ and myosin light chain kinase [E. coli BL21 (DE3)-RIL cells that were transformed with the empty pET28a(+) or pGEX-6P-1 vectors. The control samples liberated a negligible amount of radioactive phosphate from the substrate that was considered as background and was subtracted from the radioactivity measured with the recombinant protein preparations. Phosphatase activity was expressed in international units: 1 U of enzyme liberates 1 μmol phosphate in 1 minute. Protein concentration was measured [The protein phosphatase activity of the preparations was assayed as described previously . The radn kinase . In contmeasured  with bovppz1 (MATa his3Δ1 leu2Δ met15Δ ura3Δ ppz1::kanMX4) phosphatase mutant S. cerevisiae strain. In control experiments the isogenic BY4741 (MATa his3Δ1 leu2Δ met15Δ ura3Δ) strain was transformed with the empty plasmids. The transformation, as well as the culturing and testing of the transformants in drop tests were performed as earlier [ppz1 cells and we selected 19 h culturing in the presence of 50 mM LiCl for the subsequent investigations.YCplac111 and YEplac181 plasmid constructs were transformed into the  earlier . The LiC earlier  with theTo identify potentially important segments in CaPpz1 its translated protein sequence was analyzed by structure prediction tools . Accordihttps://web.expasy.org/myristoylator/). Note that the essential Arg that is required for enzyme activity is located at position 262. The ANCHOR software [S. cerevisiae Ppz1 was mapped to a specific region of the catalytic domain [The first amino acid in the translated coding sequence is the initiating Met, which is eliminated from the mature protein, and the second amino acid is Gly, a potential N-terminal myristoylation site (software  predictec domain . Since Hhttp://elm.eu.org/search.html) predicted a WDR45 binding motif and several phosphorylation sites for CDK1 and GSK protein kinases within the deleted region. As a negative control we utilized the R262L mutant that was rendered catalytically “dead” by the exchange of the essential Arg residue in the catalytic domain [C. albicans versus E. coli and S. cerevisiae a maximum of five unintentional Ser to Leu mutations were introduced into the expressed proteins  was assayed with the radioactive protein substrate.As expected the N-terminal domain alone and the R262L mutant were inactive. The del67-108 and del120-142 mutations did not cause any significant change in the specific activity in comparison to the wild type phosphatase. Deletion of amino acids 1–16 as well as the G2A point mutation within this region significantly reduced the activity of CaPpz1. In contrast, the del25-43 mutant had a moderately elevated activity. It can be hypothesized that the 25–43 peptide region may directly interact with the C-terminal catalytic domain, and its elimination may lift the inhibitory effect of the N-terminal tail on the catalytic activity. However, without knowing the 3D structure of full length CaPpz1, it is hard to confirm the mechanism and predict the significance of this moderate change in the S. cerevisiae mutant lacking the orthologous ScPPZ1 gene. Therefore, we analyzed the in vivo effects of CaPpz1 mutations by assessing their ability to complement the ppz1 deletion mutant. In our experiments the full length CaPpz1 and its mutants were expressed in ppz1 cells under the control of the ScPPZ1 promoter either from low copy number centromeric YCplac111-based plasmids or from high copy number episomal YEplac181-based constructs and the caffeine sensitivity as well as the salt tolerance of the transformants were tested.For revealing the physiological roles of mutations described in S. cerevisiae ppz1 deletion mutant can be attributed to the involvement of ScPpz1 in the cell wall biosynthesis and in the cell integrity pathway [C. albicans CaPpz1 in the ppz1 mutant yeast cells has been reported [The caffeine sensitivity of the  pathway . Complemreported . Based oppz1 strain, and the complementations by the del25-43, del67-108, and del120-142 CaPpz1 deletion mutants were similar to those of wild type CaPpz1. On the other hand, del1-16, G2A, Nter and R262L mutants as well as the Cter domain were almost ineffective. To test the possibility that the small effects of some expressed proteins were associated with the low efficiency of expression from centromeric plasmids, we repeated the experiments with high copy number YEplac181 episomal plasmid-based constructs . That is why we changed the qualitative detection method and switched to the liquid cultures that can be characterized quantitatively by measuring their turbidity at 620 nm  resulted in a decreased CaPpz1 activity  than the recombinant wild type enzyme [Our activity . In corre enzyme . ConsequS. cerevisiae ScPpz1 is less active than the full length enzyme [S. cerevisiae Ppz1 [On the other hand, the elimination of the whole N-terminal domain resulted in a radical elevation of the CaPpz1 phosphatase activity . This fah enzyme . There ah enzyme  measurediae Ppz1 , howeverC. albicans  bound more effectively to the C-terminal domain than to the full length version of the phosphatase according to several in vitro protein-protein interaction assays [S. cerevisiae ScPpz1 was treated with trypsin, indicating that the proteolytic digestion eliminated some N- or C-terminal peptides which repressed catalytic activity. Thus it is likely that the N-terminal region plays an inhibitory role in PPZ phosphatases. It should be noted that the externally added N-terminal domain did not affect the activity of either the full length CaPpz1 or its separate C-terminal catalytic domain (K. Petrényi unpublished results). Thus the covalent linkage connecting the two domains is imperative for the suggested regulation of CaPpz1 activity by its N-terminal tail.The hypothesis of spatial hindrance of CaPpz1 activity by the Nter was suggested earlier  and is sn assays . Finallyn assays  reportedppz1 mutant. In agreement with these results the large Δ17–193 deletion of ScPpz1 [From our complementation experiments Figs  and 4 wef ScPpz1   may be important since the del25-43 mutant (in which a nearly completely overlapping peptide region was deleted) exhibited higher phosphatase activity, however, we were not able to demonstrate the effect of this deletion in the LiCl sensitivity tests. Due to these circumstances we confirmed the functional significance of the 1–16 peptide region but did not find convincing evidence for the functioning of additional predicted protein binding regions in the N-terminal domain of CaPpz1.From our structure-function study we conclude that both the enzyme activity of the C-terminal domain and the proper localization of the enzyme by its N-terminal domain are essential for the physiological functions of the CaPpz1 phosphatase. The first 16 amino acid residues, including the Gly2 myristoylation site, of the CaPpz1 phosphatase are especially important as the deletion of this region or the G2A mutation significantly decreased the specific activity of the enzyme t et al.  indicati species  Figs. ThS1 Text(DOCX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S1 FigA. Purification of recombinant wild type CaPpz1. Bacterial cell extract was separated to supernatant and pellet by centrifugation and the GST-tagged CaPpz1 was purified from the supernatant on Glutathione Sepharose in batch mode. The bulk of bacterial proteins which did not bind to the resin (unbound proteins) were removed by centrifugation. Weakly bonded proteins were eliminated by washing in six steps. The first, second, fifth, and sixth wash fractions were tested in the gel. The resin with bonded GST-tagged protein was treated with Prescission protease. Fractions containing the cleaved off CaPpz1protein were eluted with five portions of the protease buffer. The third fraction (boxed) was used for further assays. The efficiency of the cleavage was demonstrated by the analysis of an aliquot of Glutathione Sepharose resin before and after protease treatment and elution. The SDS-PAGE analysis of a representative preparation is shown. St. indicates PageRuler Pre-stained Protein Ladder that was used for the estimation of the apparent molecular mass (Mr) of selected bands. The apparent Mr of CaPpz1 is 58.9 kDa that is somewhat more than the theoretical 55.5 kDa value. B. Purification of the N-terminal domain (Nter) of CaPpz1. His-tagged Nter was purified on Ni-NTA-agarose. The supernatant and pellet resulting from centrifugation of the cell extract, unbound proteins, as well as the wash and eluted fractions were analyzed by SDS-PAGE as in C. Testing the purity of mutant CaPpz1 proteins. Recombinant proteins G2A, Cter, del1-16, del25-43, del67-108, and del120-142 were purified on Glutathione Sepharose as the wild type CaPpz1 phosphatase (panel A). Three eluted fractions and the residual resin after cleavage and elution were subjected to SDS-PAGE analysis. The boxed fractions were used for phosphatase assays. The R262L mutant protein was purified on Ni-NTA-agarose as the Nter domain (panel B) and three eluted fractions as well as the residual resin were analyzed by SDS-PAGE. Apparent (red) and theoretical (black) molecular masses (Mr) of the recombinant proteins are given below the panels. The theoretical Mr was calculated with the ExPASy compute pI/Mw tool https://web.expasy.org/compute_pi/. Boxes indicate the band of the recombinant proteins in the fractions that were used for the phosphatase assays. A representative of at least three independent preparations is shown in the figure.(TIF)Click here for additional data file.S2 FigA. Wild type BY4741 and ppz1 deletion mutant S. cerevisiae cells were cultivated without any addition (blue bars), and after transformation with empty or CaPpz1 coding YCplac111 (green) and YCplac181 (red) plasmids. The turbidity (OD620) of triplicate cultures was determined after 19 h incubation. The means of two independent experiments is shown. B. The ppz1 mutant (triangles) and BY4741 control (circles) yeast strains were transformed either with empty (empty symbols) or with CaPpz1 harboring (full symbols) plasmids and were cultivated in the presence of increasing concentrations of LiCl. After 19 h of incubation the optical density of triplicate cultures was measures at 620 nm. The relative growth of the samples cultivated in the absence of LiCl was taken as 100%. The means of two independent experiments is depicted.(TIF)Click here for additional data file.S3 Fighttps://www.ebi.ac.uk/Tools/msa/clustalo/). Green background indicates deletions and pink highlights point mutations. Yellow insert marks the deletion in ScPpz1 that was studied by Minhas et al, 2012. The light blue line above the sequences labels the C-terminal domain of CaPpz1, dark blue extensions mark the limits of the same domain in ScPpz1. Stars under the sequences indicate amino acid residue identities, spot show similarities.The amino acid sequences that were investigated by Clotet et al., 1996, Minhas et al., 2012, and in the present work were aligned by the Clustal Omega multiple sequence alignment tool ((PDF)Click here for additional data file.S4 Fighttps://iupred2a.elte.hu/) for protein disorder (red lines) and protein binding regions (blue lines). The graphs were aligned along the conserved catalytic domains. Green bars and a yellow insert indicate deletions, pink stars show point mutations, and blue bars delimit the C-terminal domains as in panel A. Full black lines connect structurally related elements and dashed black lines connect the borders of related regions.Protein sequences shown in S3 Fig were analyzed by the IUPred2 and ANCHOR2 combined web interface ((TIF)Click here for additional data file.


Defects in the crystalline structure of quartz facilitate the connection with the alkali hydroxides, since under a high alkalinity condition , the Si-O bonds of quartz are easily broken. This study set out to investigate the influence of the deformation structures of quartz on its susceptibility to the alkali–silica reaction. A granite, a protomylonite, and a mylonite were selected for this study. Using optical microscopy, the quartz grains contained in these rocks were quantified and their texture characterized. The quartz samples extracted from the rocks were analyzed by magnetic nuclear resonance, to evaluate their potential for dissolving silica as well as changes in their atomic scale before and after the reaction with alkali hydroxides. These analyses were compared with the results of the accelerated mortar bar test. The study showed that the quartz with intense undulatory extinction and deformation bands denotes the most favorable condition to the development of the alkali–silica reaction. However, on an atomic scale, the slightly deformed grains were highly prone to react. Thus, in a high alkalinity condition, over a long period of time, any quartz tends to develop the alkali–silica reaction, regardless of the deformation degree of the grain. Quartz is the main mineral responsible for triggering the alkali–silica reaction (ASR) in concrete structures ,2,3. ThiTherefore, the same grain could have zones that are more and less reactive, and which are directly related to the thermodynamic stability of the tensioned quartz to the alkaline solution as it can attack the mineral in different ways, in accordance with its degree of deformation. Thermodynamically unstable quartz with high free energy in its crystalline networks is found in very deformed granites and in metamorphic rocks . HoweverThis study evaluates the susceptibility of quartz to react with alkaline solutions, as a function of degree of deformation with quartz grains.The samples used in this study are the same as those of Tiecher et al. . A speciThe Pelotas Batholith is a plutonic complex with approximately 270 km of length and 70–110 km of width that crops out in the eastern portion of the South-Riogrande Shield and continues into Santa Catarina and Uruguay . It compIn the outcrop, it was possible to identify three degrees of deformation of the rock, which were called M1, M2, and M3, where M1 represents the rock with the least deformed minerals, M2 the rock with intermediate deformation, and M3 the rock with the most deformed minerals.In order to evaluate the influence of the quartz deformation on the products formed by ASR, without the interference of other silicates present in the samples, the quartz grains were extracted from the matrix of the rocks.First, samples were ground to obtain grains smaller than 0.15 mm, because this is the average size of most minerals from the rocks. In this process some grains can be subdivided, but their crystalline structure does not change; in this way it is assumed that the behavior and results obtained will not be affected by the comminution. After that, the minerals were separated magnetically to extract those containing iron (predominantly micas).3), adding acetone (CH3(CO)CH3-99.5%, density 0.788 g/cm3) until the higher density mineral sank into the balloon and the mineral with lower density floated. The density of the quartz is 2.65 g/cm³, and the feldspar varies from 2.55 to 2.63 g/cm³ [The separation between quartz and feldspar (K-feldspar and plagioclase) was done using the density difference in a volumetric flask. The ground rock was immersed, without the micas, in bromoform  from Geoscience Institute of Federal University of Rio Grande do Sul (BR). Petrographic analysis by optical microscopy was performed together with the quantitative modal analysis in order to quantify each different quartz microstructure, which reflects the different degrees of deformation of the quartz. The thin section description was made with assistance of an optical microscope model Leica-DM4500 P LED  and the optical images were obtained using the digital camera Leica Model DFC-495 .To evaluate the development of ASR within the samples, the accelerated mortar bars test, recommended by ASTM C 1260 , was useThe NBR 9848 test  was perf29Si, to verify on the atomic scale the influence of the quartz deformation structures on the susceptibility of ASR occurring. Samples were evaluated before and after the alkaline attack in a 1 M solution (KOH) for 3 days at 80 °C.Nuclear magnetic resonance spectroscopy (NMR) measures the intensity of absorption or emission of electromagnetic radiation at certain frequencies. When the technique is used to evaluate solids, it is possible to determine the structure of the compounds. To measure an NMR spectrum, a reference material is used as the standard. In this study, the frequency range studied was The chemical shift (δ) from the NMR to the silicate group is determined by the number of Si–O–Si and/or Si–O–Al bridges, in addition to weaker perturbations that are produced by cations coordinating the other oxygen atoms present. The tetrahedra species are identified with the nomenclature QnmAl, where n is the number of oxygens in Si–O–Si bridges  and m the number of oxygens in Si–O–Al bridges.In order to verify if the separation of the quartz from the different rocks (with different intensities of deformation) was carried out appropriately, i.e., to verify if there is predominance of quartz after minerals are separated, X-ray diffraction (XRD) with Rietveld refinement was carried out. The data acquisition time was 16 s at each step of 0.02°, from 2 to 72° . A Siemens Bruker-AXS diffractometer , model D5000 with goniometer θ-θ was used, with Cu–Kα radiation (λ = 1.5418 Ǻ) being the graphite monochromator. The operating conditions of the X-ray tube were 40 kV and 25 mA.Scanning Electron Microscopy (SEM) was carried out in a Jeol 6610-LV  at the Isotopic Laboratory of the Geosciences Institute of Federal University of Rio Grande do Sul (BR). Backscattered Electron Imaging combined with energy dispersive spectrometry (EDS) were employed to assess the compositional change of quartz crystals after the alkaline attack. For SEM-EDS analysis, thin laminates of the mortars submitted to the ASTM C 1260 accelerated assay were used in order to evaluate the dissolution of the aggregates used in the accelerated test.2 content of the granite and the mylonites and that the whole composition of the rocks is not greatly affected by the deformation process, which is confirmed by the petrographic observations.Rock chemical analysis  show theMineralogical composition of M1, M2, and M3 was identified, characterized, and quantified qualitatively through petrographic analysis.According to the petrographic analysis, M1 is a coarse-grained monzo to sienogranite, with a porphyritic, hypidiomorphic texture. The K-feldspars megacrystals has an average size of 3.0 cm, reaching 8.0 cm. The matrix comprises quartz (37.6%), K-feldspar (33.8%), plagioclase (25.2%), and biotite (3.5%) with sizes between 0.1 and 1.0 cm. As accessory minerals, zircon, epidote, allanite apatite, and opaques were identified.The quartz of M1 is characterized by the weakest deformation among the three rocks analyzed. It appears with anhedral grains with sizes between 0.2 and 0.6 cm. These crystals invariably present deformation features such as undulatory extinction and, in a few regions, deformation bands and subgrains, as shown in Sample M2 represents the transition from granite to mylonite, with decrease in grain size, definition of a spaced foliation marked by the slight orientation of platy grains and stretching of quartz a. The miIn M2, there is a reduction in the size of the grains of quartz, which are more uniform in this rock. However, they sometimes appear as large crystals (1.0 to 2.0 mm), but with deformation features, such as undulatory extinction, deformation bands, subgrains, and recrystallized crowns of quartz around the larger grains. Quartz ribbons begin to appear, separated from the other minerals of the rock. In these places, the grains are more stretched and can present features that range from intense deformation, with strong undulatory extinction and subgrains, to mosaics of new grains, surrounding feldspar porphyroclast, without undulatory extinction and which are free of deformation b.Sample M3 is a mylonite , composeThe quartz-feldspatic bands are mainly characterized by aggregates of new small grains (<0.3 mm). These new grains can have contacts that range from those which are serrated and interpenetrating, with strong preferential orientation and, moreover, deformed, with undulatory extinction, to straighter, polygonal contacts without preferential orientation, which are free of deformation.As shown by Tiecher et al. , five st- Stage 0: deformation-free grainQuartz free of defects, without deformation. The quartz grain is characterized by the absence of undulatory extinction, i.e., it extinguishes perfectly the light when the stage of the optical microscope is rotated, which shows that its crystalline reticulum is free of defects (displacements).- Stage 1: grain with undulatory extinctionThe optical characteristic of the mineral in this stage indicates that there is deformation of the crystalline reticulum. On observing quartz through petrographic microscopy, the light does not extinguish in a homogeneous way but has lighter and darker areas.- Stage 2: grain with undulatory extinction which forms deformation bandsDeformation increases and the undulatory extinction becomes more pronounced, creating well-defined zones within the crystal . These zones/bands are called deformation bands and represent regions where the grain is more fragile.- Stage 3: grain with undulatory extinction which starts to form subgrainsWhen deformation increases, the deformation bands are amplified, i.e., the defects in the crystalline reticulum of the mineral grow, thereby creating regions where the chemical bonds tend to break more easily. Since subgrains have well-defined walls, this leads to their developing more easily within the grain of origin.- Stage 4: recrystallized grainIn the last stage of the deformation process, subgrains are completely separate and form new grains without defects in a process called recrystallization. Recrystallized grains have the same characteristics as those of Stage 1, i.e., without deformation of the crystalline reticulum, but they are of smaller size.The influence of textural characteristics of quartz to AAR was evaluated isolating this mineral, i.e., quartz grains were extracted from the matrix, as described in The quality of the analysis by Rietveld can also be observed graphically in 29Si NMR spectra of 308). The peak at −105.2 ppm corresponds to phases containing sodium and aluminum, i.e., plagioclase (NaAlSi3O8) and the narrow peak at −87.4 ppm is indicative of the presence of muscovite   2O6). All peaks referring to the Si–O–Al sites are grouped in the spectra and identified by the QnmAl nomenclature.The All NMR spectra of the pure samples and KOH-attacked samples, for which there is a wide base, corresponding to quartz in an amorphous network , which iIn the amorphous portion of the spectra of all samples, the Qn sites have different Si–O–Si connectivities, corresponding to the species Q0, Q1, Q2, Q3, and Q4, similar to what the study by Leemann  concludeNMR analyzes performed by Tambelli et al.  show thaThe Qn site presents a distribution of isotropic chemical shifts caused by the 29Si structural disorder, which gives rise to Gaussian-type line shapes in the NMR spectra. The groups Q0, Q1, Q2, Q3, and Q4 may be present in amorphous or crystalline areas . The Q4 groups, which form the structure of the silica (quartz), have a chemical shift with values around −110 ppm. Studies evaluating the products formed by the ASR show that it has a layered structure .It is important to point out that these results could not be taken as true if they were obtained alone, but only after NMR analysis, since the differences observed between the samples before and after the KOH attack were small. However, there is support for the other results obtained in the study, such as the analysis by using the visible spectrophotometer method , which eIt is considered that the higher the silica dissolution, the higher the incidence of ASR. According to The expansions measured for M3 were lower than the other samples. M3, as shown previously, is the rock with the greatest deformation, which contains predominantly recrystallized quartz grains (Stage 4), these grains do not have deformation of the crystalline reticulum, that is, they take longer to react . Figure In order to evaluate if the differences between the expansions of the M1, M2, and M3 rocks can be considered significant, it was take the variance analysis (ANOVA) of the influence of the type of the rock on the expansions test was performed .p-value, it was verified that the type of rock influences significantly the expansions, as do the age and the interaction between the type of rock and age. Thus, M2 expanded/reacted more intensely than M1, which expanded more than M3, in the same way it was observed in the NMR and by the visible spectrophotometer . Slightly deformed quartz, predominant in the M1, effectively contributes to the occurrence of ASR, together with the grains of Stage 2, which are also expressive in this sample.According to In the sample M3, the percentage of grains with Stage 2 are 15% higher than M1, but expansions measured for M3 were lower. The recrystallized grains  are less susceptible to the alkaline attack. Grains of the Stages 3 and 4 result from more intense deformations undergone by the rock. However, their expansions were less than the others’.2). From this model, the expansions were recalculated and results were obtained by varying each stage of deformation alone and keeping the other stages constant. Thus, it was possible to trace the behavior curves for the degrees of deformation, as shown in Based on the quartz with different stages of deformation which were quantified in this study and the results of the accelerated mortar bar test at 30 days, it was possible to obtain a model of the behavior (Equation (1)), which represents 70% of the experimental data (RCorrelation coefficient (R²) = 70%.The curves obtained by Equation (1) are valid only to interpret the behavior of the expansions arising from M1, M2, and M3. The results are similar to those of the assays. Thus, A petrographic analysis was performed on mortars submitted to the accelerated mortar bar test, where it was verified that the alkaline solution effectively dissolves the quartz first. SEM with EDX enabled it to be verified that there was a predominance of the plagioclases and K-feldspars in the fragments exposed to the alkaline solution a,b,f,g. On observing the rock fragments from the accelerated mortar bar test, partially dissolved quartz was detected, which presented voids within the grain itself. This situation is illustrated by The dissolution of the quartz in the mortars resembles what occurs in nature, when alkaline fluids come into contact with acidic rocks. Mexias  reveals In the literature, numerous studies observe the relationship between rock characteristics and their susceptibility to silica dissolution ,8,37,38.Ponce and Batic  verifiedThe main contribution of the present study is the evaluation of the silica dissolution, promoted by the alkaline attack, made using quartz extracted from the rocks. Samples evaluated have quartz with different stages of deformation; thus, it was possible to verify the importance, for the dissolution of silica, of intensely deformed grains, forming bands and subgrains (Stage 2). It has also been observed that recrystallized grains contribute less to the dissolution of silica.A good example of how alkaline hydroxides penetrate into the quartz grains, promoting ASR, is illustrated below by the micrographs published by Mesquita . The autIn order to better explain the relation between the quartz characteristics for the development of ARS, four stages of deformation were established by using petrographic analysis. The analysis was performed to evaluate the silica dissolution of the different textures of quartz performed on the grains extracted from the rocks. This showed that the presence of displacement walls in the quartz (Stage 2) facilitates the reaction with the alkali hydroxides. The displacement walls are regions in the grains where the chemical bonds between Si and O are brittle and can be viewed under optical microscopy. The same behavior was observed when the evaluation was made using the accelerated mortar bar test in which the rock which was used did not have the extracted quartz.29Si, it was possible to verify that the poorly deformed quartz (Stage 1) shows the same characteristics as the most deformed grains (Stages 2 and 3) do on a molecular scale. In addition, the analyses indicated that, after the alkaline attack, there is a greater reduction in the number of crystalline sites in sample M2, in which there are largely grains of Stage 2, thus suggesting that Stage 2 can react faster than Stages 1 and 3, respectively, thereby contributing more to the occurrence of the ASR.Using NMR analysis by direct polarization of Further investigations will be performed using XANES (X-ray Absorption Near Edge Structure) and EXAFS (Extended-Ray Absorption Fine Structure), through the Synchrotron Energy, both in grains extracted from rocks and in grains of pure synthetic quartz. The synthetic quartz will be used as a reference parameter in order to evaluate whether the results obtained in the extracted samples are influenced by the presence of micro impurities present.


Covalent organic frameworks (COFs) have emerged as a new class of crystalline porous materials prepared by integrating organic molecular building blocks into predetermined network structures entirely through strong covalent bonds. The consequently encountered “crystallization problem” has been conquered by dynamic covalent chemistry in syntheses and reticular chemistry in materials design. In this contribution, we have reviewed the progress in the crystallization of COF materials and their hydrogen, methane and carbon dioxide gas storage properties for clean energy applications. In recent decades interest in organic materials with permanent nanometer scale pores has grown very quickly due to their specific properties and broad applications in gas storage, gas separation, drug delivery, energy conversion, catalysis, and optoelectronics. Scientists have designed and constructed a large number of porous materials, such as zeolites, metal-organic frameworks (MOFs), mesoporous silica, organosilica, microporous polymers, porous carbons, covalent organic frameworks (COFs) and organic molecular cages. These porous materials can be designed with variation of components, functionalities, pore metrics and skeletons. During the past decades, construction of persistent organic porous materials via covalent bonds is attracting increasing interest due to their unique stability and advanced applications. However, the wide pore-size-distribution and disordered extended structures of these amorphous networks restrict their further applications. Therefore, COFs as a class of covalent porous crystalline polymer in which the organic building blocks are covalently integrated to form extended ordered crystal structures, makes them different from the amorphous porous organic polymers ,3,4,5,6.G‡), kinetic intermediates dominate initially due to a fast rate of formation the kinetic products, which can be trapped during quenching of the reaction and form amorphous products with the gain of entropy from their randomness of arrangement, meanwhile the thermodynamic products have the lowest overall Gibb’s free energy (ΔG°), so that given the opportunity the reaction will re-equilibrate towards the global minimum to form the most stable and probably crystalline products. It is also possible that the kinetic products can also be transformed into thermodynamic products by using certain thermo-treatments for recrystallization. It is common in COF chemistry that amorphous products are initially obtained until the synthetic conditions are optimized by varying the crystallization times, reaction temperatures, reaction solvents, and catalysts . The discovery process may change the activation energy of thermodynamic products and their reaction rates to accelerate the crystallization of COFs. On the other hand, rational design of building blocks may also increase the correctness of the assembly of molecular components, thus enhance the crystallinity of the obtained products. It is generally believed that 2D COFs are easier to form having less probability of resulting in networks than the 3D ones with more structural diversity.In order to achieve higher reversibility for efficient error correction during crystallization, dynamic covalent chemistry (DCC) has been well applied in the construction of COFs. To stitch discrete molecular building blocks into periodic networks, DCC enables strong covalent bond formation accompanied by the assembly of molecular building blocks into predetermined spatial arrangements ,8. ConsiFrom the chemistry viewpoint, the difficulty of constructing COF materials can be classified by the difference of bond energy of the linkages between the molecular building blocks . It is e3O3), dioxaborole (-BO2C2), and spiroborate (-O4B¯).Featuring light-weight compositions and high crystallinity, boron-based COFs have been intensively studied including understanding their crystallization mechanism, to the designing of new topologies, achieving high surface area, and the utilization in gas storage applications. The successful construction of such crystalline materials benefited from the decent reversibility of the B-O linkages, including boroxine  COFs, COF-102 and COF-103 by self-condensation of tetrahedral (3D-Td) nodes, tetra(4-dihydroxyborylphenyl)methane (TBPM) and its silane analog (TBPS), respectively (2 g−1 for COF-102 and 4210 m2 g−1 for COF-103) and pore size distributions of 11.5 Å (COF-102) and 12.5 Å (COF-103).The choice of solvent and temperature to maximize the error correction associated with the boroxine ring-forming reactions can optimize the crystallinity of the boroxine-based COFs. COF-1 with two-dimensional (2D) architectures, was prepared by the self-condensation of 1,4-phenylenediboronic acid (BDBA) under solvothermal conditions, to produce an extended staggered layered structure with hexagonal pores of 15 Å diameter and a BET surface area of 711 mectively  [11]. ThMicrowave heating can accelerate the reaction times and obtain much cleaner COFs with high yields. Cooper and co-workers demonstrated the synthesis and purification of COF-5 and COF-102 by microwave heating. The microwave synthetic method reported by Cooper and co-workers was 200 times faster than solvothermal methods reported by Yaghi et al. without changing the physical properties of COFs .The cross-condensation of polybornic acids with catechols opened the way to stich different building blocks into one framework . The popBy using the tetrahydroxybenzene and its derivatives, 2D COFs can be prepared by stitching tritopic boronic acids together. The reactions were conducted in the mixture of tetrahydrofuran (THF) and methanol (MeOH) by stirring under an inert atmosphere ,16.2·g−1 and the pore volume decreased from 0.69 to 0.052 cm3·g−1, as the chain length increased. Interestingly, modification of the pore interior with increasingly larger alkyl groups causes a decline in nitrogen uptake but an increase in the molar amount of hydrogen adsorbed. Once submersion in aqueous media, the porosity of alkylated COFs decreased by about 25%, while the non-alkylated COFs were almost completely hydrolyzed, losing virtually all porosity. In addition, the degree of crystallinity decreased by about 40% for alkylated COFs and 95% for non-alkylated COFs. By using microwave synthesis, Bein et al. extended the framework into materials having large 4 nm openings . The synthesized ICOFs (ICOF-1 and ICOF-2) show good thermal stabilities and excellent resistance to hydrolysis, remaining nearly intact when immersed in water or basic solution for up to two days. The SEM images support the single-crystalline morphology of ICOF-1 and ICOF-2. The PXRD results also show multiple sharp peaks, which indicate the orderliness structures in the framework. However, the crystal structures of ICOFs have not been characterized. These ICOFs also have high BET surface areas up to 1259 m2·g−1 and adsorb a significant amount of H2  and CH4 . The existence of permanently immobilized ion centers in ICOFs enables the transportation of lithium ions with room-temperature lithium-ion conductivity of 3.05 × 10−5 S·Lcm−1 and an average Li+ transference number value of 0.80 ± 0.02.Researchers have shown great interest in exploring new methods to enhance the chemical stability of boron-containing COFs. Recently, the condensation of diols with trialkyl borate in the presence of basic catalysts has been explored for the synthesis of spiroborate-linked ionic COFs in which the negatively charged borons are located on the edges with different cations as counter ions. Zhang and co-workers  construcTo avoid the structural fragility of boron-containing COFs, imine-based COFs were discovered by a reversible condensation of polytopic anilines with polybenzaldehydes or polyketone with elimination of water by a catalyst of aqueous acetic acid . General2·g−1 and a pore size of 7.8 Å, which provides better hydrolytic stability compared to the boron-containing COFs. Moreover, the broad range of multifunctional amines and aldehydes provides a large number of structural possibilities for imine-based COFs.The first imine-linked COF was developed by Yaghi and co-workers in 2009 . They co2·g−1. The crystal structure of the COF-320 solid was elucidated by single-crystal 3D electron diffraction  centers in COFs and creating an intramolecular [O-H···N=C] hydrogen bond  30]. Th. Th30]. The formation of crystalline structures via relatively weak interactions between planar layers has been well defined and designed in crystal engineering. Jiang and co-workers synthesized a series of 2D COFs locked with intra-layer hydrogen-bonding (H-bonding) interactions  31]. Th. Th31]. In addition to H-bonding interactions, self-complementary π-electronic forces also provide new opportunities for enhancing the crystallinity of COFs. Jiang and co-workers reported a synthetic method to control the crystallinity and porosity of COFs by managing interlayer interactions based on self-complementary π-electronic forces .They controlled the crystal structure of CuP-Ph COF by introducing 2,3,5,6-tetrafluoroterephthalaldehyde into the COF structure. Fluoro-substituted aromatic units in different ratios were integrated into the edge units, which will induce the self-complementary π-electronic interactions in the COFs. This interaction increases the crystallinity and the porosity of COFs by maximizing the total crystal stacking energy and minimizing the unit cell size. This work provides a new pathway to improve the crystals of COFs by controlling the interlayer interactions . C2-phenyl edges soften the polarization through resonance and increase the stability and high crystallinity of the COF. Compared to the TPB-TP-COF and TPB-DHTP-COF, the methoxy-functionalized TPB-DMTP-COF has much higher crystal stacking energy, showing high crystallinity and high BET surface area. Through the introduction of chiral centers, (S)-Py moieties, onto the walls of the open channels, they obtained chiral COFs which can be used for heterogeneous asymmetric organocatalysis of asymmetric Michael reactions. The Jiang group also investigated the proton conduction properties of this highly crystalline and stable COF and explored the potential applications in many areas, including fuel cells, electrochemical sensors, electrochemical reactors and electrochromic devices 2[C6H4(CO)2]3, 1’ and [(C6H4NH)4][C6H4(CO)2]2, 2’ without losing their crystallinity or topology. FT-IR and 13C CP-MAS NMR spectra indicated the successful conversion of COF 1 and COF 2 to COF 1’ and COF 2’. PXRD results demonstrated the high crystal structure of COF 1’ and COF 2’. The reduction of BET surface area of COF 1’ and COF 2’ compared to COF 1 and COF 2 due to the increase of framework mass and decrease of pore volume. This method offers a new pathway to overcome the usual crystallization problem in COF chemistry . Th. Th36]. ic route .The amidhemistry .Like the reactions of imine-based COFs, the reaction of benzaldehydes and hydrazide groups could yield hydrazone-linked COFs . HydrazoDichtel et al. reported the exfoliation of the COF-43 in various solvents to yield few-layer 2D polymers. The obtained exfoliated COF-43 polymers showed an apparent loss of crystallinity by PXRD, but no apparent changes in its covalent linkages and high aspect ratios .2·g−1. When illuminated with visible light, the Pt-doped COF continuously produces hydrogen from water without signs of degradation. This new application of COFs in photocatalysis to open new pathways for heterogeneous photocatalysts x%-H2P-COFs) into an outstanding carbondioxide-capture material, carboxyl group modified 2D COF ([HO2C]x%-H2P-COFs) [2C]x%-H2P-COFs show micropores between 1.4-2.2 nm instead of the mesopores of [HO]x%-H2P-COFs (2.5 nm). From the results of x%-H2P-COFs have low CO2 capacity between 23-32 cm3·g−1 at 273 K and 1 bar. However, [HO2C]x%-H2P-COFs exhibit a dramatically enhanced CO2 adsorption between 49–89 m3·g−1 under the same conditions.Functional groups inside the channel walls of COF materials can enhance the CO monomer . The tri2P-COFs) . The x-H2P-COFs. The CO2 adsorption capacities of [CH≡C]x-H2P-COFs and [EtNH2]x-H2P-COFs enhanced with the increase of BET surface area and pore size. However, [MeOAc]x-H2P-COFs, [AcOH]x-H2P-COFs, [EtOH]x-H2P-COFs and [EtNH2]x-H2P-COFs show much higher CO2 adsorption capacity than those of [CH≡C]x-H2P-COFs. The CO2 adsorption capacities are in order of [CH≡C]x-H2P-COFs ≈ [Et]x-H2P-COFs ≪ [MeOAc]x-H2P-COFs < [AcOH]x-H2P-COFs ≈ [EtOH]x-H2P-COFs < [EtNH2]x-H2P-COFs, which is due to the interaction between the functional group and CO2 molecule. These results indicate that the CO2 adsorption performances are highly related to the pore surface of the COF materials.Jiang et al. also carried out a similar strategy to modify the pore surface with different functional groups through click reactions ,67. FromThe past decade has been witnessed the blooming COF chemistry and their applications. Their unique features, such as flexible molecular structure design, permanent porosity, low density, modest to high crystallinity and controllable pore size from ultraporous to mesoporous, and the diversity of available building blocks, easily make them targets for structural and functional design, and promise their great potential as materials for application in various areas. However, there are still a lot of challenges in the design and synthesis of COFs.First, compared to the high ordered structure of MOF materials, further investigation on the control of crystal growth, the skeletons, and pores is still of great importance. Currently, thermodynamically controlled solvothermal reactions are widely used to synthesize COF materials. The investigations reveal that the formation and growth of crystal nuclei to improve the crystallinity of COFs is quite important. Such studies provide a general pathway for the preparation of high crystallinity COFs. Moreover, control over the defect sites, the morphology and the stacking mode of COF’s structures are also important and effective methods to construct precise crystal COF structures are necessary. At present, no direct analytical tools can be used to visualize and analyze the stacking and topological structures of COFs. Thus, the creation of single-crystal COFs an unattainable target.SBET over 6000 m2·g−1.Meanwhile, through the existing approaches, the methods to increase the surface area and porosities of COF materials are a big bottleneck. The surface area and porosities are the most important parameters for COF materials for gas adsorption and gas separation applications. How to design COFs structures which can improve both surface area and these properties is the key to realize the potential application of COFs in these fields. For the 2D COFs, spatially enlarging the interlayered spaces would increase the surface areas. For 3D COFs, the construction of highly crystalline frameworks would lead to very high BET surface areas that can reach the theoretical


Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A’s localization is integral to its function.We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals.We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals.By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder. Protein degradation through the ubiquitin-proteasome pathway (UPP) is important for a wide range of functions in the nervous system , 2. In UTwo major classes of E3s have been defined: HECT (homologous to E6-AP carboxyl-terminus) domain E3s and RING finger E3s. UBE3A, the first E3 discovered in the HECT class , is of sDespite its clinical importance, our current understanding of UBE3A’s function in the brain remains limited. UBE3A has been implicated in regulation of Golgi acidification  and mitoCrucial for the elucidation of UBE3A’s functions is to determine where the protein actually lies within the cell. Previous immunohistochemical studies in rodents found a broad distribution of UBE3A, but emphasized its nuclear expression in mature neurons, consistent with a proposed role for UBE3A in the regulation of transcription , 36–38. All procedures regarding human tissue were performed with the approval of the UCSF Committee on Human Research and the University of Maryland Institutional Review Board. Nine small blocks from postmortem temporal cortex were obtained from the UCSF Neurodegenerative Disease Brain Bank (NDBB) and the NIH NeuroBioBank, including autopsy materials from all three adults with AS currently available for study. Details are listed in Table The ultrastructural integrity of all postmortem tissue was seriously compromised. In the best preserved samples, most subcellular structures were identifiable, but the tissue contained abnormal holes, neurons were vesiculated, cytoplasm was disrupted, nucleoplasm appeared abnormally condensed, membranes were distorted, and mitochondria were swollen Fig. , b. The 2 and 2.5% DMSO, or 4% PFA in 0.1 M sodium cacodylate + 0.1% CaCl2 and 2.5% DMSO. Following fixation, the samples were stored in PO4-buffered saline (PBS) at 4 °C. Sections were cut at 50 μm on a Vibratome and collected in cold PB.Choosing to focus on well preserved material, we obtained 9 surgical biopsies. Because of limited tissue availability, all nine were from patients undergoing surgery to remove epileptic foci in the hippocampus Table . The temIn contrast to the postmortem tissue, the ultrastructural integrity of the biopsy samples was generally good, and in many cases, comparable to that obtained from perfusion-fixed mice Fig. , f. SincUbe3am−/p+), which maternally inherit the Ube3a knock-out allele, were generated by mating wild-type (Ube3am+/p+) male mice to female mice with paternal inheritance of the Ube3a knock-out allele (Ube3am+/p–), which themselves are phenotypically normal [All procedures related to the care and treatment of animals were in accordance with institutional and NIH guidelines, and all animal use protocols were reviewed and approved by the UNC Institutional Animal Care and Use Committee. AS model mice  raised against a peptide corresponding to mouse UBE3A isoforms 1 and 3 a.a. 315–415) and isoform 2 (a.a. 336–436). Importantly, this immunogen shares 100% sequence identity with human UBE3A isoforms 1 (a.a. 318–417), 2 (a.a. 341–441), and 3 (a.a. 338–437). We used this antibody because we had previously verified its specificity against AS model and UBE3A KO mice [5 and isoTo identify GABA, we used a rabbit polyclonal antibody  developed using GABA-BSA as the immunogen. The antibody was affinity-purified using the immunogen. It showed positive binding with GABA and GABA-KLH in a dot blot assay and negative binding with bovine serum albumin (BSA) .To identify GFAP, we used a rabbit polyclonal anti-GFAP  developed against purified bovine GFAP. This antibody recognizes a 51 kDa band on immunoblot of rat spinal cord.To identify Oligodendrocyte Transcription Factor 2 (Olig2), we used a rabbit polyclonal anti-Olig2  developed using a recombinant mouse Olig-2. This antibody specifically labels cells of oligodendrocyte lineage , consist2O2 in phosphate-buffered saline . After preincubation in 10% normal donkey serum (to block secondary antibody binding sites), sections were incubated in UBE3A (1:500 to 1:5000) overnight on a shaker at room temperature.Free-floating sections were treated for 30 min in 1% sodium borohydride, followed by 30 min with 3% HFor immunoperoxidase microscopy, sections were then incubated overnight on a shaker at room temperature in biotinylated secondary antibody  and for 4 h in ExtrAvidin-peroxidase complex ; peroxidase was histochemically visualized with diaminobenzidine. Processed sections were mounted on gelatin-coated slides, air dried, and cleared with xylene before being coverslipped with D.P.X. Mountant .For immunofluorescence, antigenic sites were visualized with donkey IgG conjugated to DyLight 549 . For double labeling, the second primary antibody  was applied overnight and visualized by a secondary antibody conjugated to Alexa 488 . Some sections were then counterstained with DAPI  to visualize nuclei, and with NeuroTrace 640–660  to visualize neuronal somata. Sections were examined with a Zeiss LSM 880 confocal microscope with Airyscan.To assess structural preservation, sections were processed for reduced osmium according to the Knott protocol , 45. Bri2O2/PBS, then 0.025% Triton X-100/PBS, then blocked with 10% normal goat serum (NGS), and incubated overnight in primary antibody . Sections were rinsed, and blocked again in 2% NGS, then incubated overnight with biotinylated goat-anti mouse IgG , rinsed, and incubated for 2 h in 1.4 nm gold particles conjugated to streptavidin . After rinsing, binding was stabilized by treating 30 min in 2% glutaraldehyde/PBS; sections were washed again and transitioned into 0.05 M sodium acetate (to remove phosphate and chloride ions), followed by silver enhancement with HQ Silver . After rinsing in sodium acetate and transitioning to PB, sections were post-fixed in 0.5% osmium tetroxide in 0.1 M PB for 45 min, rinsed and transitioned to 0.1 M maleate buffer, pH 6, and then incubated for 45 min with 1% uranyl acetate in maleate buffer .For immunohistochemistry, sections were treated with 1% sodium borohydride/PBS, followed by 3% HAfter processing, sections were dehydrated in a graded ethanol series and infiltrated with a low viscosity epoxy resin  and flat-mounted between sheets of ACLAR® fluoropolymer  within glass slides. Sixty-nanometer sections were cut and mounted on 200 mesh copper grids. For double labeling with GABA, nickel grids were pretreated 15 min at 60 °C in 0.01 M citrate buffer, pH 6, rinsed in water, blocked in 1% BSA in Tris-buffered saline, pH 7.6, with 0.005% Tergitol NP-10, and incubated overnight at 21–24 °C room temperature with the primary antibody . Grids were rinsed, blocked in 1% normal goat serum in TBS, pH 8.2, with Tergitol NP-10, and incubated in secondary antibodies for 2 h . Grids were then rinsed in buffer, then water, and counterstained with 1% uranyl acetate in water, followed by Sato’s lead. Electron microscopy was performed with a Philips Tecnai transmission electron microscope.Biopsy specimens are subject to an unavoidable delay ; only a very weak background staining could be detected in these mice Fig. , f. We cTo compare the level of nuclear UBE3A between excitatory neurons and GABAergic neurons, we acquired 58 random fields of human cortical layers II/III  from material quadruple-stained for UBE3A, GABA, NeuroTrace, and DAPI. To minimize artifacts arising from limited penetration, confocal images were acquired just below the section surface. For each neuron, the intensity of UBE3A staining in the nucleus (defined by DAPI) was computed using FIJI . To contTo examine the quantitative relationship between staining for UBE3A and DAPI, we performed intensity correlation analysis as described by Li et al. , using FThe overall pattern of staining was consistent across biopsy samples from all 9 subjects. UBE3A was found in neurons throughout the depth of cerebral cortex Fig. –c. Stainn = 51) higher in pyramidal neurons than in GABAegic neurons. In addition, colocalization was observed in fine processes and puncta distributed through the neuropil  and inhibitory (GABAergic) neurons of the human cerebral cortex. Epilepsy, a common comorbidity in AS, is thought to reflect a disrupted balance between excitatory and inhibitory neurotransmission. We recently demonstrated in an animal model that selective deletion of UBE3A may also serve important functions in glia. For example, flies with glia-specific overexpression of Dube3a (the fly UBE3A homolog) display a robust seizure phenotype, suggesting a possible role for glial UBE3A in Dup15q pathophysiology . Our finWe found that UBE3A is expressed in multiple subcellular compartments. In neurons, it concentrates in euchromatin-rich domains within the nucleus, where it is positioned to regulate the expression of active genes, possibly exerting cell-wide global effects. A recent study shows that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1 , leadingUBE3A labeling was also associated with mitochondria. Loss of UBE3A has been linked to key enzymatic deficits in the mitochondrial respiratory chain , 18. A rThe enrichment of UBE3A that we observed in both excitatory and GABAergic axon terminals and (to a lesser extent) in dendritic spines is of special interest since both Dup15q and AS mouse models show synaptic deficits , 25, 62.The overall pattern of UBE3A immunoreactivity we found in human temporal cortex is generally consistent with that seen in previous studies of rodent cortex, reinforcing the idea that mice offer a valuable model in which to study the role of UBE3A in the brain. Mouse models of AS display many Angelman-like phenotypes, including learning and memory deficits, motor dysfunction, and seizures, while transgenic mice expressing UBE3A at increased dosages manifest several core autistic behavioral traits . NeverthThat UBE3A is expressed at high levels in both excitatory and inhibitory neurons in the human cerebral cortex points to the many ways that its dysregulation might disrupt circuit function, while its concentration at synapses and in nuclei offers intriguing functional clues. UBE3A in axon terminals presumably regulates the function of individual synapses; in contrast, its concentration in euchromatin-rich nuclear domains suggests that UBE3A may also mediate global effects on neuronal physiology, by regulating gene transcription. This information identifies axon terminals and nuclei as two distinct potential targets for future clinical intervention.


Chaetomium thermophilum (CtBGL) was determined at a resolution of 2.99 Å. The structure showed the three-domain architecture found in other β-glucosidases with variations in loops and linker regions. The active site catalytic residues in CtBGL were identified as Asp287 (nucleophile) and Glu517 (acid/base). Structural comparison of CtBGL with Protein Data Bank (PDB)-deposited structures revealed variations among glycosylated Asn residues. The enzyme displayed moderate glycosylation compared to other GH3 family β-glucosidases with similar structure. A new glycosylation site at position Asn504 was identified in CtBGL. Moreover, comparison with respect to several thermostability parameters suggested that glycosylation and charged residues involved in electrostatic interactions may contribute to the stability of the enzyme at elevated temperatures. The reported CtBGL structure provides additional insights into the family GH3 enzymes and could offer new ideas for further improvements in β-glucosidases for more efficient use in biotechnological applications regarding cellulose degradation.Beta-glucosidases (β-glucosidases) have attracted considerable attention in recent years for use in various biotechnological applications. They are also essential enzymes for lignocellulose degradation in biofuel production. However, cost-effective biomass conversion requires the use of highly efficient enzymes. Thus, the search for new enzymes as better alternatives of the currently available enzyme preparations is highly important. Thermophilic fungi are nowadays considered as a promising source of enzymes with improved stability. Here, the crystal structure of a family GH3 β-glucosidase from the thermophilic fungus  Beta-glucosidases (β-glucosidases) are key cellulolytic enzymes that catalyze the hydrolysis of cellobiose to glucose. They are terminal enzymes of the cellulase system as they act during the final step of the cellulose degradation. Although beta-glucosidases do not act directly on cellulose, their activity is important to circumvent the inhibitory effect of cellobiose on endoglucanases and cellobiohydrolases. Consequently, inclusion of β-glucosidases in cellulase preparations can synergistically increase the hydrolytic efficiency of other cellulolytic enzymes .Beta-glucosidases  have been found in the glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, GH30, and GH116 of the CAZy database [Aspergilus aculeatus [Rasamsonia emersonii [Trichoderma reesei of BGL1 has been deposited in the Protein Data Bank (PDB), but no paper has been published yet. Recently, the structure of the filamentous fungus Neurospora crassa Cel3A (NcCel3A) was reported at 2.25 Å resolution [Hypocrea jecorina (a teleomorph of Trichoderma reesei) [Aspergilus niger [Aspergillus fumigatus, and Aspergillus oryzae [Despite their extensive use in biotechnological application, structural data on fungal β-glucosidases are still scarce. Many fungal BGLs are classified as part of the GH3 family, one of the largest CAZy families with over 13,600 annotated protein sequences. Two fungal β-glucosidase structures currently available are produced by the mesophilic fungus mersonii , respectus niger , Aspergis oryzae .Hordeum vulgare) exo-1,3-1,4-glucanase ExoI (HvExoI) [6 sandwich domain where the acid/base glutamate resides. Subsequent studies, however, revealed variations in the modular architecture of GH3 β-glucosidases with the first domain to adopt a collapsed/incomplete triosephosphate isomerase (TIM)-barrel domain and the presence of a third domain with a fibronectin FnIII-like fold. The function of the third domain is still unknown, although it has been suggested to stabilize the incomplete TIM-barrel domain [Kluyveromyces marxianus and Pseudoalteromonas sp. BB1 β-glucosidases [The first structure of a β-glucosidase, that of barley ((HvExoI)  showed aosidases ,11.Myceliophthora thermophila has been found to act as an efficient biocatalyst in alkyl glycoside synthesis [Thermophilic fungi have attracted considerable attention in recent years as an alternative reservoir of thermostable cellulases for cellulose degradation ,13. Impoynthesis .Chaetomium thermophilum (CtBGL) at a resolution of 2.99 Å, and compare it with other β-glucosidases of the GH3 family. This is the second structure of a β-glucosidase produced by a thermophilic fungus, and therefore, it is expected to provide further insights into the structure–function relationship of this family of enzymes in high-temperature settings.Here, we report the crystal structure of a β-glucosidase from the thermophilic filamentous fungus CtBGL was refined using data up to 2.99 Å resolution. The final Rcryst and Rfree (5% of the reflections excluded from refinement) were 0.201 and 0.252, respectively  and A. fumigatus (AfβG) [A. aculeatus (AaβG) [NcCel3A [ReCel3A [CtBGL as a crystallographic dimer that involves the −Y, −X, −Z+ ½ symmetry-related molecule.The crystal structure of ectively . The str POLYGON . The ref POLYGON . Residues (AfβG) , A. aculs (AaβG)  and NcCe[ReCel3A . The solCtBGL has the highest structural similarity with NcCel3A (PDB id 5nbs), as indicated by the root mean square deviation (rmsd) of 0.60 Å for 831 aligned residues (sequence identity 72%), followed by Aspergilus fumigatus AfβG , ReCel3A  and Aspergillus aculeatus AaBgl1 . Lower similarities were found with Hypocrea jecorina HjCel3A .Structure-based sequence alignment  showed tCtBGL  with a 25-residue (residues 351–376) insertion previously described as a hydrophobic linker responsible for the activation of ReCel3A and AaBgl in organic solvents [CtGBL Phe364 (Phe352 in NcCel3A) and CtGBL Trp367 (Trp355 in NcCel3A) are the most conserved. Moreover, loop II contains residues Phe366, Trp367, and Trp377 that line up one side of the substrate-binding site, and they are also found in NcCel3A  with variations in other β-glucosidases, such as ReCel3A and AaBgl1. It has been suggested that this loop is stabilized by interactions with the N-glycans from a neighboring glycosylated Asn residue (Asn57 in NcCel3A) [CtBGL, the equivalent residue is Asn72, which is also glycosylated (see below).The structure of CtBGL  consistsCtBGL second domain, an (α/β)6 sandwich fold, is structurally well conserved among all the GH3 enzymes. It consists of residues His396 to Gly596 and includes loops III and IV that encompass residues Gly433–Val467 and Ala513–Asn536, respectively . Moreover, the architecture and location of loops III and IV, which constitute one site of the active site cavity in between loops I and II, are also found conserved. Ser458, Asp444, and Glu517 are found pointing downwards from the loop regions and towards the active site.ectively . Loop IICtBGL structure is followed by a second linker region . This linker region exhibits an extended structure, which almost resembles a boundary that separates domain I and domain II from domain III. The extended linker region probably plays a role in stabilizing loops I and IV.Domain 2 of the TnBgl3B structure [NcCel3A, ReCel3A, and AaBgl1, but it is absent in TnBgl3B and HjCel3A  residues. Also, conserved in loop V is Asn720, which was found to be N-glycosylated.The FnIII-like domain 3 was first observed in the GH3 family in the tructure . It cons HjCel3A . SeveralCtBGL structure in 10 positions, all in Asn residues . As observed before in this class of β-glucosidases, all glycosylation sites in CtBGL were also located on one face of the molecule  without any attempt of enzymatic cleavage. It is possible that, in these cases, the enzymes were subjected to endoglycosidase activity during the expression stage. The other seven N-glycosylation sites in CtBGL ranged from two to eight monosaccharides. The N-glycan at CtBGL Asn329 was located in domain I of the enzyme and consisted of eight monosaccharides . Extra densities that could accommodate another two monosaccharide molecules were present in the electron-density map, but they were not modeled owing to lack of clarity. It was, nevertheless, the largest N-glycan structure in CtBGL and was involved in forming multiple H-bonds with the enzyme residues at Thr293, Asp730, Val295, Gly734, and Tyr733 and a pi-sigma interaction with Tyr733 residue. The structural equivalent Asn residue in AaBgl1 (Asn322) had a long-length glycan with eight Man and two GlcNAc that were also involved in extensive stabilizing interactions with domain I. N-glycans at positions Asn72 and Asn720 were located in domains I and III, respectively, with loop V residues in between them. They both participated in interactions with protein residues. The N-glycan at Asn72 was found to interact in conventional H-bonding interactions with Ala90, Tyr716, Tyr718, and in pi-sigma interaction with Tyr715 from domain III. The N-glycan at Asn720 interacted with Arg710 via H-bonding and Tyr704 via weak van der Waals interactions. A long chain glycan moiety at Asn259 at the outer surface of the enzyme was found to interact via H-bonding with Ser35, Glu36, and Asp229.A total of 27 glycan moieties were found in CtBGL. This Asn bears only two monosaccharide molecules, whereas, for comparison, the equivalent Asn residue in AaGL1 has seven monosaccharides with the terminal ones able to reach the adjacent subunit. The second Asn residue that participates in dimer interface contacts is not present in CtBGL. The lack of these contacts could therefore contribute to reduced strength of intermolecular interactions in the crystal lattice, resulting in further instability of the CtBGL crystals.Notably, the protein glycans were also shown to exhibit a potential binding affinity for polysaccharides such as cellulose  and aromβ-d-glucose (BGC) was fitted at the active site based on residual electron density observed in electron density Fo–Fc difference maps. The source of the β-d-glucose is most likely the growth medium used as no β-d-glucose was used during crystallizations or soaking. Two catalytic residues were identified, Asp287 (nucleophile) at the N-terminal TIM-barrel domain and Glu517 (acid/base) at the sandwich α/β domain II. Both catalytic residues were found conserved in the GH3 family members  was much shorter and antiparallel, which creates an active site much wider and accessible when compared with GH3 enzyme structures with complete TIM-barrel fold [The active site is located in a shallow pocket near the interface of the first and second domain. A molecule of  members . The corCtBGL has been found to be thermostable at 50 °C and to retain half of its activity after incubation at 65 °C for 55 min [CtBGL exhibits comparable thermostability, as previously reported [A. fumigatus β-glucosidase has been found highly thermostable and able to retain most of its activity for at least 19 h at 65 °C [r 55 min . The enzreported . In contat 65 °C . HjCel3A from the mesophilic fungus Hypocrea jecorina had the lowest SAS (22812 Å2), owing to the smaller number of residues and the lack of loop V. Similarly, loop V was absent in TnBgl3A and the SAS value was reduced. The SAS values for the other β-glucosidases, including CtBGL, were quite similar, i.e., around 28100 Å2.Protein thermostability is usually hard to predict and there is no a common mechanism yet available ,25. SeveHjCelA showed reduced numbers of positively and negatively charged residues, despite its high thermostability with an optimum temperature at 90 °C and an unfolding temperature of ~88 °C depending on the enzyme concentration [AfβG, despite its high thermostability, was also characterized by a similar content of charged residues, suggesting that the strength of individual ion–pair interactions may play a key role.Charged residues in the structures can contribute to structural integrity and, in turn, to thermostability. The negatively (Asp and Glu) and positively (Arg and Lys) charged residues may provide a stability profile of the structure . The numntration . The numHjCel3A samples with different degree of N-glycosylation revealed the same melting temperature (74.0 ± 0.2 °C), thus suggesting that the effect of glycosylation may be case-specific and other factors could play a role. Interestingly, TnBgl3B that lacks glycosylation exhibited thermostability, most likely as a result of the high numbers of charged residues. Nevertheless, the moderate glycosylation in CtBGL could not be ruled out as a contributing factor to the limited stability of the enzyme compared to other more thermostable β-glucosidases characterized by extensive glycosylation. Further studies are, however, required to better understand the role of glycosylation in β-glucosidases and in protein stability.Finally, the glycosylation pattern found may also contribute to the thermostability of the enzyme by promoting interactions with amino acid residues. It has been shown that glycosylation enhances the solubility, reduces the aggregation, and increases the thermal stability of proteins ,29. The Chaetomium thermophilum CT2 was carried out as previously described [Pichia pastoris GS115 cells and purified by ion-exchange chromatography on a DEAE-Sepharose  to homogeneity as judged by SDS-PAGE. β-Glucosidase activity was assayed with salicin using the Miller’s method and also detected in native polyacrylamide gel using 4-methylumbeliferyl-β-d-glucopyranoside [Protein expression and purification of the β-glucosidase from escribed . Brieflyranoside . The act® Ultra Centrifugal Filters   in 10 mM HEPES–NaOH, pH 7.0 buffer. Crystals were obtained by the hanging-drop vapor diffusion method at 16 °C using a well solution of 35–45% v/v MPD . The drops were prepared by mixing 2 µL of protein solution with an equal volume of well solution. The crystals grew as octahedra to a maximum size of approximately 0.06 × 0.06 × 0.08 mm3 within a period of 1 month.Prior to crystallization, the enzyme solution was concentrated to ~10 mg/mL with AmiconP41212/P43212. Assuming one molecule in the asymmetric unit, the Matthews coefficient VM [3/Da corresponding to a solvent content of ~77%.Data were collected on the X13 beamline at EMBL-Hamburg (c/o DESY) from a single crystal under cryogenic (100 K) temperature using a MARCCD detector. The presence of MPD in the crystallization was sufficient for cryoprotection, thus no additional cryoprotectant was needed. One hundred and fifty diffraction images were collected from a single crystal with a rotation range of 0.45° per image. Data processing was carried out with XDS . The crycient VM  is 5.4 ÅAspergillus aculeatus β-glucosidase in complex with castanospermine  was used as a search model after pruning side chains with Sculptor [P41212. Refinement was carried out using simulated annealing (1000 K) in Phenix with maximum likelihood as target function. The refinement was alternated with model visualization and rebuilding using Coot 0.8.9 [Rfree with 5% of the reflections used for the calculations [Initial phases were obtained with molecular replacement using Phaser , as implSculptor  based onot 0.8.9 . Tight rulations . High-reulations . Data coCtBGL and deposited PDB structures. Structure-based sequence alignment was performed with UCSF Chimera 1.13.1 [http://bioinformatica.isa.cnr.it/ESBRI/). SAS area was calculated using PDBePISA [http://web.expasy.org/protparam/) ProtParam tool [PDBeFold  was empla 1.13.1 . ESPripta 1.13.1  was usedPDBePISA . The valram tool .CtBGL structure was determined at 2.99 Å resolution and refined to good stereochemical and refinement numbers, despite the limited resolution. The structure exhibited a three-domain architecture with two linker regions as other β-glucosidase structures. Variations in the length of the first linker regions and the third domain were identified. CtBGL showed the highest structural similarity with NcCel3A. The catalytic residues at the active site were identified as Asp287 (nucleophile) and Glu517 (acid/base). CtBGL showed a low number of glycans at glycosylation sites compared to other heavily glycosylated GH3 β-glucosidases. Charged residues and the glycosylation pattern are suggested as potential contributing factors for the thermostability properties of CtBGL. The analysis presented in this study could offer new ideas towards further improvements in β-glucosidases for better use in biotechnological applications.The


Nematostella vectensis to understand the mechanisms promoting cellular specialization across animals.Understanding the drivers of morphological diversity is a persistent challenge in evolutionary biology. Here, we investigate functional diversification of secretory cells in the sea anemone N. vectensis and show that adult gland cell identity is acquired very early in development. A phylogenetic survey of trypsins across animals suggests that this gene family has undergone numerous expansions. We reveal unexpected diversity in trypsin protein structure and show that trypsin diversity arose through independent acquisitions of non-trypsin domains. Finally, we show that trypsin diversification in N. vectensis was effected through a combination of tandem duplication, exon shuffling, and retrotransposition.We demonstrate regionalized expression of gland cell subtypes in the internal ectoderm of Together, these results reveal the numerous evolutionary mechanisms that drove trypsin duplication and divergence during the morphological specialization of cell types and suggest that the secretory cell phenotype is highly adaptable as a vehicle for novel secretory products. The development of new tissue layers provides the opportunity to spatially segregate cell types enabling the compartmentalization of different functions. Cnidarians are diploblasts, comprised of an internal endodermal epithelium separated from an external ectodermal epithelium by a largely acellular matrix called mesoglea. Anthozoans  are unusual among cnidarians in their possession of internal tissues (pharynx and mesenteries) that arise by secondary epithelial fold morphogenesis following completion of gastrulation . AdditioNematostella vectensis, the starlet sea anemone, has become a valuable model for studies of animal body plan evolution [Nematostella have focused largely on these endodermal functions [N. vectensis [N. vectensis [volution –6; yet, unctions –9. The eunctions  and two ectensis , 12. Tryectensis . A previectensis , suggestectensis  may beliN. vectensis, and to characterize the evolutionary history of a large (super)family of proteases expressed abundantly in the mesenteries. Building on a previous study using RNA-seq to characterize the expression profile of the mesenteries in N. vectensis [N. vectensis is partitioned into different regions associated with distinct morphologies and functions. Additionally, we show numerous lineage-specific expansions of trypsins and that trypsin diversification arises through novel domain acquisition. Finally, we propose a model by which the expansion of trypsins may have promoted specialization of gland cell subtypes in cnidarians.We sought to understand the evolutionary mechanisms promoting functional diversification at the cell and tissue levels in the mesenteries of ectensis , we showN. vectensis during feeding for evidence of morphological and functional variation  and electron lucent (mucus-secreting) gland cells Fig. a–f. The N. vectensis [We previously identified numerous genes encoding different classes of proteases to be upregulated in the adult mesentery of ectensis . Using iN ≥ 1 read), we examined more fully the co-expression of the large superfamily of trypsin proteases and found 6727 cells expressing at least one trypsin gene. Nearly, 50% of the trypsin-expressing cells (3282/6727) appear to express only a single trypsin, while the remaining cells exhibited co-expression of up to 24 trypsins  for all sequences containing a significant Trypsin or Trypsin_2 domain using hmmsearch  and constructed domain architecture diagrams for each protein . Six domains were found to be represented in high abundance (≥ 10%) in the tryptome: DIM, ShK, Lustrin_cystein, Sushi, MAM and SRCR  of ein Fig. . Of the ein Fig. ”, 28 encRCR Fig. a. The DIN. vectensis, we searched for proteins with these same domain architectures in representatives from all domains of life . Two domain architectures were found to be present across taxa: those with only a trypsin domain, and those with a trypsin and a PDZ domain  both have at least 20. Surprisingly, there was little conservation in trypsin domain architecture across animals. The tryptome of N. vectensis had more trypsin domain architectures in common with other actiniarians (sea anemones) than with any other animal group; however, we still identified 3 trypsin architectures unique to N. vectensis that were absent event from Edwardsiella lineata (a representative of the genus sister to Nematostella). Two of these (NVJ_105271 and NVJ_199428) represent unique associations between trypsin and other conserved domains  and the other (NVJ_105548) exhibits a novel arrangement of trypsin and its associated MAM domains , followed by trypsins in the immune/coagulation group (N = 10), the “pancreatic” group  (N = 5), and intracellular trypsins (N = 2). Trypsins from the non-catalytic clade lack associated domains completely. Four trypsin-associated domains  were found in the immune/coagulation clades from both N. vectensis and H. sapiens, the CUB domain was found in chymotrypsins from both taxa, and the PDZ domain is restricted to the intracellular clade of trypsins in both taxa; surprisingly, there were no other domains found in common between N. vectensis and H. sapiens trypsins from the same clade  include secreted trypsins, membrane-bound trypsins, and trypsins with divergent sequence that have likely lost their catalytic function, suggesting that spatial and functional specialization has evolved multiple times in different lineages of trypsins.To understand the mechanisms generating trypsin diversity in ome Fig. . The tryN. vectensis are associated with trypsins  of the ShK domains in ins Fig. a. To detels Fig. b. Despitlianthus . What ro/82 of thsis Fig. b suggestN. vectensis tryptome have only a trypsin domain  encodes three trypsin domains, all of which form a monophyletic group suggesting this gene structure arose through tandem duplication of the trypsin domain  and trypsin-WSC (NVJ_105271), and one association was found only in Edwarsiidae (Nematostella + Edwardsiella): trypsin-Lustrin_cystein (NVJ_164017). The WSC domain is present throughout eukaryotes . These two associations represent extreme cases whereby trypsin diversity in N. vectensis arose through acquisition of both young (Lustrin_cystein) and old (WSC) domains.There is no relationship between the age of the domain and the origin of its association with trypsin Fig. b. Two trNVJ_19942 and trypN. vectensis  in our TEM or in situ hybridization results Figs. . Indeed,erm Fig. . This isendoderm . These oendoderm . We consN. vectensis  and two paralogs of Factor B , each of which was expressed in the endoderm (gastrodermis) of juvenile polyps [N. vectensis. One trypsin (NVJ_127465) was not reported in the single-cell dataset [N. vectensis [Beyond their roles in digestion and tissue remodeling, trypsins are an important component of the innate immune system. In vertebrates, immune trypsins play a role in blood coagulation and are part of the complement system which recognizes foreign particles . In symbe polyps . We founNVJ_12746 was not N. vectensis  of genes encoding secreted proteases enabled nuanced variation in the function of these secretory cells before the increase in anatomical diversity Fig. .Fig. 9Se2 and processed for transmission electron microscopy as described previously [N. vectensis [Adult polyps were immobilized for 10 min in 7.5% MgCleviously . Samplesectensis .N. vectensis, we first searched the JGI protein models (indicated throughout by NVJ_X) using the default settings with hmmsearch  and two target HMMs: Trypsin (PF00089) and Trypsin_2 (PF13365). This approach yielded 99 putative trypsin-domain containing proteins with an E-value ≤ 1e−05 [https://www.geneious.com) and manually examined models for duplicate predictions using the JGI genome viewer. Based on these analyses, we removed 27 sequences, resulting in a final set of 72 curated trypsin protein models .To identify trypsin-domain proteins from  ≤ 1e−05 . Where m ≤ 1e−05 . Based o ≤ 1e−05 , we founN. vectensis by searching for non-Trypsin domains in the amino acid sequences using hmmscan (HMMER 3.1b2) and the complete Pfam-A database . Hmmscan identifies regions of similarity between protein queries and domain models (protein profiles) derived from numerous proteins within the family from a range of animals [E-value ≤ 0.05 that were found in a protein containing a significant Trypsin (or Trypsin_2) domain. Domains that overlapped by ≤ 20% were both retained; when the overlap was > 20% the domain with the lower E-value was retained. In addition to domain analysis, we manually searched an alignment of the corrected set of trypsin protein models from N. vectensis for the conserved residues that comprise the trypsin catalytic triad (necessary for inferring protease activity): H-57, D-102, or S-195. Finally, we searched the corrected amino acid sequences for signal peptides and transmembrane domains using SignalP v4.1 [We examined the domain architecture of trypsin proteins from  animals . Followi animals , we ran alP v4.1  and TMHMalP v4.1 , respectCapitella teleta, Branchiostoma floridae, Homo sapiens), 10 cnidarians , three non-planulozoan animals , five non-metazoan eukaryotes  and a combined database of representative archeaea and bacteria . Protein models were predicted from transcriptome data previously for N. vectensis, E. lineata, A. pallida, A. elegantissima, A. alatina, A. vanhoeffeni, and P. carnea [R. renilla and C. cruxmelitensis were predicted from the transcriptome data reported by Kayal et al. [https://github.com/josephryan/2019-Babonis_et_al_trypsins).To characterize the origin of trypsin domain architecture, we used hmmscan with the same approach described above to identify and characterize trypsins from representatives across all domains of life . All trees were constructed using a maximum likelihood framework with RAxML and IQ-TREE [https://github.com/josephryan/2019-Babonis_et_al_trypsins).To understand the diversification of animal trypsins, we built a phylogeny using predicted proteins from  IQ-TREE –48. We uN. vectensis. The two remaining trypsin proteins (NVJ_23745 and NVJ_203589) were recovered using the Trypsin_2 HMM (note: the Trypsin_2 HMM recovered only 89% (64/72) of the curated trypsins). To understand the evolutionary relationships of these two proteins to the rest of the trypsin family, we generated another phylogeny using the same procedure as above and an alignment built using the Trypsin_2 HMM. This best tree recovered using the Trypsin_2 HMM is provided in Additional file B. floridae for ease of viewing and re-ran the full analyses. All tree files and alignment files are available on our Github site .Using the Trypsin HMM, we recovered 97% 70/72) of the curated trypsin proteins from  of the cN. vectensis has undergone lineage-specific expansion of trypsins or if the common ancestor of all cnidarians had an equally diverse tryptome, we built a phylogeny of trypsin proteins from cnidarians only using a subset of the proteomes listed above. Specifically, we used four species of anthozoans  and four medusozoans . We then pruned all non-Nematostella taxa from this tree using Phyutility v.2.2.6 [N. vectensis trypsins only. To examine the evolutionary history of ShK domains from N. vectensis, we used hmmsearch with the ShK HMM and a custom script (as above) to identify and align all ShK domains from the predicted proteome. We then used the approach described above to produce a phylogeny of ShK domains.To evaluate whether  v.2.2.6  to generAdditional file 1. Gland cells of the external ectoderm. a–c Mucus cells  in the tentacle ectoderm. d Zymogen cell  in the body wall ectoderm. White arrows point to cnidocytes, black arrowheads point to electron dense apical vesicles in cells adjacent to gland cells. a, b SEM, c, d TEM. Scale bars: black—5 µm, white—10 µm.Additional file 2. Phylogeny of Trypsin_2 domains across animals. Protein models for N. vectensis are shown. NVJ_203589 and NVJ_23745 were not detected by the trypsin HMM and do not appear in Fig. Additional file 3. Human trypsin domain architecture mapped on the animal trypsin phylogeny. Proteins with multiple trypsin domains are polyphyletic; in such cases, the diagram points to a single trypsin domain and the position of the other trypsin domains is indicated by symbols. N. vectensis proteins characterized by in situ hybridization are indicated by arrows (this study), †[study), †, or *[12Additional file 4. Excel file tabulating: presence/absence of signal peptides and transmembrane domains in N. vectensis trypsins, amino acid sequences for trypsin catalytic domains for all taxa, pfam IDs for all domains, and a summary of single-cell expression of trypsins published previously [eviously .Additional file 5. All trypsin domains are encoded by multiple exons in N. vectensis (excluding the NVJ_128003 and NVJ_216003) but many of the associated domains are encoded by a single exon (indicated by triangle). Domains that span intron/exon boundaries by ten or fewer nucleotides were considered to be encoded by a single exon.Additional file 6. Trypsin protein domain architectures from all taxa examined in this study.Additional file 7. Trypsin protein IDs from all taxa examined in this study.