UBE3A is an E3 ubiquitin ligase encoded by an imprinted gene whose maternal deletion or duplication leads to distinct neurodevelopment disorders Angelman and Dup15q syndromes. Despite the known genetic basis of disease, how changes in copy number of a ubiquitin ligase gene can have widespread impact in early brain development is poorly understood. Previous studies have identified a wide array of UBE3A functions, interaction partners, and ubiquitin targets, but no central pathway fully explains its critical role in neurodevelopment. Here, we review recent UBE3A studies that have begun to investigate mechanistic, cellular pathways and the genome-wide impacts of alterations in UBE3A expression levels to gain broader insight into how UBE3A affects the developing brain. These studies have revealed that UBE3A is a multifunctional protein with important nuclear and cytoplasmic regulatory functions that impact proteasome function, Wnt signaling, circadian rhythms, imprinted gene networks, and chromatin. Synaptic functions of UBE3A interact with light exposures and mTOR signaling and are most critical in GABAergic neurons. Understanding the genome-wide influences of UBE3A will help uncover its role in early brain development and ultimately lead to identification of key therapeutic targets for UBE3A-related neurodevelopmental disorders. UBE3A becomes silenced on the paternal allele due to the paternal-specific expression of an anti-sense transcript (UBE3A-ATS) originating from the unmethylated allele of SNRPN. UBE3A is the imprinted gene implicated in the maternal-specific effects of 15q11.2-q13.3 deletion or duplication disorders  prevents UBE3A catalytic inhibition by disrupting protein kinase A (PKA) phosphorylation inhibition toward itself and other substrates , which are critical for learning and memory, rhythmic activity, and sleep  with a unique 3′ untranslated region containing microRNA (miRNA) binding capabilities. Ube3a1 knockdown increased activity of miR-134, which regulates plasticity, suggesting that Ube3a1 lncRNA acts as a competing endogenous RNA, or “RNA sponge” for miR-134. In rat neurons, Ube3a1 transcript sequestered miRNAs from the miR379–410 cluster, which contains miR-134, thereby regulating translation of miR379–410 targets in dendrites. During development, increased neuronal activity and subsequent increased Ube3a1 RNA levels buffered miR379–410 activity allowing progression to spine maturation (Valluy et al., Ube3a1 lncRNA may help regulate the spatiotemporal control of mRNA translation within dendrites. Many questions remain about the regulation and function of Ube3a1 including its imprinting pattern, if the paternally expressed UBE3A-ATS is required for Ube3a1 expression, and its relevance in human AS.Recently, Finally, we have begun to explore the chromatin-related genome-wide effects of UBE3A dysregulation in human brain and neurons. We previously observed that elevated UBE3A in Dup15q syndrome had widespread effects on the neuronal methylome that converged in the dysregulation of chromatin and synaptic gene pathways (Dunaway et al., via the UBE3A-ATS and direct influences on chromatin dynamics and genome-wide DNA methylation including regulation of other imprinted genes. Understanding the functions of UBE3A in a neurodevelopmental context will improve the study of its associated disorders and may lead to enhanced therapeutic options at key targets and pathways (Figure UBE3A has long been linked with ASD and is causal in AS etiology, however how UBE3A leads to disease phenotypes is not well understood. More recently, UBE3A genome-wide functions may enlighten additional gene pathways relevant to neurodevelopmental disorders Table . Recent s Figure .SL, JL, and DS all participated in the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation.Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis.FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-β sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments.These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.The online version of this article (10.1007/s11095-019-2599-2) contains supplementary material, which is available to authorized users. Human factor VIII (FVIII) is an essential plasma glycoprotein in the blood coagulation cascade, serving as a co-factor for factor IXa in the conversion of factor X to factor Xa ,2. A defRecombinant protein technology has generated recombinant FVIII (rFVIII) products to treat haemophilia A by protein replacement. Products differ mainly in glycosylation  and the All protein-based drugs, including FVIII, bear a certain risk to aggregate during manufacturing and shelf storage, and a susceptibility to mishandling during treatment ,11. In t2-A1-a1-A2-a2-B-a3-A3-C1-C2-COOH. Different intra- and extracellular processing of the B-domain causes different heterodimeric molecular species to circulate in plasma. Thus, FVIII contains a constant-sized light chain (LC) (a3-A3-C1-C2) and a heavy chain (HC), minimally composed of the A1-a1-A2-a2 domains, but variable in size due to the presence of some or all of the adjacent B-domain  on protein stability and aggregation. We studied the structural characteristics of the B-domain, and compared FL-rFVIII, pdFVIII and purified rFVIII molecular species with variable B-domain content with regard to their aggregation behaviour upon physical stress. Based on our observations, we built a schematic model of FL-rFVIII and BDD-rFVIII aggregation and suggest a new role for the B-domain and molecular heterogeneity in ensuring the stability of the FVIII molecule.Human FL-rFVIII (0.23 mg/ml) used was an intermediate material from a production line for a commercial rFVIII product expressed in Chinese hamster ovary (CHO) cells and was provided by Baxalta Innovations GmbH, a Takeda company, Vienna, Austria. Vials of historical lots of commercially available human rFVIII as well as commercially available lyophilised human plasma FVIII product were provided by Baxalta Innovations GmbH, a Takeda company, Vienna, Austria. Chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA.Von Willebrand factor (VWF)-free pdFVIII was purified from a commercially available lyophilised plasma FVIII product, which was provided by Baxalta Innovations GmbH, a Takeda company, Vienna, Austria. Multiple vials of the commercially available lyophilised human pdFVIII product were reconstituted and pooled to achieve a homogeneous starting material. FVIII was captured on an anti-FVIII affinity column and further processed using strong cation exchange chromatography. Another purification step on a strong anion exchange resin was performed for buffer exchange and to increase the FVIII concentration.rFVIII molecular species with different degrees of B-domain truncation were isolated from an intermediate material of a commercially available human FL-rFVIII product expressed in CHO cells, which was provided by Baxalta Innovations GmbH, a Takeda company, Vienna, Austria. A high-resolution anion exchange chromatography step with a flat gradient was used to pre-separate the entities. Pools with enriched sub-species were generated and further purified by preparative size-exclusion chromatography (SEC), followed by concentration and buffer exchange on a strong anion exchange resin.SDS-PAGE was carried out using Novex NuPAGE . Samples were mixed with 0.5 M iodoacetamide and incubated for 30 min at 37°C. NuPAGE lithium dodecyl sulfate (LDS) sample buffer and NuPAGE reducing agent were added to the reaction mixture and incubation was continued for 30 min at 37°C. Samples and Precision Plus unstained protein standard  were loaded onto a 7% Tris-acetate mini gel. Electrophoresis was performed for 90 min at 150 V. Protein bands were visualised with SilverQuest silver staining kit (Thermo Fisher Scientific).BioAnnotator from Vector NTI Advance 11  was used to calculate the average hydropathicity of the B-domain.2 and NaCl). The reaction was stopped with ice-cold phosphate buffer, pH 2.3, containing 100 mM Tris(2-carboxyethyl)phosphine and 3.3 M urea, and by subsequent snap freezing in liquid nitrogen. Samples were digested using a high-performance liquid chromatography (HPLC) column  packed with pepsin-agarose from porcine gastric mucosa (Sigma-Aldrich) and desalted on a C18 pre-column (ACE). Peptic peptides were subjected to liquid chromatography coupled to mass spectrometry (MS) using a HALO C18/1.8 μm column . Peptides were eluted by an acetonitrile gradient and analysed on an Orbitrap XL MS . Peptic peptides were identified by three independent liquid chromatography-MS/MS analyses of a non-deuterated sample using the same procedure as for the deuterated samples.Local amide hydrogen/deuterium exchange (HDX) kinetics were followed after 3, 10 and 30 s; 2, 10 and 60 min; 3 h; and 3 days of incubation. All HDX reactions were performed at 22°C, except for the 3-s reaction (6°C). Human rFVIII containing 70% B-domain (B70-rFVIII) was mixed with deuterated buffer  on an automated coagulation analyser . The reference standard was commercially available FL-rFVIII , calibrated against the World Health Organization international standard.FVIII samples were dialysed against phosphate-buffered saline containing 0.9 mM CaCl2 and 0.5 mM MgCl2. To ensure reproducibility, all experiments were performed at least twice.All FVIII samples were dialysed against phosphate-buffered saline containing 0.9 mM CaClAll FVIII samples with protein concentrations of either 0.122 μM for high-performance size-exclusion chromatography (HPLC-SEC) or 0.61 μM for dynamic light scattering (DLS) were incubated at 25, 30, 35, 40, 45 or 50°C for 20 h in polystyrene microplates  covered with plate sealers in a Synergy H4 Hybrid Reader  with 20 s medium shaking every 10 min. Samples were subsequently frozen at −80°C until analysis.All FVIII samples (0.122 μM) were incubated at 45°C for 24 h in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker . Samples were withdrawn after various time intervals and immediately frozen at −80°C until HPLC-SEC analysis.To prepare seeds, FVIII samples (0.122 μM) were incubated for 2, 5, 8 or 18 h at 45°C in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan). Native FVIII samples (0.122 μM) were mixed 1:1 with the corresponding seeds and time-dependent aggregation at 45°C was initiated. Samples were stored at −80°C until HPLC-SEC analysis.Samples (0.244 μM) were hand agitated for 10 min in a disposable Omnifix syringe . Shear stress was induced by injecting the solution through ‘winged infusion sets with needle protection’  from Terumo Europe, Leuven, Belgium, after which all samples were stored at −80°C until flow cytometry-based particle analysis.DLS was performed using a Malvern NanoZetasizer ZSP . Samples (0.244 μM) were centrifuged  at 10,000 rpm for 5 min and filled into a ZEN0040 disposable microcuvette. Operation temperature was set at 25°C with an equilibration time of 2 min. The angle was set to 173° backscatter to determine the hydrodynamic diameter of a protein and thus, the effective size of proteins. A minimum of three runs per sample were measured to obtain an average result.2, 400 mM NaCl and 0.05% NaN3, pH 7.0. A sample volume of 100 μl (0.122 μM protein concentration) was mixed with 3 μl Thioflavin T  and subsequently loaded onto the column. To monitor the elution of the protein with fluorescence detection, the excitation and emission wavelengths were set to 280 nm and zero order, respectively. ThT fluorescence was monitored with 440-nm excitation and zero-order emission. Peaks eluting with the void volume (retention time 18.0–21.2 min), with retention times of 21.2–27.0 min and 27.0–43.0 min, were designated as soluble protein aggregates, oligomers and monomers, respectively. The amount of each was calculated as a percentage of the total area of all peaks in the chromatogram. ThT binding was calculated as the ratio between ThT and intrinsic protein fluorescence signals. The protein-based gel filtration standard (Bio-Rad) was analysed in between samples to monitor optimal column performance. All samples were analysed in random order.HPLC-SEC was performed using a 7.8 × 300 mm TSKgel G4000SWxl column  and a 6 × 40 mm TSK guard column (Tosoh Bioscience) coupled to an HPLC 1260 infinity system . SEC was carried out under isocratic conditions at a flow rate of 0.3 ml/min using a buffer consisting of 50 mM Tris-HCl, 5 mM CaClkoligo [h−1]) were derived by fitting data to the one-phase association model according to: y = y0 + (plateau-y0)*(1-exp[−koligo*x]); y = oligomer amount (%); x = time (h); y0 = y value when x is zero; plateau = y value at infinite times. Aggregation rates (kagg [h−1]) were derived by fitting data to the Boltzmann sigmoidal model according to: y = ymin + (ymax-ymin)/(1 + exp[(x1/2-x)/(1/kagg)]); y = aggregate amount (%); ymin = y during lag phase; ymax = y after aggregation ended; x = time (h); x1/2 = time at half-maximum y . Kinetic rate constants for oligomerisation . The HC of B70-rFVIII, B20-rFVIII and BDD-rFVIII exhibited apparent molecular weights of 150, 110 and 90 kDa on the SDS-PAGE gel, due to varying amounts of B-domain  of FL-rFVIII, B70-rFVIII, B20-rFVIII and BDD-rFVIII were 0.13 ± 0.02, 0.12 ± 0.05, 0.35 ± 0.24 and 0.70 ± 0.24 h−1, respectively. Aggregation curves had sigmoidal shapes with varying aggregation rates (kagg), depending on B-domain content  = 0.11 ± 0.00 h−1, kagg(B70-rFVIII) = 0.18 ± 0.02 h−1, kagg(B20-rFVIII) = 0.21 ± 0.07 h−1 and kagg(BDD-rFVIII) = 0.21 ± 0.08 h−1. Aggregation pathways of FL-rFVIII and BDD-rFVIII diverged the most  = 0.24 ± 0.04 h−1 and kagg(pdFVIII) = 0.15 ± 0.03 h−1. Consistent with these oligomerisation and aggregation rates, the loss of monomers in BDD-rFVIII was faster than in FL-rFVIII and pdFVIII . BDD-rFVIII had a significantly higher concentration . The sub-visible protein-containing particle concentration of B-domain truncated molecular species depended on the B-domain content of the test species and was higher for B20-rFVIII  than for B70-rFVIII  , 70% (B70-), 20% (B20-) or 0% B-domain were the main rFVIII species found in FL-rFVIII and pdFVIII in the present study. Jankowski Interestingly, FL-rFVIII produced in a baby hamster kidney cell line exhibits a slightly different protein profile regarding band intensity and migration level than CHO cell-derived FL-rFVIII and pdFVIII . In geneFL-rFVIII and pdFVIII demonstrated the lowest propensity to form aggregates. The aggregation tendency increased with decreasing B-domain content of the rFVIII molecular species. Detailed time-dependent analysis of oligomerisation and aggregation under thermal stress revealed diverging pathways for different FVIII samples. The slow oligomerisation of thermally stressed FL-rFVIII and pdFVIII nearly inhibited aggregation. The kinetics of oligomer and aggregate formation were more rapid with BDD-rFVIII. ThT-positive cross-β sheet-rich structures were detected in thermally induced BDD-rFVIII oligomers and aggregates, but less so or not at all in B-domain-containing FVIII. It is likely that cross-β sheets in BDD-rFVIII oligomers trigger extensive aggregation and facilitate homologous seeding of BDD-rFVIII aggregation, but not in the other FVIII samples.Based on these observations, we built a schematic model describing the diverging pathways of BDD-rFVIII and FL-rFVIII oligomer and aggregate formation Fig. . While tSupported by a previous study in which the B-domain had little to no effect on the overall FVIII secondary structure in solution as measured by far UV circular dichroism spectroscopy , the preHeterogeneity, as shown in FL-rFVIII and pdFVIII, causes lower sequence similarity between FVIII species. Sequence diversity in proteins has already been demonstrated in previous studies as being essential in reducing aggregation susceptibility and seeding processes. Wright and co-workers’ findings on the multi-domain protein titin showed that the co-aggregation efficiency among different domains decreases markedly with decreasing sequence identity. Furthermore, they claimed that maintaining a low sequence identity between proteins is an important evolutionary characteristic that strongly inhibits aggregation in the crowded environment of a living system . SimilarUsing agitation and shear stress to mimic mishandling of FVIII therapeutic products, and facilitation of the interaction of proteins with silicone oil, which is often present on the interior surfaces of syringes ,51–53, sN-glycosylation sites are distributed within the B-domain


Epigenetics is a rapidly growing field in drug discovery. Of particular interest is the role of post‐translational modifications to histones and the proteins that read, write, and erase such modifications. The development of inhibitors for reader domains has focused on single domains. One of the major difficulties of designing inhibitors for reader domains is that, with the notable exception of bromodomains, they tend not to possess a well‐enclosed binding site amenable to small‐molecule inhibition. As many of the proteins in epigenetic regulation have multiple domains, there are opportunities for designing inhibitors that bind at a domain–domain interface which provide a more suitable interaction pocket. Examination of X‐ray structures of multiple domains involved in recognising and modifying post‐translational histone marks using the SiteMap algorithm identified potential binding sites at domain–domain interfaces. For the tandem plant homeodomain–bromodomain of SP100C, a potential inter‐domain site identified computationally was validated experimentally by the discovery of ligands by X‐ray crystallographic fragment screening. Genetic information is contained in chromosomes which are made of chromatin, principally a combination of DNA and histone proteins. The repeating unit of chromatin is the nucleosome, which is composed of DNA wrapped around an octamer of histone proteins. In eukaryotes each nucleosome consists of two copies each of histone 2A (H2A), histone 2B (H2B), histone 3 (H3), and histone 4 (H4). N‐Terminal histone tails protrude from the nucleosome and post‐translational modification (PTM) of these tails control access to the nucleosomal DNA. PTMs of histone tails therefore play an important role in regulating gene expression. This process of epigenetic regulation is dynamic, and various writer‐ and eraser‐enzyme families are involved in adding and removing post‐translational modifications to histone tails. Although many different modifications of histones are known, acetylation and methylation of lysine found on the H3 and H4 tails are amongst the most extensively studied. The combination of PTM on histone proteins has been shown to be important in transcriptional regulationA second aspect of the hypothesis states that “modifications on the same or different histone tails may be interdependent and generate various combinations on any one nucleosome.” The predicted interdependence of histone tail modifications has been validated by the discovery of proteins that recognise multiple marks through multiple domains,There have been many recent reports of inhibitors for reader,As well as the development of inhibitors for pharmaceutical use, there have been a number of inhibitors designed to be used as chemical probes.Despite these successes, there are still many families of protein domains involved in reading, writing, or erasing histone tail modifications with very poor inhibitor coverage. Of these many are predicted to be targets with intrinsically low ligandability.There are increasing reports of X‐ray crystal structures of multiple domains involved in recognising and modifying post‐translational marks on histone tails. These include examples of multiple reader domains from the same protein,We used SiteMapSiteMap works by creating a grid of points separated by 1 Å. Each point is then evaluated to determine whether it overlaps with any protein atoms, points that overlap with protein atoms are removed. Further points are removed if they are insufficiently enclosed by the protein or if they have a too small van der Waals interaction energy with the protein. Points are then grouped into sites, and the enclosure, size, and hydrophilicity of the site calculated. These values are combined into a single scoring function called the SiteScore.The structures used in this study were identified using ChromoHub.The domain combinations in Figure SiteMap was used to calculate a SiteScore for every multi‐domain combination identified. In general it was possible to identify potential binding sites in most structures, and in some cases these were found at domain–domain interfaces Figure . SpecifiKD 431 μm) and a K36‐trimethylated H3.3 peptide, residues 19–42 (H3.319–42K36me3 KD 56 μm).https://www.rcsb.org/structure/4N4G) with SiteMap identified the BRD as the most ligandable site (SiteScore=1.01). However Wen et al. described this BRD as “unlikely to be a histone acetyl‐lysine‐binding module” due to the presence of a tyrosine residue at the position where asparagine is commonly found in acetyl‐lysine binding BRDs.The structure of the BRD‐PWWP domain of tumour suppressor ZMYND11 has been solved with a bound H3.3 peptide Figure .39 Wen eA binding site at the BRD‐PWWP interface where H3.3 S31 binds is identified as having a SiteScore of 0.85. This is at the lower end of what would be considered ligandable.Our structure set contains seven Tudor domain containing proteins. All seven of these examples contain a multiple Tudor domain, with one example (UHRF1) containing a tandem Tudor and a PHD. In most of these cases, only one of the multiple Tudor domains contains the arrangement of aromatic side chains commonly known as an aromatic cage that is typically found in Tudor domains and plays a crucial role in binding to methylated lysines.The tandem Tudor domain of DNA repair factor TP53BP1 (tumour protein p53 binding protein 1) is an illustrative example of the case described above. Our analysis by SiteMap reveals a site with a SiteScore of 0.98 containing the aromatic cage of N‐terminal Tudor and extending to an area at the interface of the two Tudor domains, Figure  A. This https://www.rcsb.org/structure/3DLM) does not show a well formed aromatic cage in any of the three Tudor domains. However, closer inspection reveals that the second and third Tudor domains contain suitable residues to form an aromatic cage, the formation of which could be induced by binding of a tri‐methylated lysine. The first Tudor domain also contains two aromatic residues but this potential aromatic cage is blocked by a lysine side chain. The triple Tudor domain contains a potential ligand binding site between the second and third Tudor domain  and low hydrophilicity (0.88).The histone methyltransferase SETDB1 contains three contiguous Tudor domains. The available crystal structure  identifies the H3 binding site as having a site score of 0.95. Although this site is large (size score 175) it is as well quite open, with an enclosure score of 0.61. Although it would be considered difficult to design a conventional ligand for this type of pocket, this pocket would be a suitable candidate for techniques designed to target protein–protein interactions.https://www.rcsb.org/structure/2KWJ identifies a site with a mean SiteScore of 0.81. Similarly to KAT6A, the site identified in DPF3 is large and open, with a mean size score of 72 and a mean enclosure of 0.59. This suggests that a PHD–PHD is more ligandable target than a single PHD  of PHD–PHDs. The structures of DPF3 and KAT6A show that the tandem PHD module interacts with residues 1–14 of H3, with H3 binding at the domain–domain interface. Tandem PHDs have been shown to be acetyl‐lysine binders; this acetyl‐lysine recognition takes place at a site formed by the domain–domain interface.There are 20 structures containing a PHD and another domain in our structure set. The most common partner domain being a C‐terminal BRD (8 examples). PHD–BRDs have been shown to act together in substrate recognition.Amongst the group of structures with a globular arrangement of the PHD and BRD is SP100C. Analysis of this structure by SiteMap reveals the presence of a novel potential ligand binding site at the interface of the PHD and BRD domains Figure  B. This m.In order to further investigate this newly identified potential binding site of SP100C, we pursued structural and ligand binding studies on the tandem BRD–PHD construct.1 binds deeper into the pocket than aniline 2. It exploits hydrophobic interactions with surrounding residues. Oxadiazole 1 is positioned to form a hydrogen bond with the backbone carbonyl of residue I871 via its primary amine  PEG 20000 and placing the drops over 20 μL reservoir solution. Crystals appeared in 6–7 days. Crystal soaking was performed by liquid droplet transfer using a TTPLabtech Mosquito® HTS. Ethylene glycol was added for cryoprotection using the Mosquito® to a final concentration of 25 % (v/v) . The SP100C crystals diffracted to 1.6–2.0 Å resolution in space group C2, with typical unit‐cell parameters a=127.4 Å, b=45.4 Å, c=84.0 Å, β=102.0° and with one SP100C molecule in the asymmetric unit.SP100C crystals were grown in SWISSCI 3 Lens crystallisation sitting‐drop plates at 4 °C by mixing 50–100 nL of 10 mg mLX‐ray diffraction data were collected on beamline I04‐1 at Diamond Light Source and were processed using the Diamond autoprocessing pipeline, which uses xia2,1 and 2 can be accessed using PDB IDs https://www.rcsb.org/structure/6G5N and https://www.rcsb.org/structure/6G5P, respectively.The atomic coordinates and structure factors for the crystal structures of SP100C with compounds The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Oryza sativa), a Thai black rice, contains polyphenolic compounds which have antioxidant properties. The objective of this study was to investigate physicochemical properties of Hom Nil rice flour (HN) and its application in gluten free bread by using Hom Mali 105 rice flour (HM) as the reference. The results demonstrated that HN flour had significantly higher average particle sizes (150 ± 0.58 µm), whereas the content of amylose (17.6 ± 0.2%) was lower than HM flour . Furthermore, HN contained higher total phenolic compounds (TPC) (2.68 ± 0.2 mg GAE/g flour), total anthocyanins (293 ± 30 mg cyanidin-3-glucoside/g flour), and the ferric reducing antioxidant power (FRAP) (73.5 ± 1.5 mM FeSO4/g) than HM flour (TPC = 0.15 mg GAE/g flour and FRAP = 2.24 mM FeSO4/g flour). In thermal properties, the onset temperature (To), the peak temperature (Tp) and the conclusion (Tc) temperature of HN flour were similar to the values of HM flour. However, HN flour had lower enthalpy change (ΔH) than HM flour. The results showed that HN flour had lower swelling power and higher solubility than HM flour at the temperature between 55 °C and 95 °C. In pasting properties, HN flour also showed lower peak, trough and breakdown viscosity than HM flour. In addition, the bread samples prepared by HN flour had higher value of hardness and lower value of cohesiveness than the bread prepared from HM flour. Taken together, the findings suggest that HN flour could be used as an alternative gluten-free ingredient for bread product.Hom Nil ( It has been reported that overconsumption of dietary carbohydrate has been associated with increased risks of obesity and metabolic diseases . Rice is quality . The com quality . In food quality . In addi quality . Black r quality .Oryza sativa), a Thai black rice, has been reported as a good source of antioxidants, which contains phenolic compounds [Hom Nil  were randomly purchased from a local farmer community of Yasothorn Province, Thailand during May–June 2016. Each batch of the sample was prepared by pooling and mixing of the three different areas of purchasing. Folin & Ciocalteu’s phenol reagent, gallic acid, 2,4,6-Tri (2-pyridyl)-s-triazine (TPTZ), amylose from potato and other chemical reagents were purchased from Sigma-Aldrich .Rice flour was ground using dry-milling method with an Alpine Pin Mill  with voltage 50 Hz and speed 4800 rpm. The percent yield of Hom Nil (HN) and Hom Mali 105 (HM) flour was 97.5%. The rice flour was screened into particle sizes of 125–180 µm using sieve of 125 µm and 180 µm sizes. The flour was stored in sealed ethylene-vinyl alcohol copolymer (EVOH) bags at −20 °C until further analysis. The moisture contents of HN and HM and RB were 11.9% and 12.9%, respectively.The particle size distributions of flour were measured using a Laser particle size analyzer  with dry dispersion module according to Kraithong et al. [The content of amylose in rice flour was determined according to a modified method of AACC . Rice flλ510 − Aλ700) pH 1.0 − (Aλ510 − Aλ700) pH 4.5 and total monomeric anthocyanins were expressed as cyanidin-3-glucoside.The extraction was performed according to the modified method of Shen et al. . In brie3 90 µL of FRAP reagent was mixed with 10 µL of the rice extract at a ratio of 10:1:1. The reaction mixture was incubated in the dark at room temperature for 30 min and the absorbance was read at 595 nm. The FRAP value was calculated from the calibration curve of FeSO4. The assay of ferric reducing antioxidant power (FRAP) of flour was determined according to Benzie; Strain  with min®, Selb, Germany) according to a previous study [The measurement of differential scanning calorimetry (DSC) for degree of gelatinization on rice flour samples was performed using a differential scanning calorimeter (Netzsch DSC 204F1 PhoenixThe sample (0.25 g) was heated in 10 mL distilled water in a water bath at 55, 65, 75, 85, and 95 °C for 30 min with constant mixing and then cooled to room temperature. The samples were centrifuged at 2000 rpm for 20 min. The precipitated and the supernatant part were dried at 105 °C and weighed in order to calculate swelling power and water solubility of rice flour according to the modified method of Leach et al. .The pasting property of rice flour (3 g flour based on 14% moisture in 25 g distilled water) was determined using a Rapid Visco Analyser  according to a previous study . The floThe rice bread from HN and HM flour was prepared according to a previous study . The fol3/g.Texture analysis was measured according to a modified method of Kang et al.  using a p < 0.01 was considered statistically significant.Data were expressed as mean ± standard error of mean (SEM) with three or five independent batches, in which duplicates were tested for each assay. The statistical significance of the results was evaluated using an independent t-test to analyze the mean and the differences between the means. The analysis of variance carried out with Duncan’s multiple test was performed to conduct significant difference for swelling power and solubility. The value of As shown in Previous studies demonstrated that the large particle size of rice flour (100–200 µm) provided preferable bread properties with less hardness and high specific volume ,28,29. TThe amylose content of HN was significantly lower than HM b. Accord4/g flour and 293 ± 30 mg cyanidin-3-glucoside/g flour, compared to those in the HM flour (TPC = 0.15 mg GAE/g flour and FRAP = 2.24 mM FeSO4/g flour). The current study indicates that pigmented rice demonstrated higher values of phenolic compounds and antioxidant activity than white rice. These findings are similar to previous studies [Total phenolic content (TPC) and ferric reducing antioxidant power (FRAP) are shown in  studies ,40 that  studies . Therefoo), peak (Tp) and conclusion (Tc) were similar to those of HM. In general, differential scanning calorimetry (DSC) has been widely used to evaluate the thermal properties of starch, especially starch gelatinization. The endotherm for disordering of amylopectin crystallites is clarified by onset (To), peak (Tp) and conclusion (Tc) temperatures. The values of gelatinization temperature of HN are the similar range to other rice cultivars such as brown rice [As shown in own rice  and glutown rice . Howeverown rice . In contown rice . Accordiown rice ,45,46,47own rice . Furtherown rice . FurtherThe result showed that swelling power (SP) and solubility (SB) of all rice flours significantly increased with an increase in temperature from 55 °C to 95 °C a,b. HN hDuring starch gelatinization, the hydrogen bonds between the hydroxyl groups in the double helices of starch molecules are disrupted and allowed the hydroxyl groups to form new hydrogen bonds with water molecules, resulting in the swelling of starch granules. This increases the accessibility of starch molecules to leach out from the inner part of the granules and then increases the solubility of starch molecules ,50. The The pasting characteristics of the flour are shown in Texture profiles and specific volume of HN and HM bread are shown in 3/g for HN and 2.45 ± 0.00 cm3/g for HM, which consistent with a previous study reporting that preferable values ranged 2.35–2.41 cm3/g [The bread crumb appearance of gluten free bread from HN and HM are shown in 41 cm3/g .Hom Nil rice flour (HN) contained higher total polyphenolic and total anthocyanins as well as antioxidant activity than HM flour. The larger particle size and lower amount of amylose content was found in HN. In addition, HN flour demonstrated lower peak, trough and breakdown viscosity when compared to HM flour. Moreover, HN flour had lower swelling power and higher solubility than HM flour. Finally, the bread prepared by HN flour had higher value of hardness and chewiness, whereas it demonstrated lower value of cohesiveness, adhesiveness and springiness than the bread prepared from HM flour. These findings might be related to low amylose content and large particle size of HN flour. It suggests that HN could be an alternative gluten-free ingredient for bread products.


Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent β-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains.Uropathogenic  E. coli must resist mechanical forces to remain attached to the epithelium. Here the authors use single-molecule force spectroscopy to demonstrate a hierarchy of mechanical stability among the pilus domains and show that the oxidoreductase DsbA also acts as a folding chaperone on the domains.The pilus type 1 of uropathogenic  Escherichia coli (UPEC), one of the most common and recurrent infections1, bacteria use long appendages called pili type 13 to attach to cells of the bladder epithelium. For successful attachment, the mechanical integrity of the pilus is crucial. The pilus is composed of four different subunit types, FimA-FimF-FimG-FimH, and it is believed that all subunits and particularly their intermolecular interactions, play an important role in the attachment of the bacterium by providing mechanical resistance to the whole pilus. However, we still do not have a complete quantitative understanding of the intermolecular interactions responsible for the mechanical design of the pilus.Bacteria initiate infection by mechanical anchoring to tissues. In the case of uropathogenic 4. Thousands of FimA subunits form the pilus rod, a helical structure with spring-like properties5 connected to the tip fibrillum composed by FimF-FimG-FimH  to investigate the mechanical interactions and formation of the pilus domains. We find a hierarchical mechanical organization of the pilus, in which the resistance of the subunits to force decreases from the pilus rod to the tip. We have complemented the AFM measurements with Steered Molecular Dynamics (SMD) simulations, which give an atomic-level view of the unfolding process. This extraordinary resistance is achieved by a folding process of pilus subunits assisted by DsbA and FimC. We monitor this process on single Fim domains confirming the previously described process22, results in an autonomously folded domain with no disruption of the original hydrophobic interaction. In Fig. 23 2, with X being H, G, F and A, or the array F-G-H.For our AFM measurements, we designed polyprotein constructs containing the pilus type 1 subunits, including the complementing β-strand from the preceding domain in the pilus chain. Given that this β-strand does not establish a covalent bond, in our constructs, we linked the β-strand to the preceding subunit through a flexible linker Fig. . This β- 23 Fig. . We gene−1. The AFM force-extension experimental data is characterized by a typical sawtooth pattern where each peak corresponds to the unfolding of one domain, except the last one, which corresponds to the detachment of the protein from the cantilever. In all the measurements, we identified four identical peaks corresponding to the unfolding of the I91 domains. The additional peak observed in each trace corresponds with the unfolding of the self-complemented Fim domains  vs unfolding force plots for each pilus domain. To estimate ΔLc, we fitted the peaks using the worm-like chain model of polymer elasticity24 , which is in agreement with the theoretical length of about 44 nm estimated by considering 0.4 nm per residue and 5 nm for folded domain26. The measured length corresponds to the length of the protein in the oxidized state, i.e., with the disulfide bond formed being unbreakable at these forces. Next in the chain is FimF for which we estimated an unfolding force value of 418 ± 4 pN and a contour length of 43 ± 2 nm, again corresponding to the length of the oxidized domain. For FimG, we obtain 431 ± 4 pN for unfolding force and 40 ± 2 nm for contour length, consistent with our previous report27. Finally for FimH, we determined two values, corresponding to the two subdomains that compose FimH , and the lectin subdomain (FimHL). For FimHP the rupture force is 362 ± 6 pN and 38 ± 1 nm of contour length, and for the FimHL, we measured an unfolding force of 130 ± 5 pN and a contour length of 40 ± 2 nm. Interestingly, we collected traces displaying an intermediate. Now FimHL is splitted into a small peak of 6 ± 1 nm and 98 ± 8 pN, preceding a bigger one of 36 ± 1 nm and 107 ± 5 pN of force, which are treated individually . We attribute this force to the rupture of the β-strand interaction, confirming the high mechanical stability of bacterial adhesion proteinsimH Fig. , which aP. The final value for FimHL subdomain does not correspond to β-strand complementation rupture since this domain is covalently linked to FimHP. These values establish a hierarchical mechanical design, making the pilus domains mechanically weaker as they are closer to the tip 2 to be tested in the AFM. Each domain is in the oxidized state and was β-strand-complemented as we did with the individual domains. The limitation of this particular polyprotein construct is that the unfolding of multiple domains may compete with detachment from cantilever and/or surface, due to the high mechanical resistance of each domain. To minimize such possibility, we perfomed the experiments in the force-ramp mode. In this mode, the force is linearly increased from −10 to 400 pN at a constant loading rate of 10 pN s−1, in a totally controlled way by the feedback loop. The unfolding forces of all domains are lower and the full unfolding can be captured more easily29.In the case of the array FimF-FimG-FimH, they do not seem to establish interactions beyond the β-strand complementation. However, other than the crystal structure, there is no experimental evidence that rules out the possibility of quaternary contacts. In order to investigate such possibility, we constructed a polyprotein with chained domains (I91)L with step size of 33 ± 1 nm and unfolding force of 62 ± 4 pN. Lower forces than those in force extension mode are expected given the lower loading rate. Following this, we detect the four equal I91 domains. The more stable domains FimHP, FimF, and FimG unfold later at forces beyond 200 pN. FimHP unfold before which can be distingushed by the step size of 32 ± 1 nm and unfolding force of 238 ± 7 pN. Following this, we have the unfolding of FimG and FimF. These domains can also be distinguished by the step size; 37 ± 1 nm for FimF and 33 ± 1 nm for FimG  structures for the four Fim domains, in each case covalently linking the domain to the donated β-strand of the preceding one with the addition of the same linker as used in experiments. The proteins were placed in a simulation box with explicit TIP3P water and salt ions. After equilibration, the ends of the protein were stretched at a rate of 1 m sD structu9. The SecYEG pore is about 6 nm long and <2 nm in width. This implies that each preprotein has to traverse the pore in an extended state before reaching the periplasm, which requires the preproteins to be reduced31. The proteins reach the periplasm where interaction with DsbA and FimC occurs 2-FimG-(I91)2 to monitor the complete unfolding and refolding process of the FimG subunit using its oxidized and reduced form in the absence and in the presence of the FimC protein  and reduced FimG (FimGSH-SH) is shown, both in the presence and in the absence of the chaperone FimC. As we increase the quenching time, the probability of refolding increases for FimGS-S. Our FimG domain is self-complemented with the donor β-strand of FimF, therefore, it may be able to fold also in the absence of an external help. In the presence of FimC, the probability of refolding of FimGS-S is higher, becoming noticeable from 30 to 60 s of quenching time . This finding correlates with the force-extension data obtained for reduced and oxidized FimG, where the reduced protein showed lower mechanical stability than the oxidized one.We also estimated the unfolding rates of reduced and oxidized FimG at 300 pN. The graph shown in Supplementary Fig. 19. To measure the refolding of FimG in the presence of DsbA, we performed force-clamp experiments following a similar protocol than for FimC, but using DsbA to monitor oxidation of disulfide bonds , the force was again quenched to 0 pN for 45 s, before repeating the same force protocol. The resulting force-clamp traces indicate two unfolding patterns depending on the initial redox state of the FimG. A ~33 nm step corresponds to the unfolding length of oxidized FimG  cleaves the disulfide bond of the partially unfolded FimG, an additional ~12 nm step appears accounting for the sequestered residues. The sum of both steps gives the total length of 45 nm. The trace in Fig. To prevent non-oxidative release of DsbA from its substrateimG Fig. . If a reWe thus investigated the unfolding and refolding behavior of FimG in the presence of both FimC and DsbA. These measurements intend to reproduce the conditions that pilus domains encounter in the periplasm. Here, the force protocols were identical to those used in the case of FimG and DsbA. Figure L subdomain. Given that FimHL is the only domain in the chain that does not incorporate a complementing β-strand from a different domain, it is also the only one that can potentially unfold and refold in the pilus upon force application. Any other domain unfolding would result in β-strand disconnection and pilus rupture. In the outer space, unfolding of FimHL could be possible under extreme forces. This implies that FimHL will have to refold quickly in order to recover its functionality for attachment. To test this idea, we have investigated the folding kinetics of FimHL and compared to that of FimHP. Using the polyprotein (I91)2-FimH-(I91)2 2 Fig. , we havemHP Fig. .Fig. 6Fo36. Our findings also highlight the fact that pilus domains are mechanically weaker as we approach the tip fibrillum. FimA was found to be the highest mechanically stable protein in the pilus. The strongest interaction between FimA domains is perhaps mandatory since it is the most present interaction in the pilus. Pilus dissasembly due to rupture of FimA–FimA interaction could lead to cell detachment, hence it makes it plausible that the β-strand complementation strength has evolved in such a way that the most numerous interaction is the strongest. This high mechanical stability was also reported for the shaft pilins FimA and SpaA from Gram-positive bacteria37. Very recently, the mechanical stability of the staphylococcal adhesin SdrG was shown to reach 2 nN25. All these findings indicate that the main components of bacterial pili display high mechanical stability.Here we report a mechanical characterization of the type 1 pilus domains complemented with their cognate β-strands. This protein design resembles the interactions present in a real pilus. Using an AFM, we are able to study their mechanical integrity. We found that the pilus domains exhibit high mechanical stability, among the highest known for proteins38. In order to reduce the tension on this interaction, the pilus rod unravels at forces below 100 pN acting as a shock absorber30. These two interactions may explain the adhesion of the bacterium, but do not seem to justify the mechanical resistence of the whole pilus. Given that the β-strand interactions are the only non-reversible interactions in the pilus, they must be higher than the catch-bond and rod uncoiling forces. The elevated mechanical resistance of the β-strands actually allows for the pilus to behave as a shock absorber because it prevents rupture and secures the spring-like function of the pilus. In addition, we have discovered mechanical signatures that suggest an additional quaternary interaction between the domains of tip fibrillum similar to that found on the FimA domains in the pilus rod. However, this interaction does not seem to impact the mechanical stability of the domains, still dominated by the β-strand complementation.Bacteria exposed to shear forces need to distribute the mechanical stress along the pilus. Catch-bonds between FimH and the mannosylated glycoprotein of a bladder cell break at forces of 60–180 pN, depending on pulling conditions38, all the donor β-strands and pilin domain interactions are very strong. This hierarchy of mechanical forces suggests a mechanism for pili dettachment: first, the pilus rod will completely unravel and then the catch-bond between FimH and the mannose will be broken. The latter process will lead to cell detachment but preserving the structure of the pilus, which will remain intact for a new attachment in the bladder epithelium. Considering that bacteria surface is surrounded by a large number of pili39, this strategy seems to be the best suited for bacterial attachment. Our data also reveal that disulfide-bonded domains are mechanically more stable than the reduced ones. As we previously reported for FimG27, the disulfide bond acts as a mechanical lock that pushes the unfolding of the domain through a higher energetic unfolding pathway. The position of this disulfide bond is conserved not only for the subunits of the type 1 pilus but also among other subunits from different pili, making it an important mechanical feature in proteins used by bacteria for attachment40.While uncoiling or detaching the pilus require low forcesThe SMD simulations give us insight into the events taking place during the unfolding of the domains, showing a sequential ripping of the hydrogen bonds keeping the donor β-strand in place. A map of the persistent hydrogen bonds present in each domain from simulation  of the pilus type P44. Further experiments exploring the peptide-binding ability of DsbA showed that the mixed disulfide intermediate between DsbA and its substrate was stabilized by non-covalent interactions45. Besides, the chaperone-like activity of DsbA on proteins lacking cysteines on their sequence was demonstrated46. Even though the chaperone activity of DsbA was already suggested, our experiments are the first demonstration on its native substrate. One could expect that if both FimC and DsbA have chaperone activity; the combination of the two of them would have a synergistic effect on the folding of FimG. However, our experiments clearly show that the combination of both has a similar effect on the refolding of FimG than just DsbA by itself, highlighting that DsbA seems to be the principal chaperone.In the case of DsbA, in our experiments, we not only see the formation of disulfide bonds catalyzed by DsbA, we also observe that DsbA increases FimG refolding in a greater extent than FimC does. This finding is a totally new aspect describing the folding of pilus proteins. DsbA would assist Fim proteins to fold not only due to the entropic and enthalpic contributions derived from the disulfide bond formationOur results complement previous observations to render a complete view of the mechanical architecture and maturation of the pilus. This knowledge will help to develop new therapeutical mechano-drugs that target the strength of the donor-strand complementation. Nowadays traditional antibiotic treatments are becoming less effective due to the appearance of resistant strains. Targeting some of the main actors involved in pilus mechanochemistry could be another therapeutic approach.Escherichia coli cells (Life Technologies). These genes were cloned in the pQE80L plasmid (Qiagen) with the exception of DsbA, which was cloned in pET11a plasmid, both kindly provided by Julio Fernandez’s lab. The gene sequence of the pilus subunits was built including their cognate donor-strand sequence following their C-terminal end  with a four aminoacid sequence (DNKQ) in between acting as a linker as it was previously done11 (Supplementary Note). Two copies of the I91 domain (former I27) from human titin protein were placed flanking the pilus subunits as it was explained above. The last C-terminal I91 domain contains two cysteine residues in its C terminal sequence, allowing polyprotein immobilization on the gold surface.The genes encoding the proteins used in this article were synthesized and codon-optimized for expression in E. coli competent cells (Agilent Technologies) were transformed and grown in LB media at 37 °C up to OD600 ~0.6. Then the protein expression was induced adding 1 mM of IPTG and the culture was incubated overnight at 37 °C. After cell pelleting through centrifugation, cell lysis was performed mechanically with a french press and the His6-tagged polyproteins were purified by affinity chromatography using a Talon column (Clontech). Disulfide bond formation was triggered by oxidation with 0.1% of H2O2 overnight at room temperature. Reduced non-disulfide proteins were obtained incubating with 10 mM DTT overnight at room temperature. Protein purification was followed by a size exclusion chromatography in a Superdex 200HR column . The buffer used was HEPES 10 mM pH 7.0, NaCl 150 mM, and EDTA 1 mM. Protein purity was addressed with SDS-PAGE and the proteins were aliquoted and freezed until use.BL21 (DE3) 47. Further purification steps included anion exchange chromatography in 5 mL HiTrap Q FF column  and size exclusion chromatography35.In the case of DsbA, we used a different purification protocol. After protein expression induction with 1 mM IPTG, the culture was incubated overnight at 20 °C. Instead of mechanical disruption of the bacterial cells, an osmotic shock was induced in order to perform the periplasmic extraction of DsbA−1 (Bruker MLCT) and 6 pN nm−1 (Bruker OBL-10). The proteins under study were incubated for 10 min over custom-made gold surfaces at a final concentration of 0.1–1.0 g L−1 and the buffer used was HEPES 10 mM pH 7.0, NaCl 150 mM, and EDTA 1 mM in all the experiments.Atomic force microscopy (AFM) experiments were conducted in a commercial Atomic Force Spectroscope (Luigs & Neumann). The cantilevers used in these experiments were calibrated using the equipartition theorem and they had typical spring constants of 15 pN nm−1, applying first a force of ~1 nN on the cantilever. Force-extension data were analyized using the worm-like chain for polymer lasticity24. The contour lentgh increments are estimated as the difference between the fittings in the asymptote, which corresponds to the fully unfolded domain. Force-ramp experiments were done at 10 pN s−1 pulling speed for 30 s. Force-clamp experiments were conducted with the FimG construct in its reduced and oxidized form and in the presence of FimC and/or DsbA, at 10 and 100 μM, respectively. Folding experiments in the presence of FimC were conducted applying two consecutive force pulses of 160 pN for 2 s and 300 pN for 20 s, followed by a quench pulse at 0 pN of different time lengths in order to allow the refolding of the protein. Then, the same two force pulses were applied again in order to check the folding of FimG. Oxidation and folding experiments in the presence of DsbA were conducted applying either three consecutive pulses of 160 pN (2 s), 300 pN (5 s), and 100 pN (2 s), or two consecutive pulses of 160 pN (2 s) and 300 pN (7 s), then followed by a quench pulse of 0 pN. After the quench pulse of 0 pN for 45 s, the force pulses were applied again. For the combined experiments with FimC and DsbA, the same pulse protocols used for DsbA alone were applied. Experiments conducted on the FimH construct were carried out using a four-pulse protocol with three unfolding forces, 60 pN (5 s) to monitor FimHL unfolding followed by a 160 pN (3 s) pulse for I91 and a 250 pN (7 s) for FimHP unfolding. Thereafter, the force was quenched to 0 pN to trigger refolding during 10 s and then the three-force pulses were applied again. In the case of force-ramp experiments conducted on the pilus tip polyprotein (FimH-FimG-FimF), the force was linearly increased from −10 to 400 pN at a speed of 10 pN s−1. The traces obtained in both force-extension and force-clamp modes were collected and analyzed with a custom-written code in Igor Pro 6.37 (Wavemetrics), available to the scientific community upon request. All the figures were generated using Igor Pro, Adobe Illustrator CS6 (Adobe), PyMOL (Schrödinger), and VMD49.Force-extension experiments were conducted at 400 nm sRefolding probability was calculated as the ratio between the number of trajectories showing refolding and the total number of trajectories collected for a specific quenching time and experimental condition. The standard deviation was calculated assuming data as a binomial distribution.50 code (version 4.6.5). Modelization of these structures was carried out based on previously resolved 3D structures accessible on the Protein Data Bank . Each subunit was separated together with the donor β-strand from the next one, and the DNKQ linker was manually inserted between the two to form a single chain, equivalent to what was used in the force spectroscopy experiments. After the insertion, the proteins were placed in a box of dimensions 7 × 7 × 28 nm. The principal axis of the proteins was aligned with the z direction. Explicit water solvent and 0.15 M of NaCl were then added to the box. The CHARMM27-CMAP51 force field was used for the protein and TIP3P52 for the solvent. The systems were initially prepared by a steepest descent minimization followed by an equilibration run of 100 ps using velocity rescaling with a stochastic term53 to ensure a physically reasonable configuration. For the case of FimF, for which no structure complemented with FimA has been reported, the donor β-strand of FimA was manually inserted together with the linker and a long equilibration run was performed to ensure the correct bonding between the subunits. For the case of FimH, only the pilin domain was used. Each protein was then pulled along the z direction of the box at 1 nm ns−1 for 10 ns with Nosé-Hoover temperature coupling. We use a spring constant of 1000 kJ mol−1 nm−2. The pulling reference groups were the terminating nitrogen and carbonyl carbon atoms along the main chain from the N and C-terminal aminoacids of the donor Fim-linker-β-strand complex. During the run, both the protein and solvent were kept at 300 K; no pressure coupling was applied. The simulations used a time step of 0.001 ps, periodic boundary conditions in all directions, the smooth particle mesh Ewald54 (SPME) method for long-range electrostatics, and a real-space cutoff for van der Waals interactions of 1.5 nm.All-atom simulations were carried out on the self-complemented proteins with the GROMACSThe pulling was performed up to the removal of the donor β-strands from the protein fold. The interaction of FimH with the donor β-strand of FimG was also tested without the linker, in order to validate our experimental approach with the covalent attachment. In this case, the reference pulling groups were the N-terminal aminoacid nitrogen atom from the FimH pilin domain and the C-terminal aminoacid carbonyl carbon from the donor β-strand of FimG. For all the proteins, the hydrogen-bonding pattern was analyzed during the course of the equilibration and pulling simulations. This was done based on the instantaneous O–H distances and accounting for regular fluctuations in time.The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Novel crystalloid solutions containing polyethylene glycol polymers (PEG-20k) produce dramatic resuscitation effects but dose-dependently produce a hypocoagulative state. The objective of this study was to examine possible mechanisms of this effect. Based on previous thromboelastography data, we hypothesize the effect is largely due to platelet interactions with the polymers.Whole citrated blood from healthy volunteers was diluted ex-vivo 10% with crystalloids and tested for coagulation and platelet function. The specific tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and von Willebrand factor (vWf) activity, thrombin generation, thromboelastography with and without platelet mapping, platelet flow cytometry, and erythrocyte sedimentation rate.Fibrinogen and vWF activities, PT, and aPTT were not affected by PEG-20k dilutions. Thrombin activity was mildly suppressed with PEG-20k (TTP- 20%). Platelet mapping demonstrated significantly greater % inhibition of both ADP and arachidonic acid-induced platelet aggregation with PEG-20k, but direct ADP-activated gpIIa/IIIb (PAC1) and P-selectin (CD62P) binding site expression was not altered. Mild dose-dependent suppression of TEG-MA was seen with PEG-20k using platelet poor plasma. Erythrocyte Sedimentation Rates (ESR) were dramatically accelerated after dilution with 10% PEG-20k, which was competitively blocked by smaller PEG polymers, suggesting nonspecific PEG-20k cell binding effects.PEG-20k creates a mild hypocoagulative state in whole blood at concentrations ≥10%, which may be due to platelet-PEG interactions at the IIb/IIIa interface with lesser effects on fibrin polymerization. This interaction may cause a functional thrombasthenia induced by nonspecific platelet surface passivation by the PEG polymer. Trauma is the number one cause of death for people under 44 years of age in the US and the third leading cause of death overall for all age groups. Trauma accounts for about 30% of all life-years lost in the US, compared to cancer 16%), heart disease 12%), and HIV (2%) [6%, heart%, and HICrystalloid-based intravenous (IV) solutions are available for pre-hospital use because they can be safely transported and stored but they are generally limited in their effectiveness. Only a fraction of infused crystalloid volume stays in the intravascular space and the use of low volume crystalloids has minimal effects on pressure and perfusion , 6. The Recently, polyethylene glycol (PEG) polymers of specific molecular weight ranges have been used in crystalloid solutions to act as highly effective low-volume resuscitation (LVR) solutions , 12–14. k and α-angle, which are measurements influenced by fibrinogen cross-linking. The PEG-20k effects on TEG parameters were significantly different, relative to those of normal saline and hetastarch, and appeared in a dose-dependent fashion.In a set of experiments recently published , we descTherefore, the aim of this study was to characterize the mechanism of the dose-dependent hypocoagulopathy findings in the TEG parameters observed with PEG-20k solutions. To that end, we systematically studied a battery of coagulation and platelet function parameters in blood samples obtained from healthy volunteers diluted 10% with PEG-20k solution. From our previous TEG analysis, we hypothesize that the hypocoagulable state induced by PEG-20k solutions on whole clotting blood is related mainly to interferences of the polymer with platelet function.9/L for platelet-dependent thrombin generation assays. The remaining PPP was used for the analysis of platelet-independent thrombin generation, PT, aPTT, fibrinogen, and vWF concentrations. Platelet counts were performed with an automated cell counter . The time between blood draw and analysis was less than two hours. Normal values have been previously described [This study was done under the approval of the VCU Institutional Review Board . Each blood donor for this study provided written consent. Whole blood (15-ml) was drawn into citrated vacutainer collection tubes from 12 healthy consented volunteers (18–50 years of age) of both sexes that were free of all medications and tobacco. All volunteers donated blood under a VCU approved IRB protocol. The blood was diluted 10% in the lab with solutions of 10%, 7.5%, and 5% PEG-20k in saline (0.9% NaCl), 6% Hextend, or a 0.9% NaCl solution that served always as a paired dilutional control for all test solutions. Immediately after the dilutions, the samples were analyzed for coagulation parameters. One mL of citrated whole blood aliquots was used for TEG analysis using kaolin as activator, and the remaining citrated whole blood was centrifuged at 180 x g for 10 minutes to obtain platelet rich plasma (PRP). Platelet poor plasma (PPP) was obtained by double centrifugation of the remaining plasma at 2000 x g for 10 min at room temperature. PRP was then diluted with autologous PPP to yield a final platelet count of 150 x10Fibrinogen, PT, aPTT, and von Willebrand factor antigen (vWF) function were measured in plasma using standard assays  on the STA Compact analyzer  according to manufacturer’s instructions.The kinetics of thrombin generation was assessed in PRP and in PPP according to methods previously described by Hemker, et al . BrieflyThromboelastography with platelet mapping was determined using a TEG 5000  using the intrinsic pathway activator kaolin (Haemonetics Corp.) and recalcification to 10 mmol/L final calcium concentration. The TEG 5000 reported time to onset of clot formation (R), which positively correlates with thrombin generation; the time to reach a predetermined level of clot stiffness (K) and the clotting angle (α-angle), which correlates with fibrin polymerization; the maximal amplitude (MA) or stiffness, representing clot strength. Platelet mapping was done using the TEG-5000 instrument and a platelet mapping kit (Haemonetics Corp.) that tests the platelet component to clot formation (MA) on TEG . BrieflyTo test the effects of PEG-20k on just the fibrin component of clot formation, TEG was performed on platelet poor plasma. Citrated whole blood was drawn from six healthy volunteers as before and the plasma was obtained by centrifugation at 2500 x g for 10 minutes. The PPP was diluted 10% with solutions of PEG-20k at 0%, 5%, 10%, and 15%. Standard TEG using kaolin activation was performed as before on each sample immediately after PEG-20k dilution and compared to the 10% dilutional volume control (0% PEG-20k) using saline.Platelet activation was also quantified using flow cytometric analysis. Briefly, flow cytometry was performed on a BD Biosciences device  using citrated whole blood according to current standards from the European Working Group on Cell Analysis . CD41a cThe erythrocyte sedimentation rate was measured in diluted citrated whole blood using the Sediplast Westergren ESR system tests . About 1 ml of whole blood was drawn up into a 10 cm Westergren ESR tube, which was held in the vertical position for 75 minutes. The rate of red blood cell sedimentation was measured as the migration (in mm) of the red cell column down the tube under the force of gravity. Blood samples diluted with 10% volume of saline (volume control) were compared to 10% dilution with PEG-20k solutions and PEG-20k solutions with other test compounds.All statistical analysis was performed using GraphPad Prism version 6.07 for Windows . Data groups were analyzed for outliers using the nonlinear regression ROUT method with Q = 1%, the maximum desired false discovery rate. Normality of Gaussian distribution was then assessed using the D’Agnostino-Pearson ombinus K2 method. Most data were then analyzed by the non-parametric ANOVA Kruskal-Wallis test with the Mann Whitney U test for multiple comparisons of means. The data are presented as either mean (standard deviation) or as median with interquartile ranges. The ESR competitive inhibitor data were analyzed by nonlinear regression analysis. A p-value < 0.05 was considered statistically significant. There was no difference in fibrinogen concentrations due to dilution with PEG-20k (Panel A). However, there was a small but statistically significant decrease in vWF activity observed for the PEG-20k (Panel B) samples but the levels in this group were still within the normal range.The plasma concentrations of fibrinogen and von Willebrand factor (vWF) activity obtained from healthy volunteers that had been diluted 10% with either PEG-20k (10% w:v) or a saline dilution control are shown Fig 2 shows the PT (Panel A) and aPTT (Panel B) when plasma was diluted by 10% with either PEG-20k or saline. This is the theoretical dilution that occurs when the solutions are administered to shocked patients. These data illustrate that PEG-20k has no effect on neither PT, nor aPTT when the activator reagents included a combination of kaolin and rabbit brain phospholipids. However, when the activator for this test contained micronized silica instead of kaolin and rabbit brain phospholipids, there was a very significant prolongation in the aPTT in the PEG-20k diluted plasma samples, relative to the saline controls, suggesting that a silica-PEG-20k interaction exists that interferes with initiation of the intrinsic pathways cascade. So while PEG-20k did not have any significant effects on coagulation, it dramatically prolongs the aPTT times when micronized silica is used as an activator. This interaction has recently been observed in PEG-conjugated compounds including PEGylated factor replacement products used for patients with hemophilia [mophilia –25. ThisFig 3 shows thrombin generation in PRP and PPP when the samples were diluted with either the PEG-20k or saline control. The presence of PEG-20k in the sample showed no effect on all CAT parameters except a slight but significantly prolonged thrombin generation lag time, but only in the PRP samples.Fig 4. Dilution with 10% PEG-20k caused a significant decrease in the ADP and AA-induced aggregation response, relative to the saline control. This is expressed as the inhibition (%) of the maximal response observed in the absence of PEG-20k or the saline vehicle.The results of TEG platelet mapping, showing the platelet contribution to clot formation under platelet stimulation with either ADP or arachidonic acid (AA), is shown in Fig 5. ADP activation of platelets in PRP induces a rapid expression of glycoprotein IIb /IIIA complexes and P-Selectin that are detected by specific binding of antibodies to PAC1 and anti-CD62P, respectively. While there were significant increases in both PAC1 (88.5%) and CD62p (59.7%) antibody binding to ADP-activated platelets, compared to the non-activated state with saline dilution, the effect was not different when PEG-20k was used as the diluent (87.4% increase for PAC1 and 62.5% increase for CD62p).The flow cytometry data are presented in Fig 6. Following removal of the platelet components by centrifugation, the platelet poor plasma component was assayed in a standard TEG experiment using kaolin activation. The MA and Angle components of TEG are reported for varying doses of PEG-20k from 0–15 mg/ml with a constant volume dilution of 10% for all tests. The absolute baseline clot and kinetics are smaller compared to values obtained from volunteer whole blood [The effects of PEG-20k on just the chemical phase of blood coagulation is shown in le blood . The clo. The ESR was significantly and dose-dependently increased with 7.5% and 10% PEG-20k solutions diluted 10% with whole blood (Panel A). Addition of 10% PEG-20k (weight to volume) induced a 150 fold increase in the rate of erythrocyte sedimentation, compared to the saline control at the same 10% volume dilution. In another study, the ESR sedimentation effect of 10% PEG-20k could be competitively inhibited by the addition of shorter chain PEG polymers of 1k, 4k, and 8k (8k shown in panel B).Finally, in an attempt to understand the cell binding effects of PEG-20k in blood, we used the erythrocyte sedimentation rate (ESR) as a model for what may be happening in the platelet fraction with PEG-20k PEG-20k, a new LVR crystalloid solution has recently been developed that induces profound tolerance to the low volume state when compared to other commonly used solutions. In preliminary testing using thromboelastography  it was dThis study essentially rules out PEG-20k-related effects causing coagulation protein deficiencies. For example, the concentrations of fibrinogen were not different with PEG-20k diluted blood compared to the saline controls, and the fibrinogen remained within the normal ranges. An effect of PEG-20k on fibrinogen shape or charge leading to altered catalytic rates cannot be ruled out and may be a possible avenue to explore in future studies. PEG polymers are known to camouflage active surface molecules on cells  so maybeThrombin generation is an important component of blood clotting and should be evaluated when a coagulopathy is identified since it represents the final common pathway. Furthermore, platelet dependent or independent thrombin activity may be a more important measure of coagulation than PT and aPTT times . ThrombiThe most likely explanation for the slower clot propagation and decreased α-angle and k revolves around the axis of fibrinogen binding to activated platelets. For example, the flow cytometry data showed no difference in platelet receptor expression , there is still a smaller component (10–20%) attributable to the fibrin component of clot formation. Since PEG-20k was seen to dose dependently interfere with MA on TEG using only platelet poor plasma, we must conclude that a smaller component of the PEG-20k effect on clot strength in clotting whole human blood is due to this fibrin component. The polymer may interfere with activated factor XIII-induced cross-linking and fXIII-fibrin interactions, thereby weakening the clot strength. This is supported by the known interference with other colloids between fXIII and fibrin to limit cross linking and weaken clot strength , 34. SimIn conclusion, this study has expanded our search for a mechanistic explanation for the identified effects of PEG-20k solutions on whole blood coagulation observed in healthy volunteers and trauma patients. PEG-20k seems to have less effect on the intrinsic and extrinsic coagulation pathways and on the availability of critical non-catalytic proteins such as fibrinogen and vWF. The effects of PEG-20k solutions on clot formation suggest potential interference by PEG-20k with fibrinogen binding and polymerization on the platelet thereby mimicking a state of mild functional thrombocytopenia, platelet passivation, or thrombasthenia with similar effects on fibrin cross-linking. It may be possible to chemically modify the PEG-20k functional groups to mitigate these effects.S1 File(PZF)Click here for additional data file.S2 File(PZF)Click here for additional data file.S3 File(PZFX)Click here for additional data file.S4 File(PZFX)Click here for additional data file.


The objective was to use modified version of WHOQOL-BREF to assess the well-being and QOL of dental students of King Saud University based on four major domains.The questionnaire related to the survey was distributed to all dental students (N= 782) who were enrolled from 1st year to 5th year in College of Dentistry, King Saud University in the fall of 2018. The questionnaire comprised of four domains having different set of questions i.e. Physical domain, Psychological domain, Environmental domain and Social relationship domain. Two stand-alone questions related to  were also part of the WHOQOL-BREF questionnaire. Cronbach’s Alpha was used to assess the reliability of the WHOQOL-BREF domains. Paired t-tests were conducted to compare the means of the four domains and chi-square. Chi-square test was used to find association of demographic characteristics with four domains and two stand-alone questions.The overall quality of life and satisfaction with health of the dental students was found to be satisfactorily favorable with environmental domain and moderately favorable with social relationship and physical health domains. Physical health domain with psychological domain was statistically significant  whereas physical health domain with social relationship and environmental domain was also found to be statistically significant . Respondents, who highly rated their overall quality of life and satisfaction with health, had higher domain scores.In the present study, overall quality of life and satisfaction with health of dental students in King Saud University was found to be satisfactorily favorable. A sense of wellbeing, social functioning and emotions, all are related to Quality of life (QOL). The concept of QOL is so multidimensional, multicultural and complex that no universally accepted definition has been established yet.9Multiple factors are associated for high stress levels, burnouts and reduced personal achievement among dental students. These may comprise of demanding curriculum, fewer leisure time, amount of assignments, strained relationships, deadlines and new ethical challenges.14Due to high prevalence of stress, anxiety pressure among dental students there is an increased attention to address their happiness, wellness and well-being.st to 5th year during their classroom lectures.The Ethical committee of King Saud University approved the study with reference number 5847-2. The questionnaire related to the survey was distributed in classrooms to all dental students (N= 782) who were enrolled from 1st year to 5th year in College of Dentistry, King Saud University in the fall of 2018. All the respondents were assured about the confidentiality of personal information. All surveys were distributed in one week to all the students from 1In the present study a modified version of WHOQOL-BREFResponses were composed by means of the professional and encrypted version of Survey Monkey . All the statistical analysis was performed using statistical package for social sciences . Descriptive analysis was used to express categorical data as percentage and continuous variables as the mean and standard deviation (SD). Cronbach’s Alpha was used to assess the reliability of the WHOQOL-BREF domains. In addition, paired t-tests were conducted to compare the means of the four domains. Correlation coefficients were computed between the four domains and the two stand-alone questions on overall quality of life and satisfaction with health. Finally, Chi-square test was used to find association of demographic characteristics with four domains and two stand-alone questions.nd year dental students (D2) 90 (16.90%).  and more than three-fourth of the respondents 526 (98.7%) were single. The maximum number of participants involved in the study were third year students (D3) 118 (22.1%) and the minimum number of responses were from 216.90%). A summary of descriptive statistics and Cronbach’s alpha for the WHOQOL-BREF domains and two separated items Overall Quality of Life & Satisfaction with health was assessed in the study and are shown in Cronbach’s alpha was used to assess the reliability of the WHOQOL-BREF and the four domains, with values greater than or equal to 0.70 denoting satisfactory reliability. Social relationship showed moderately good reliability having Cronbach’s Alpha score of 0.64. Whereas, environmental domain displayed Cronbach’s score of 0.77 displaying satisfactory reliability. Furthermore, for physical domain, Cronbach’s score was 0.56 signifying good reliability. However, fair reliability was observed in psychological domain 0.36. .In addition, paired t-tests were conducted to compare the means of four domains. Physical health domain with psychological domain was statistically significant  whereas physical health domain with social relationship and environmental domain was also found to be statistically significant . However, mean difference of social relationship with environmental domain was higher than mean difference of psychological and environmental domain. .Correlation coefficients were computed between the four domains and the two stand-alone questions on overall quality of life and satisfaction with health. These correlations ranged from 0.245 to 0.542, and the differences were statistically significant (p<0.01). Respondents who highly rated their overall quality of life and satisfaction with health, had higher domain scores. The correlation between the two stand-alone questions was also statistically significant (p<0.001): a higher overall quality of life rating was observed with Psychological Domain (r=0.542). Association of demographic characteristics with overall quality of life & satisfaction with health was assessed in the present study. Overall quality of life and satisfaction with health was not significantly (p > 0.05) associated with demographic characteristics. However, psychological domain was statistically associated  with all three demographic variables. No association of demographic characteristics was observed with other three domains.The present survey provides a unique assessment of QOL of dental students at College of Dentistry, King Saud University using WHOQOL-BREF questionnaire. The type of survey was distinctive, as it assesses the impact of stress on the oral health quality of life of dental students in an institute in Saudi Arabia.The overall quality of life of the students in the current study was considerably favourable and their satisfaction with health was between neutral and good. Dental students who rated high quality of life and satisfaction with health were found to have high domain scores as well. The current study displayed an improvement of QOL of students as they progressed from first to fifth year. These results were in harmony with a study by Andre et al., who didn’t express any decline in QOL among dental students among the course of four years.18In the current study the physical health domain mean scores [19.77(3.74)] were comparable to psychological domain mean scores [17.64 (3.04)]. Whereas, the environmental domain means scores was found to be highest [33.72(5.24)]. These results indicate that these domains have had a greater impact on dental students’ quality of life. A study by Andre et al., displayed physical health and social relationship to be dominant component in QOL.19Compared to the male, female students exhibited high scores in environmental domain and social relationship domain. These results were in concurrence with studies by Andre et al.,21th and 5th year compared to previous years. Furthermore, psychological domain adheres to enjoyment of life, self-esteem, bodily image, which also showed improvement with advancing years. This type of escalating trend in psychological domain and quality of life scores speculated by authors can be due to a shift of didactic curriculum to more patient focused clinical curriculum and move from assessment-based competencies to competencies which are assessed on patient experiences.Among the dental students in the present study there is a gradual increase in quality of life, psychological scores and physical health domain scores with progressing years. Physical health domain consisted of sleep, work capacity and sleep activity in daily lives. Authors believe that the upward trend seen in physical domain with progressing years is due to a shift to comprehensive patient care and more clinical experiences which is practiced more often in College of Dentistry, King Saud University in 4Interestingly, in the present study married students displayed high scores in all domains and high satisfaction with QOL and health compared to single students. This trend can be the outcome of social maturity, companionship and spousal support which occur as a result of marriage. A study by Henning et al., showed low distress in married medical students.24The study has limitations based on its small sample size and study design. A similar larger scale study with increased sample size and representation of all dental colleges in the city may give a better understanding on factors having influence on QOL of dental students. Further, since our study was a cross-sectional design it did not follow the progress of cohort students from first to fifth year. Subsequently, QOL is multi ethnic and multi-cultural and other demographic variables should be accounted for in the future studies i.e. ethnicity, socioeconomic status, race, family business and age. In addition, it is recommended that further studies should be focused to evaluate the diverse concept of quality of life among dental students and practitioners by probing the WHOQOL-BREF domain scores.Overall quality of life and satisfaction with health was found to be satisfactorily favorable with environmental domain and moderately favorable with social relationship and physical health domains. Surprisingly, a gradual increase in quality of life, psychological scores and physical health domain scores were found in dental students with progressing years. Married dental students displayed high scores in all domains and high satisfaction with QOL and health compared to single students. Encouraging better QOL of dental students is not only important for dental schools, but has a massive impact on dental profession as a whole and translates this into a healthier patient care.


Arabidopsis thaliana, possessing over 100 type I and type II MADS TFs. All MADS TFs contain a core and highly conserved DNA binding domain called the MADS or M domain. Plant MADS TFs have diversified this domain with plant-specific auxiliary domains. Plant type I MADS TFs have a highly diverse and largely unstructured Carboxy-terminal (C domain), whereas type II MADS have added oligomerization domains, called Intervening (I domain) and Keratin-like (K domain), in addition to the C domain. In this mini review, we describe the overall structure of the type II “MIKC” type MADS TFs in plants, with a focus on the K domain, a critical oligomerization module. We summarize the determining factors for oligomerization and provide mechanistic insights on how secondary structural elements are required for oligomerization capability and specificity. Using MADS TFs that are involved in flower organ specification as an example, we provide case studies and homology modeling of MADS TFs complex formation. Finally, we highlight outstanding questions in the field.MADS transcription factors (TFs) are DNA binding proteins found in almost all eukaryotes that play essential roles in diverse biological processes. While present in animals and fungi as a small TF family, the family has dramatically expanded in plants over the course of evolution, with the model flowering plant,  Allowin6GG) . The com6GG) . Type I tructure . The I atructure . Adding wo sites ,33, unliwo sites . For exawo sites ,36. Eachwo sites ,6,20,37.wo sites ,39.Fig. In this review, we describe the overall architecture and structure of Type II MIKC MADS TFs, with a focus on the oligomerization domain based on the recent structural characterization of the SEP3 K domain and extensive mutagenesis studies . We furt22.1H. sapiens and A. thaliana). In addition to binding DNA, most plant M domains contain a predicted bipartite nuclear localization sequence (NLS) with the consensus sequence (K/R)(K/R)X10(K/R)3–5 [APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG) chimeras possessing the M domain of SRF (yeast) or MEF2 (human) are able to complement the appropriate mutant phenotype as well as the native gene, and can reproduce the phenotypes exhibited by plants expressing 35S::AP1, 35S::AP3, 35S::PI and 35S::AG, respectively [in vivo binding specificity, 2) other MADS TF partners in the plant MADS heteromeric complexes may compensate for the switch in M domain of a single partner and/or 3) the oligomerization domains  strongly contribute to DNA-binding specificity, perhaps via allosteric interactions. It is likely that a combination of these factors plays a part in determining DNA-binding specificity and function. For example, in vivo specificity studies using ChIP-seq demonstrated enrichment of non-CArG box motifs for MADS TF binding sites, suggesting a role for ternary factors [In vitro and in vivo studies of the K domain have also demonstrated a role in DNA-binding specificity and in vivo function for the plant MIKC MADS [The MIKC MADS TFs are modular multi domain proteins with highly conserved secondary structures A. The DB(K/R)3–5 ,44. The (K/R)3–5 . Domain ectively . These d factors . Domain IKC MADS .2.2in vitro domain swaps, band shift assays, computational models and yeast 2-hybrid studies [The I domain, a small (~25–30 amino acid) linker domain between M and K domains A, is inv studies ,52. Thisin situ proteolysis. While the H2 helices form a protein-protein interface for a single helix to bind, this interface is not sufficient to bind two H3 helices and is unlikely to mimic plant MIKC dimers which would present two helices. The different conformations for MEF2 are likely the result of truncated constructs and crystal packing due to inherent flexibility between H2 and H3 and may have limited physiological relevance.In the mammalian MADS box containing proteins, such as MEF2B, adjacent to the M domain is a small domain, consisting of two α-helices, called H2 and H3, that helps to stabilize the M domain by sandwiching the β sheet adjacent to the DNA-binding α-helix C. The pr1SRS and 6BYY), we generated two M domain  models for SEP3, called SEP3-M1 and SEP3-M2, respectively  and K2  helices, for example, were shown to be important for AP3/PI heterodimerization, based on extensive mutagenesis studies and yeast 2-hybrid assays ,60. Furtvia their N-terminal α-helices to form a dimer and two dimers form a tetramer via their C-terminal α-helices. Thus, the biological assembly of SEP3 in the crystal structure is a homotetramer or a dimer of dimers. Sequence alignments of representative type II MADS TFs from Arabidopsis reveal that the amino acids in the kink region are variable but contain glycines and/or proline residues, resulting in a “break” between the α1 and α2 helices . The leucine zipper is typical for coiled coils, which can be well predicted by bioinformatic algorithms, such as MultiCoil [The K domain of SEP3 has a characteristic leucine zipper heptad repeat pattern, (abcdefg)ultiCoil . Using MultiCoil ,20. DespultiCoil , shows tin planta using a splice variant of SEP3 that lacks residues 161–174 in the α2 helix, which showed abrogated tetramerization activity in vitro. The transgenic plant expressing the tetramerization mutant of SEP3 in the sep1sep2sep3 triple mutant background showed defects in flower meristem termination [in vitro and in vivo with accompanying changes in regulation of target genes.Based on the positions of hydrophobic residues in the heptad repeats of the K domain, recent studies have experimentally assessed the effects of these residues by means of mutagenesis coupled with size exclusion chromatography and electrophoretic mobility shift assays (EMSAs) ,40,42. Smination . Taken t2.3.2via the K domain to form a tetrameric complex. Based on extensive EMSAs studies and small-angle x-ray scattering experiments [in vitro and in vivo and is one of the most promiscuous complex-forming MADS TFs [versus intra molecular coiled coils to form (i.e. tetramerization), although this remains to be experimentally confirmed.A fundamental question remains as to what determines whether a MADS TF dimer will form an intramolecular coiled coil or recruit a second MADS TF dimer eriments , it is bMADS TFs . Hub proMADS TFs . AncestrMADS TFs . The SEPMADS TFs ,39,69. T2.4co-repressors TOPLESS and SIN3-ASSOCIATED POLYPEPTIDE P 18 (SAP18). This motif is present in over 20 MADS TFs from Arabidopsis [The C-terminal domain of plant MIKC TFs has a variable primary sequence with few conserved structural motifs based on secondary structure prediction. However, some short amino acid motifs are present which likely play roles in target gene regulation and/or higher order complex formation. For example, the EAR motif (ethylene-responsive element binding factor-associated amphiphilic repression motif) with a consensus sequence LxLxL or DLNxxP is recognized by the bidopsis . A direcbidopsis . Direct bidopsis . Interesbidopsis . The preAP3 or PI lacking the entire C domain were able to rescue ap3 and pi mutant plants, respectively [For certain MADS TFs, the C domain does not appear to be required for function. For example, ectively . This fuectively . Whether3via subtle changes in the energetics and kinetics of coiled coil formation. Initial formation of K domain α-helices for each monomer may depend on stabilizing hydrogen-bonding and salt bridge interactions, as has been experimentally shown for some coiled-coil forming proteins by NMR, time-resolved CD spectroscopy and mutagenesis [4OX0) residues on the hydrophilic face of the amphipathic K domain α-helices form a hydrogen-bonding network (R162/Q158/N155 and D152/Q148). In addition, the amino acids at these positions vary, suggesting a possible mechanism for controlling α-helix folding and stability. Additional amino acid substitutions on the hydrophobic face in each monomer α-helix in turn will stabilize or destabilize the formation of a dimeric coiled coil. Based on sequence alignments the “a” and “d” positions in MIKC MADS TFs vary, with leucine residues at the “d” position most strongly contributing to free energy stabilization.The plant MADS TFs possess the highly conserved DBD that has exhibited little change over the course of evolution. A ubiquitous structural motif- the coiled coil- has been acquired by the MIKC plant MADS TFS that alters the oligomerization state and interaction patterns of the protein family, helping to explain the rich regulatory and functional diversity of these TFs. The K domain relies on leucine zipper-type interactions to alter complex formation in a myriad of ways agenesis . These sAlternative splicing (AS) is another factor that increases plant MADS protein diversity by affecting primarily the oligomerization domains. The MIKC MADS proteins arise from multi-exonic genes, which undergo extensive AS resulting in alterations in the I and K domains that affects the PPI network and thus function. A number of predicted AS sites for the MADS family have been annotated, and are likely to be functionally relevant based on their location in the I/K domains and cross-species conservation . Indeed,Key questions remain as to MADS TF function and specificity. How do the different oligomerization patterns affect DNA binding and are there allosteric interactions which may further tune the direct interactions between the MADS DNA-binding domain and its cognate DNA? Is heterodimer or heterotetramer formation sufficient to functionally diversify the MADS TFs or are ternary factors necessary? How important is MADS tetramer cooperativity for gene regulation? Are additional oligomerization states accessible through extended coiled coil formation? Answers to these questions remain elusive, but the availability of structural, computational, modeling and genome wide binding data provide the necessary foundation for addressing these outstanding questions in the field.Agence Nationale de la Recherche  , Grenoble Alliance for Integrated Structural and Cell Biology  CBH-EUR-GS (ANR-17-EURE-0003))  and CFR CEA thesis fellowship (AG).This work was supported by the


Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall—radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature  Arabidopsis trichomes grow into extraordinarily shaped and precisely polarized structures, which makes them a potent model of cell polarization and morphogenesis . Images were processed using the Fiji platform .2PO4 pH 9.5 and incubated overnight with shaking at 150–180 rpm. Released trichomes were collected by centrifugation . For TEM, isolated trichomes were fixed for 24 h in 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4 °C and postfixed in 2% (w/v) OsO4 in the same buffer. Fixed samples were dehydrated through an ascending ethanol and acetone series and embedded in Epon–Araldite.Leaves of 4-week-old plants were collected and incubated for 3 h in falcon tubes in a solution of acetic acid:ethanol (1:3) and then washed three times with deionized water. Washed leaves were transferred to a solution of 150 mM KHFor cloning of the expression reporter construct EXO70A1p::GFP:GFP, the multisite gateway approach was used. The EXO70A1 promoter , first NES-2xSpo20p was amplified from YFP:Spo20p-PABD  and clonLACT), YFP-C2LACT from Lat52:YFP-C2LACT [To prepare the construct for the PS marker , optimized for in vitro coupled transcription and translation, using primers listed in ® SP6 High-Yield Wheat Germ Protein Expression System (Promega), according to the manufacturer’s instructions, in a total volume of 50 μL. To verify protein expression and stability, 10 μL of the yield was used in Western blotting. Since the protein was tagged on the N-terminus, Western blot analysis would have detected products of prematurely terminated translation, but none were observed  was blocked for one hour in blocking solution . After three washes in wash solution , the strip was incubated for two hours with primary mouse anti-HA antibody  diluted 1:1000 in blocking solution. After three washes with wash solution, the strip was subsequently incubated with secondary anti-mouse antibody conjugated to horseradish peroxidase (Promega), diluted 1:20,000 in blocking solution. Finally, the strip was washed three times with wash solution and incubated for three minutes with Amersham ECL Prime Western Blotting Detection Reagent  and imaged with Bio-Rad Laboratories  Chemidoc gel imaging system. The experiment was repeated twice with consistent results.