HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a “winged-helix” (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the 26S Proteasome “lid” regulatory subcomplex, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). We noted that in structure similarity searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group, as currently thought. Here, we report extensive structure similarity analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural similarity with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated self-association into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3.The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members:  These encompass several families, including tetratricopeptide repeat (TPR), ankyrin repeat (ANK), armadillo (ARM), as well as the HEAT repeat domain family (reviewed in –4). The n (mTOR) . Of thes3 (eIF3) –9. The Eu/ecod/) , 11 grouUnlike most globular domains, helical repeat domains do not have a fixed size, with a distinct beginning and end. Instead, they consist of varying numbers of helical repeats. The larger domains have varying curvatures and tend to form solenoids. This plasticity likely contributed to the fast divergence of helical repeat domains in size, structure, sequence, and function. Sequence analyses have revealed that it is often impossible to detect statistically significant sequence similarity, even among members of the same domain family. The variable length, irregular shapes, and low/undetectable sequence similarity have made the analysis of the evolution of these domain families challenging. Sequence conservation is limited to the interfaces between adjacent helices and tends to be lower than for other domain families .The HEAT repeat domain family was first reported in 1995 –14.Saccharomyces cerevisiae (S. cerevisiae) has two of the PCI subunits: eIF3a and eIF3c; while Schizosaccharomyces pombe (S. pombe) has four: eIF3a, eIF3c, eIF3e, and eIF3m subunits [Along with another domain, MPN/Mov34, the PCI domain was discovered as a structural unit found in three multi-subunit complexes, 26S Proteosome lid, CSN, and eIF3 –18. PCI subunits , 21–26.The structure similarity between HEAT and PCI domains is illustrated in When performing DALI  searchesWe reasoned that if structure similarity between the PCI domains and individual groups of known HEAT domains is comparable to the structure similarity among the groups of HEAT domains, then the PCI domains are a group of HEAT domains that acquired a WH subdomain during evolution, after the HEAT domains had diverged from other helical repeat domain families. Conversely, if all groups of HEAT domains have greater structure similarity to each other than to the PCI domains, then the PCI domains split earlier, before the common ancestor of all HEAT domains :If the PCI domains have lower structure similarity scores with all groups of HEAT domains than the scores among HEAT domain groups, then the PCI domains split off from their common ancestor with HEAT domains before the individual HEAT domain groups diverged from each other .If the average similarity scores between PCI domains and at least one group of HEAT domains are comparable, or higher, than the lowest average similarity scores between at least two groups of HEAT domains, then the PCI domains are a group of HEAT domains that have acquired a winged helix .To account for the presence of a Winged Helix (WH) subdomain in PCI domains, but not in the HEAT domains, which could affect the structure similarity scores, we also used PCI domain structures with deleted WH subdomains. Finally, helical repeat domains pose unique challenges for structure similarity software, because they have a repetitive structure, a variable number of repeats, and varying curvature. To try to compensate partially for these factors, we also used four-hairpin fragments of HEAT and PCI domains.vice versa. The PCI domain structures were the only group that consistently appeared in the searches with high scores. All further analysis was focused on HEAT and PCI domain structures only; however, there could be other groups of domains with comparable structure similarity to HEAT domains. We selected a representative set of structures with high-resolution and avoiding close relatives, and then subdivided the representative structures into groups based on structure similarity scores. The HEAT domains fell into four groups, based on structure similarity, as well as a couple of outlier structures, which were considered as a fifth group.  HEAT repeat domain of mTOR [We performed extensive structure similarity searches with HEAT and PCI domains with known structures, using the DALI server . The seaTable in The “fou of mTOR , 33, dis of mTOR , were ouWe found that the average similarity score between Group 1 HEAT domains and PCI domains (Group 6), was 4.8 (where scores above 2 indicate similarity). This score was higher than those between Group 1 and Groups 2 (4.2) and 4 (3.6). Similar results were observed for the outlier HEAT domains (Group 5): average similarity score with the PCI domains (Group 6) was 4.6, whereas those with Groups 2 and 4 were both 3.9. The similarity score of Group 5 with Group 3 was 4.5, comparable to that with Group 6 (PCI) , Fig 3A.The same conclusion was reached when using a set of PCI domains with deleted WH subdomain, which tended to increase further the similarity scores between the PCI domains (Group 6) and the HEAT domain groups. The score between Group 1 and Group 6 was now 5.1, and that between Group 5 and Group 6 was now 4.8. Furthermore, the new score between Group 3 and Group 6 was now 4.6, comparable to that between Group 3 and Group 5 (4.5) , Fig 3B.Finally, we used a set of protein fragments with only four helical hairpins, so that all structures had the same size. Again, the average similarity score between Group 1 HEAT domains and PCI domains (Group 6), 4.6, was higher than those between Group 1 and Groups 2 (3.9) and 4 (3.7). Likewise, the average similarity score of Group 5 with the PCI domains (Group 6), 5.2, was higher than those with Group 2 (4.2), Group 3 (4.8), and Group 4 (3.5) , Fig 3C.Structure-based sequence alignment of helical hairpins from PCI and HEAT domain structures showed a preference for hydrophobic side chains at positions buried at the interface between the two helices in the hairpin and, to a lesser extent, at positions facing adjacent hairpins, as expected . No obviThe comparative structure similarity analysis of HEAT and PCI domain structures presented here shows that the PCI domains are members of the HEAT domain family. The canonical HEAT domains (Group 1) have similar structure similarity with the MIF4G (Group 3) and PCI (Group 6) domains. And in fact, the PCI domains appear more closely related to the canonical HEAT domains than do the MA3 (Group 2) and W2 (group 4) domains . SometimTo avoid possible score bias due to the length of individual domains, we repeated the structure similarity analysis using four-hairpin fragments of the proteins, which corresponds to the size of the smallest HEAT domains. The results with four-hairpin fragments confirm the results obtained with the intact domains. Using the four-hairpin fragments has its own caveats, because, while for the PCI domains, using the last four hairpins before the WH subdomain ensures that corresponding fragments are used, this is not guaranteed to be the case for the various HEAT domains, even if using the last four hairpins of each protein. However, the observation that the evolutionary trees and groups obtained using intact domains vs. four-hairpin fragments are very similar, supports the validity of this approach. Importantly, both the analysis using intact domains, and that using four-hairpin fragments yield the same results, which strongly supports the overall conclusions.Table inTable inThe MIF4G and MA3 domain groups are evolutionarily closer to each other than to the rest of the HEAT domains, while the W2 domains are closer to the MA3 domains that to the rest of HEAT domains, including the MIF4G group . These oIn conclusion, this study offers insights into the evolution of the HEAT domain family in eukaryotes and shows that the PCI domains are in fact HEAT domains, which have acquired a WH subdomain at their C-terminus. This further expands the number of proteins containing a HEAT domain, and the already wide range of functions performed by these domains. It would be interesting to trace the origins and divergence of all helical repeat domain protein families as a whole, throughout the evolution of eukaryotes.The DALI Server  was usedTable inAny non-HEAT or PCI domain portions of the structures were deleted at this stage using PyMol . StructuTable inThe evolutionary tree automatically generated by the DALI server served as a starting point for grouping the structures in the dataset. The HEAT domains fell into four groups, “classic” HEAT domains, MA3 domains, MIF4G domains, and W2 domains, all of which had previously been defined –30, 46. We calculated average structure similarity scores (Z scores) and standard deviations for every pair of groups from the pairwise Z scores between all members of the two groups, obtained using the DALI server . The aveTo evaluate the contribution of the WH subdomain in the PCI domains, we repeated the analysis using “wingless” PCI domains. The WH subdomains were deleted from the corresponding structures in PyMol . To accohttps://alignmentviewer.org/).Exhaustive PSI-BLAST  and HHblS1 Table(XLSX)Click here for additional data file.


Quercus rotundifolia), being mainly used nowadays to feed animals, however a substantial part remains in the fields without any valorization. Underexploited crops are gaining new interest, driven by food security concerns and health benefits potential as well. In the present work, it was studied the physicochemical characteristics and functional perspective of acorn flour, as an ingredient for human diet. The study included nutritional composition analysis, phenolic compounds profile through HPLC, starch content and its microstructure, fibre, and pasting properties assessment. Acorn flour presented a high content in fat, particularly monounsaturated and polyunsaturated (oleic and linoleic acids), and high minerals content in particular K. Concerning phenolic profile, rutin, catechin, ellagic acid, gallic acid, and syringic acid were identified. In regards to technological profile, fibre was mainly insoluble, with around 11%, and starch content was 50%. Its pasting behaviour revealed a high gelatinization temperature (85 °C), with low breakdown, and higher retrogradation consistency. These results show acorn flour potential as a valuable and sustainable multipurpose food ingredient.Acorn is the fruit of holm oak (  •Quercus rotundifolia) was chemical and technologically assessed.Acorn flour .•Minerals were analysed for the first time, revealing high values in particular potassium.•Phenolic acids and flavonoids were identified in particular rutin and syringic acid.•Pasting behaviour has shown high gelatinization temperature and shear stress resistance. In particular, holm oak (Quercus rotundifolia) is one of the most important trees in the unique Mediterranean woodland ecosystem named “Montado | Dehesa” , saline water (0.9% NaCl), and methanol were acquired from Merck . Folin–Ciocalteu's reagent, 3,4,5-trihydroxybenzoic acid , acetic acid, both extra pure (>99%), and sodium hydroxide were purchased from Panreac . Sodium molybdate (99.5%) was obtained from Chem-Lab . The compounds 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+), 2,2-diphenyl-1-picrylhidrazyl radical (DPPH), potassium phosphate, catechin, potassium persulfate, sodium acetate, 2,4,6-Tripyridyl-s-Triazine (TPTZ iron reagent), acetic acid, hydrochloric acid, and iron(III) chloride were obtained from Sigma-Aldrich . Additionally, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Fluka Chemika . Ethanol, ether petroleum, potassium persulfate, sodium hydroxide, methanol, hydrochloric, formic, and nitric acid, acetonitrile, iron(III) chloride, sodium acetate, sodium carbonate, sulphuric acid (98%), and potassium hydroxide (98%) were acquired from Panreac .2.2Quercus rotundifolia) was sampled from 2.5 kg buckets available in the market . Samples were collected from ten different buckets, from two different batches. Batches were correspondent to different consequent years (2018 and 2019), and acorns were harvested in two different geographical areas in the South of Portugal . The acorn flour in study is produced from Quercus rotundifolia specie acorns. In order to protect the flour from oxidation, smaller packages were prepared in food safe plastic bags and closed under vacuum.Acorn flour obtained from holm oak , through gravimetric measurements, after reaching constant weight at 130 °C. Ash Content was measured gravimetrically, incinerating samples at 550 °C in a muffle furnace L 9/R . Fat content was determined by using petroleum ether in a Soxhlet extractor Det-gras N , dried in the oven (overnight), and finally fat content was calculated gravimetrically. Protein content was assessed using Kjeldhal method (Nx6.25). Carbohydrates were determined by difference (100 - % moisture - % ash - % protein - %fat).2.4After previously determination of fat content, according to the aforementioned method, fatty acids profile was determined using the method ISO 5509:2000, with some modifications. Finally, methyl esters were prepared and then analysed in a GC-FID Agilent 7820A . Analysis were carried out in triplicate.2.5Mineral content was determined using acid digestion followed by absorbance reading in an Inductively Coupled Plasma Optical-Emission Spectrometry (ICP-OES) Thermo ScientificTM iCap Series 7000 . A sample of 0.25 g was digested with a mixture of nitric and hydrochloric acid (ratio 1:3) at 105 °C. The mixture was cooled down and when room temperature was reached, it was filtered and diluted to 50 mL with distilled water. The extracts were then analysed for K, Ca, Mg, P, S, Fe, Zn, and Mn. Analysis were carried out in triplicate.2.6Bioactive compounds extraction from acorn flour samples was performed weighting 40 mg of sample, and adding 1.5 mL of extracting solvent ) R . Samples2.6.1−1 DW)  .Ortho-diphenols in acorn flour samples were measured using the protocol as previously described in Ortho-diphenols content was quantified using gallic acid as standard, and results expressed in milligrams of gallic acid per gram of dry weight (mg GA g−1 DW).−1). After waiting 5 min, 28 μL of aluminum chloride (100 g L−1) were added and the mixture reacted for 6 min. Next, 120 μL of sodium hydroxide (1 M) were added to the mixture, the microplate was shaken for 30 s, and absorbance measured at 510 nm. Flavonoid content was quantified using catechin as standard, and results presented in milligrams of catechin per gram of dry weight (mg CAT g−1 DW)  .2.6.2•+ radicals inhibition was determined through the expression: ABTS radical inhibition was assessed with the following procedure: a sample (12 μL) or standard (12 μL) were poured into the microplate, and then previously prepared ABTS working solution (188 μL) was added. Next, the reaction was completed at room temperature, for 30 min, in the dark, and absorbance measured at 734 nm. ABTS−1 DW).Acorn flour extracts antioxidant activity was calculated by Trolox calibration curve interpolation, and the results were expressed in mmol of Trolox per gram of dry weight .DPPH antioxidant capacity was performed throughout adding in each well of the microplate, the sample (10 μL) or Trolox standard (10 μL), and DPPH (190 μL). Next, the incubation occurred in the dark, at room temperature for 30 min, and the absorbance was read at 520 nm. Free radical DPPH−1 DW).Ferric reducing antioxidant power (FRAP) was measured following the steps: the sample (20 μL) was placed in the microplate, and previously prepared FRAP working solution (280 μL) was added. Then, the reaction was performed in the dark, for 30 min at 37 °C, and mixing at 250 rpm, with an incubator PST-100HL , and finally the absorbance was read at 593 nm. The standard was Trolox, and results were expressed in mmol of Trolox per gram of dry weight , and solvent B, CH3CN/HCOOH , together with a non-polar stationary phase. A linear gradient scheme was used, with the following characteristics: : , , , , , , and . At 55 min, return to 5% of B to stabilize and prepare the column for the next sample. All of the analysis was performed at room temperature (25 °C) with a flow rate of 1.0 mL min−1. The injection volume of the samples was 20 μL. All samples were injected in triplicate. The equipment consisted of an LC pump (SRVYR-LPUMP), an auto-sampler (SRVYR-AS), and a photodiode array detector (SRVYR-PDA5) in series .The phenolic profile of acorn flour samples was assessed as described in  using a −1 DW)  .2.7g for 10 min. Finally, supernatant water was discharged, weighed again, and by gravimetrically difference, WBC was calculated. Results were expressed in g of water per g sample (g water/g sample) (The acorn flour water binding capacity (WBC) determination was performed according to AACC (1999), as described in  sample) .2.8Acorn flour total fiber (TF) and insoluble fiber (IF) content were determined by using a “Total Dietary Fiber Assay Kit” from Megazyme , following manufacture instructions. All procedures were done at least in duplicate. Soluble fiber (SF) was calculated by difference (SF = TF-IF).2.9Acorn flour starch content was determined using an enzymatic kit K-TSTA-100A , and then was measured in a spectrometer UVmin-1240 .Starch microstructural characterization was carried out with a scanning electron microscopy – SEM . Samples were observed with 400–1500x magnification.2.10The acorn flour pasting properties were performed using a Micro-doughLAB 2800 , following the manufacturer's cooking protocol with some modifications . Constan33.1−1) for acorn flour nutritional composition were: moisture 8.15 ± 0.03, ash 1.61 ± 0.01, total protein 4.28 ± 0.27, total fat 11.39 ± 0.53, and carbohydrates 74.56 ± 0.76. These results were in agreement with the values range obtained for proximal composition, in a study about variability between acorns from nine different trees, also Quercus rotundifolia, which were (in g 100 g−1): ash (1.37–1.76), protein (3.84–6.09), and fat (9.34–13.00)  .−1), such as rice (0.90), wheat (1.81), maize (2.48), sorghum (3.50), wholewheat (3.63), buckwheat (4.21), or oat (6.74). Concerning protein (4.28 g 100 g−1), acorn flour revealed a lower value than quinoa (13.48), buckwheat (12.19), wheat (11.54) and rice flour (7.33), but similar to sorghum (4.68), and higher than cassava (1.7) (Acorn flour presented the highest fat content when comparing with other common flours (11.39 g 100 gva (1.7) . Protein3.2Fatty acids profile of acorn flour is shown in Thus, acorn flour revealed a higher proportion of monounsaturated fatty acids (MUFA), mainly oleic, and in polyunsaturated fatty acids (PUFA), linoleic and α-linolenic, which are essential fatty acids. Both group of fatty acids, presents many benefits from an health point of view, such as preventing the risk of cardiovascular diseases, lowering blood serum triglycerides, and increasing HDL-cholesterol (high density lipoprotein cholesterol) levels, among others. Moreover, regarding functional advantages, acorn also presents β-sitosterol as the predominant phytosterol, and is an important source of tocopherols, exhibiting as well a profile quite similar to olive oil  41.00 Fe, 3.00 Mn, 6.5 Zn (−1), 8.1 Fe, 77.8 Mn, and 5.40 Zn. Thus, K, and P revealed quite similar results, Ca was lower, and Mg was higher, in comparison with Quercus robur acorn. Fe presented a really lower value, Mn was much higher, and Zn was similar. Quercus rotundifolia acorn flour showed to be a good source of minerals, in particular K, even when comparing with common flour sources such as wheat, oat or sorghum, which usually present lower values of these minerals. This is a relevant aspect, because potassium has an important role in muscles, and minerals in general are essential for human health, since take part in many metabolic processes , respectively 18.0 and 14.3 for acorn flour phenolic compounds , performing similar extracts to the ones in the present work, with methanol/water 80:20 (v/v). These authors found gallic acid, ellagic acid, and their derivatives. Moreover, it was also found several compounds that could not be identified in our samples, which were galloyl glucoside, valoneic acid dilactone, and their derivatives (ivatives . Despiteivatives .3.5−1 DW). Consequently, acorn flour could contribute to a reduction of the WBC when mixed with other flours, nevertheless the complexity of food matrices, along with the interactions of starch, fiber, protein, fat, and water, does not allow to establish a direct cause-effect answer.In −1 DW and 10.30 ± 1.13 g 100 g−1 DW for insoluble fiber. −1 DW and was quite lower (2.07–2.25 g 100 g−1) in Concerning total fiber, acorn flour revealed an amount of 11.40 g 100 g−1 DW, in accordance with other authors who also have studied starch, obtaining 51.79 ± 1.35 g 100 g−1 DW , corresponding to protein weakening, occurred at 71 °C with a drop of torque value to 1 mN m. Following the heating process, viscosity increased until reaching the peak (C3), consistent with starch gelatinization at 85 °C, and a torque value of 87 mN m. This higher viscosity corresponds to a maximum of swollen-intact starch granules, that with temperature increase, and continuously water absorption, granules achieve its rupture point, and viscosity starts decreasing until reach a minimum (C4), which occurred at 90 °C showing a torque value of 68 mN m. Breakdown viscosity is the difference between C3 and C4 (19 mN.m), which represents the resistance to heat and shear. In other words, when breakdown viscosity is lower, means a higher starch granules resistance to heat and shearing stress. Finally, the test finished when temperature cools (50 °C), with amylose retrogradation, and reaching the highest torque value of 129 m.Nm, which supports predicting final product texture characteristics. Setback is the difference between C5 and C4 (61 mN.m), and it is an indicator of starch ability to undergo retrogradation.In Quercus rotundifolia from Algeria (Acorn starch structure has been associated with fewer amylose leaching and lower degree of gelatinization, and also its gelatinization higher temperature showed an increase resistance to swelling in comparison with other flours. Moreover, a lower gelatinization viscosity reveals a reduced swelling capacity as well . Accordi Algeria , present4Acorn flour is an ingredient presenting good nutritional and functional characteristics to recover its use in human nutrition, in particular in bakery products, with potential to many other uses. Acorn flour presents a high fat content, particularly monounsaturated and polyunsaturated, with high antioxidant capacity, although its effectiveness in the final product should be evaluated according to each formulation and purpose, as well as bioavailability and bioaccessibility. In regards to technological aptitude, acorn flour has a high amount of fiber mainly insoluble, and presents a starch content approximately of 50%. Its pasting behaviour reveals a high gelatinization temperature and resistance to heat and shear stress, showing that acorn flour could have an important contribution to texture and structure, depending on the type of product developed. Thus, the high amount of acorns that are being wasted, have the potential to be a valuable ingredient, with many uses and applications in human nutrition, giving a new value to this resource. However this flour has been consumed in human diet for many centuries, further investigation about anti-nutrients would be recommended in a future research, in order to analyze if eventually any negative effects of acorn flour could be found.Rita Beltrão Martins: Conceptualization, Validation, Formal analysis, Investigation, Methodology, Data curation, Writing – original draft, Writing – review & editing. Irene Gouvinhas: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – review & editing, Supervision. Maria Cristiana Nunes: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – review & editing, Supervision. Luís Mendes Ferreira: Methodology, Validation, Formal analysis, Investigation, Resources, Writing – review & editing, Supervision, Funding acquisition. José A. Peres: Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition, All authors have read and agreed to the published version of the manuscript. Anabela Raymundo: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Ana I.R.N.A. Barros: Conceptualization, Validation, Formal analysis, Formal analysis, Investigation, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The formalism of mechanical compatibility of ferroelastic domains is further developed for the analysis of the geometry of the reciprocal space. The application of the algorithm to the reciprocal-space maps of tetragonal BaTiO J. Appl. Phys.40, 135–142] is adapted for the analysis of the X-ray diffraction patterns. The necessary equations are derived and an algorithm is outlined for the calculation of the separation between the Bragg peaks, diffracted from possible coherent twin domains, connected to one another via a mismatch-free interface. It is demonstrated that such separation is always perpendicular to the planar interface between mechanically matched domains. For illustration purposes, the analysis is presented of the separation between the peaks diffracted from tetragonal and rhombohedral domains in the high-resolution reciprocal-space maps of BaTiO3 and PbZrx1−TixO3 crystals. The demonstrated method can be used to analyse the response of multi-domain patterns to external perturbations such as electric field, change of temperature or pressure.Twinning is a common crystallographic phenomenon which is related to the formation and coexistence of several orientation variants of the same crystal structure. It may occur during symmetry-lowering phase transitions or during the crystal growth itself. Once formed, twin domains play an important role in defining physical properties: for example, they underpin the giant piezoelectric effect in ferroelectrics, superelasticity in ferroelastics and the shape-memory effect in martensitic alloys. Regrettably, there is still a lack of experimental methods for the characterization of twin domain patterns. Here, a theoretical framework and algorithm are presented for the recognition of ferroelastic domains, as well as the identification of the coherent twin relationship using high-resolution reciprocal-space mapping of X-ray diffraction intensity around split Bragg peaks. Specifically, the geometrical theory of twinned ferroelastic crystals [Fousek & Janovec (1969).  Domain switching, domain rearrangement and domain-wall motion may underpin/enhance the technologically important piezoelectric effect  is important for material design and properties engineering. Twinning is the subject of considerable interest in fundamental and applied science.Twinning is a common crystallographic phenomenon . Using synchrotron radiation adds the capabilities for in situ (e.g. stroboscopic) studies of domains at variable temperature and external electric field  domains alternate and connect to one another without a lattice mismatch. If formed and stable, such twin domain patterns may significantly enhance the ability of a material to respond to external perturbations and thus enable new domain-related physical properties. Specifically, we focus on the assemblies of ferroelastic domains . While the method assumes the availability of the reciprocal-space information alone, it may also assist in the interpretation of the dark-field X-ray microscopy data.3) and rhombohedral (PbZr0.75Ti0.25O3) symmetry is presented. We inspect diffraction from multi-domain ferroelectric crystals accordingly and display the means to assign different peak components to the individual domains.The article has the following organization. After the introduction of the glossary of symbols and important relationships, we recapitulate the well known formalism for mechanical compatibility of coherent patterns of ferroelastic domains in a way that is suitable for the analysis of X-ray diffraction from them. Then we analyse the orientation relationship between their reciprocal lattices and calculate reciprocal-space separation of Bragg peaks of twinned ferroelectric domains. The demonstration of the method for the identification of a coherent twin relationship in domains of tetragonal  are the basis vectors of a crystal lattice. {The terms lattice and structure are often misused in the recent materials science literature . The columns of the matrix [S] are the coordinates of ain with respect to aim:Transformation of the metric tensor. The transformation of the basis vectors (1)Twinning matrix. [T] represents a symmetry operation of the parent-phase lattice (i.e. the one built using the basis vectors ai0) that is no longer the symmetry operation of a ferroelastic phase lattice. We define [T] as a 3 × 3 matrix, which describes the transformation to the coordinate system ai0 from its symmetry equivalent e.g. 48 for a cubic lattice). The transition from a paraelastic to a ferroelastic phase is associated with the distortion of the basis vectors ai0 and m and n correspondingly. Appendix AGn] and [Gm] exists:n and m:The following group of notations is introduced for the compactness of the description of the connectivity of the lattices of domains G] = [G]n − [G]m is the difference between the metric tensors of the domains n and m. The reference to domain numbers in [ΔG] is omitted below., [V] is the 3 × 3 matrix, whose columns are the corresponding eigenvectors. The condition ΔGij = ΔGji means that λi are real and that [V] is orthogonal ([V]−1 = [V]T).Supplementary coordinate systems. The coordinate system vi is defined by the matrix [V] so that ≠ 0.:W] so thatwi can be defined with Z31 = +Λ or Z31 = −Λ.Vector coordinates. xi, vi, wi are the coordinates of an arbitrary vector with respect to the coordinate systems ai, vi and wi, so that xiai = vivi = wiwi. The following direct and inverse transformations between the coordinates apply:Supplementary metric tensors. [GV)(] are defined by the dot products GV)(] = [V]T[G][V] and [GW) are related by the matrix [S] [according to equation (1)S*]. The following relationship between [S] and [S*] holds:Reciprocal coordinates of a vector. B. We also use the notations hkl for the indices of a plane and HKL for the indices of a Bragg reflection.3.e.g. oxygen octahedra in perovskites  plane so that those have exactly the same in-(hkl)-plane two-dimensional lattice parameters. The theory of martensitic phase transformations ai to vi according to (5)V] are the eigenvectors of [ΔG] simplifies (12)e.g. λ2 = 0). Two cases may be considered:This section recapitulates the approach of Fousek & Janovec 1969, Sapriel1 = λ2 = 0, λ3 ≠ 0 yieldsv plane (the subscript v refers to the Miller indices with respect to vi instead of ai). Using (9)V13, V23, V33 to the integer numbers will give the Miller indices hkl of the mismatch-free plane.The case λ1 < 0, λ2 = 0, λ3 > 0 leads to two possible plane solutions of (14)Vi1 ± Vi3) to the integer numbers would give the Miller indices hkl of the habit planes between the domains. Alternatively, the indices of the habit plane can be expressed using the coordinate system wi correspondingly. Indeed, substituting (10)w (the subscript w refers to the Miller indices with respect to the coordinate system wi instead of ai).The case λThis formalism gives a well known result for the Miller indices of the possible habit planes between the domains of different symmetry. Some examples are presented further in Sections 64.S] [as defined by (1)aim and ain when the lattices of the domains m and n meet along their common (001)w plane. Let us first find the similar transformation matrix [Sw] between wim and win. The matching along the (001)w plane implies wm1,2 = wn1,2 so thatyi can be calculated as follows. First, consider that the determinant of the transformation matrix (|Sw| = y3) should be equal to the ratio of the unit-cell volumes; thereforeThe goal of this section is to find the transformation matrix n = [Sw]T[GW)(]m[Sw]) and (20)y1, y2 and accordingly all the elements of the matrix  [Sw]. Finally, the [S] can be found according to the equationNote that, although S] can be found by going through the following steps.Accordingly, the elements of the matrix [G]m and [G]n using (4)(i) Choosing appropriate twinning matrices and calculating the elements of the corresponding metric tensors [V]) and eigenvalues (λi) of [ΔG] = [G]n − [G]m. Mismatch-free connection of domains m and n is possible only if at least one eigenvalue is zero, while the remaining two have opposite signs.(ii) Calculating the eigenvectors  Rearranging the eigenvalues and eigenvectors in such a way that λZ] and [W] (either with Z31 = Λ or Z31 = −Λ) according to equations (5)G] are zero, [Z] is a unitary matrix.(iv) Using these eigenvalues and eigenvectors to form two pairs of matrices [GW)(]m and [GW)(]n according to equation (2)y1, y2, y3 using (21)Sw] according to (20)S] according to (25)(v) Calculating  is diagonal and λ2 = 0 we can set [V] = [I] and immediately obtain that  and [S*]:HKL Bragg peaks, diffracted from the domains 1(a) and 3(c):We now have to find the unknown coefficients B in the reciprocal coordinate system of the domain m. Note that the separation vector ΔB is always perpendicular to the domain wall.An identical analysis can be implemented for the (101) domain wall and other pairs of domains. Table 26.2.m3m / Let us assume that the lattices of the paraelastic/ferroelastic phases belong to the cubic/trigonal point symmetry group Six pairs may be formed between four ferroelastic domain variants. We will demonstrate the connection between domains 1 and 2:Vi1 ± Vi3)xi = 0 takes the formEquation (18)W] = [V][Z] will have to be introduced withGW) domain walls, the transformation matrix [y3 = 1 and applying the system of equations (23)S] = [W][Sw][W]−1:According to (21)W] = [V][Z] will have to be introduced withFor the case of the (100) domain wall, the transformation matrix [y3 = 1 and applying equation (23)S]:According to (21)m and n and corresponding separation of Bragg reflections in the reciprocal coordinate system of the domain m. As can be seen, the separation vector is always perpendicular to the domain wall.Here the following notation was introduced:7.et al., 2020et al., 2018e.g. ω) and converting three coordinates Xd Yd ω (XdYd are the coordinates of the detector pixels) to the coordinates of the scattering vector B relative to the chosen Cartesian  coordinate system. Such experiments are facilitated by the recent progress in synchrotron-based and home-laboratory X-ray sources, availability of pixel area detectors, beam conditioning systems and big-data exchange protocols , we collected high-resolution reciprocal-space maps of the diffraction intensity distribution around 102, 002, 222 and 103 reflections. The data were then represented in the form of three-dimensional diffraction intensity tables I. Here Bx, By, Bz refer to the Cartesian coordinate system such that the X axis is nearly parallel to the scattering vector, so that Bx is almost equal to the scattering vector length. Fig. 5Bx, By projections X axis . This splitting of the peaks along the X axis can be used to determine tetragonality or  the deviation of the lattice parameters from that of the cubic system.This section illustrates the recognition of a coherent twin relationship in a twinned BaTiOet al. . The lattice parameters are adjusted to achieve the optimal match between the sets of |Bobs| and |Bcalc|. Using combined analysis of the scattering vector lengths in four reciprocal-space maps, we obtained tetragonal lattice parameters a = 3.962 (1), c = 4.005 (2) Å. According to (37)Iz projections in Fig. 5Bz corresponds to the Z coordinate of the best matching sub-peak.The initial assignment of the sub-peaks to domains can be solely based on the analysis of the scattering vector lengths. The procedure was described by Gorfman  al. 2020. It inclIz, Iy and Ix projections. Figs. 6a)–6c) show the marked positions of the peaks . Moreover, it may also be used for the analysis of the connections of domains of different symmetry. Formation of the habit planes between domains is possible every time when at least one eigenvector of the matrix [ΔG] is zero. The implication of this condition in the cases when pairing of different phases  is in question will also be discussed in the forthcoming publications.We have demonstrated the procedure of domain recognition for the cases of crystals with tetragonal and rhombohedral domains. The same algorithm can be applied e.g. in the 002 reciprocal-space maps . Unpaired peaks may appear when e.g. the limited volume of the crystal is covered by an X-ray beam (because of strong absorption of an X-ray beam hiding some domains and keeping some of the peaks unpaired). This is the reason why assignment of peaks may fail in such cases. Note that an assignment may still be attempted based on the length of the reciprocal-lattice vector and the radial position of the peak in the reciprocal space. In some cases this means that suggestions of more than one domain for a single sub-peak may be possible.It is vital that the presented technique will be able to recognize domain pairs, as opposed to the individual domains themselves individually. Accordingly, some of the peaks may remain unrecognized. Such cases are apparent 11.3 crystal with tetragonal domains and a multi-domain PbZr0.75Ti0.25O3 crystal with rhombohedral domains. It can be useful for the analysis of the individual domains’ response to external perturbation .We developed the theoretical framework for the calculation of three-dimensional splitting of Bragg peaks, diffracted from a crystal with ferroelastic domains. Specifically, we extended the existing theory of domains’ mechanical compatibility to calculate the corresponding geometry of the reciprocal space. We have shown  that Bragg peaks always separate along the reciprocal-space direction that is perpendicular to the domain wall. The analytical expression for the Bragg peak separation for the cases of the entire domain wall between domains of tetragonal and rhombohedral symmetry was obtained. The formalism is illustrated using the example of single-crystal X-ray diffraction from a multi-domain BaTiO10.1107/S2053273322002534/lu5017sup1.pdfSupporting figures and tables. DOI:


Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium  Both show anti-inflammatory properties which might help in human chronic diseases like diabetes or obesity  and natural products derived thereof since the identification of erythromycin, an antibiotic identified in the early 1950s Demain, . In genepfa genes can be found in several microorganisms for which the production of PUFAs or derivatives thereof is mostly unknown  as well as shortened fabclavines (sFCL), named here as sFcl-A and sFcl-B, and polyamines  are widespread in Xenorhabdus strains and detailed analysis revealed a large chemical diversity of derivatives in each strain  polyamine biosynthesis in Xenorhabdus strains revealed a wide distribution of the fcl BGC, resulting in a large structural diversity  and 598.6334 [M + H]+ (2)  from X. budapestensis  . Compounof −4.29 . Therefo . CompouX. szentirmaii usually harbor only one FabA-like DH domain in FclD . The production of the compounds 1 and 2 were confirmed in the promoter exchange mutant of X. bovienii as well as in E. coli   Fig. .Aureispira marina the PKS-like DH domain is required for the dehydration in the early stages of arachidonic acid formation and an inactivation leads to dramatic decrease in the production titer  is an effective tool for PKS engineering to change the product spectrum DEFGH from X. bovienii. Both E. coli strains produce compound 2 , corresponding to a fabclavine hybrid, which consists of a NRPS-PKS-part from X. szentirmaii and the deoxy-polyamine from X. bovienii  beside an already described polyamine 2  polyamine biosynthesis of X. budapestensis and X. hominickii, both possessing naturally only the FabA-like DH domain in FclD. Thereby, deoxy-polyamines were produced independently of the PKS-like DH domain being covalently fused to FclC or coexpressed as stand-alone DH domain.In this work, the (fabclavine) polyamine biosynthesis in orhabdus . This adXenorhabdus, this additional DH domain seems to be another diversification mechanism during fabclavine biosynthesis (Wenski et al., Following the previously identified large diversity of fabclavine derivatives among the genus kuab006_Supplemental_FileClick here for additional data file.


Different factors are involved in the incidence, etiology, metastasis, diagnosis, and treatment of oral squamous cell carcinoma, including apoptosis inhibitor proteins. Baculoviral IAP repeat containing protein 6 (BIRC6) is one of the apoptosis inhibitor proteins contributing to cancer cells' survival in many cancer types with diagnostic and treatment importance. This study is aimed at assessing the serum level of BIRC6 in oral squamous cell carcinoma.  In this retrospective cross-sectional study, 60 serum samples were collected from 45 male and 15 female patients with a mean age of 61 years as the case group and 28 serum samples of healthy people as a control group. The serum samples were analyzed using a commercial sandwich ELISA kit.  There were no significant differences between BIRC6 serum levels in patients and healthy subjects. Moreover, we did not observe any significant relationships between BIRC6 serum levels and the patients' demographic or clinical characteristics.  There was no significant difference in serum BIRC6 levels in patients with oral squamous cell carcinoma and healthy individuals. Its use in determining the prognosis of squamous cell carcinoma or considering it a determinant marker in this type of cancer may not have a place. More in-depth studies for evaluating BIRC6 serum levels in oral squamous cell carcinoma patients are recommended for better insight into this protein's role in diagnosing, progression, and prognosis of the disease. Oral cain women . Oral sqin women . In a stin women . SCC comin women , 7. Therin women , 9.Different factors are involved in the incidence, etiology, metastasis, diagnosis, and treatment of the SCC, including the apoptosis inhibitor proteins . BaculovThe role of BIRC6 in many cancer types has been investigated, including the brain, leukemia, osteosarcoma, colon, and lung . AccordiThis study is aimed at investigating the presence of BIRC6 as a biomarker in oral squamous cell carcinoma. Determining the role of this marker in oral cancers can significantly impact the prognosis, diagnosis, and treatment of oral cancers.This study was approved by the ethics committee of Shiraz University of Medical Science (IR.SUMS.REC.1397.203). In this cross-sectional study, 60 serum samples from those affected by oral SCC, including 45 male and 15 female patients with a mean age of 61 ± 13 years as a case group, and 28 serum samples of healthy individuals, including 20 men and 8 women with a mean age of 61 ± 13 years as a control group were collected. All participants were hospitalized in the ENT units of the Shiraz Khalili hospital. SCC diagnosis was confirmed histopathologically. Participants who suffered from other malignancies by history, inflammatory, or infectious diseases (in the last three years) have been excluded from the study. The control group consisted of healthy volunteers without any history of cancer, inflammatory or infectious diseases on their own, and first-degree family in the last three years. The informed consent form was completed and signed by all the study participants at the beginning of the study.The blood samples were taken by venipuncture method and then centrifuged at 3,000 rpm for 10 min. Then, aliqouted serum samples were stored at -80°C for future analysis. After completing the sample, the BIRC6 concentrations were measured using a Sandwich ELISA kit , ELISA Kit) according to the manufacturer's protocol. The ELISA kit contains a 96-well microplate, precoated with the anti-BIRC6 monoclonal antibody. In order to perform the ELISA Sandwich assay, the manual of the MyBioSource BIRC6 ELISA (MBS760993) kit was followed.U statistic. Furthermore, the Mann–Whitney and Kruskal-Wallis statistics have been used for testing the association between serum BIRC6 concentration and clinical findings. The p value of less than 0.05 was statistically considered significant. Moreover, the correlation between age and BIRC6 concentration was evaluated using Spearman's rank correlation.We excluded 3 of 88 samples (2 cases and one control) from our calculations because the resulting concentration exceeded the kit's detection range (0.156-10 ng/ml). The BIRC6 concentrations of case and control samples were compared using the Mann–Whitney n = 60) and control (n = 28) was about 61 years, in the 35-88 years age range. The sex distribution of male and female participants of both groups was approximately 3 : 1. There were two exclusions in the case group and 1 in the control group due to highly exceeding the kit's detection range.In this retrospective study, SCC clinical samples were used. The demographic characteristics and clinical findings of patients who had been studied for BIRC6 serum levels were summarized in p = 0.788). BIRC6 serum levels were 0.20 ± 0.16 and 0.31 ± 0.41 ng/ml in men and women, respectively; however, the difference was not statistically significant between the sexes (p = 0.172). Moreover, the BIRC6 serum levels were increased by age, but this increase wasn't statistically significant . Findings showed that the mean of BIRC6 serum levels in SCC cases was 0.313 ± 0.438 ng/ml, which was higher than the control group with a 0.207 ± 0.106 ng/ml difference was not statistically significant . However, previous studies reported a significant association between the BIRC6 level of patients and healthy subjects, including colorectal cancers [This study included 85 participants  for BIRC6 serum levels. Findings did not show any significant association between BIRC6 levels of SCC patients and healthy subjects ( cancers , lung [1 cancers , and pro cancers . A study cancers . Our finAs noted, the finding did not show any association between BIRC6 serum level and clinical characteristics of oral squamous cell carcinoma, such as lymph node involvement. Although previous studies have shown a significant association between BIRC6 expression and lymph node involvement in lung cancer, lymphoma, thyroid cancer, and esophageal squamous cell carcinoma, the higher the BIRC6 serum level, the more lymph node involved in the disease. Ma and colleagues found a significant association between BIRC6 expression with cancer metastasis and lymph involvement ; howeverBecause there was no significant relationship between BIRC6 protein and clinical characteristics of this cancer type, we can conclude that BIRC6 may not have any significant role in predicting oral squamous cell carcinoma. Therefore, targeting this protein or corresponding gene may not help in the reduction of oral squamous cell carcinoma severity. This can be due to some limitations that any experimental study can suffer. Despite our precision in performing the study and quantitative measurement of the BIRC6 level using an ELISA kit, many factors such as metabolism, elimination of BIRC6 from blood and serum, and its half-life can impact our results. Moreover, the main purpose of this project was to measure the BIRC6 in blood samples as a low-aggressive low-risk method. Therefore, we recommend a more extensive investigation on BIRC6 expression in the tissue and serum with a bigger sample size of oral squamous cell carcinoma and different test types.It showed that the BIRC6 serum level did not have any significant association with age, sex, tumor size, differentiation, metastasis, lymph node involvement, and site of cancer. Moreover, we did not find any differences between patients and healthy subjects regarding BIRC6 serum levels. Therefore, for better generalization, we recommend a more in-depth study of the subject matter using a larger sample size using different molecular tests in order to understand the role of BIRC6 serum level and expression in oral squamous cell carcinoma.


Apis mellifera) was identified as an analogue model since it collects and processes propolis, which largely consists of collected tree resin. Propolis is a sticky substance used by bees to seal their hive and protect the colony against pathogens. In spite of its stickiness, honeybees are able to handle and manipulate propolis with their mandibles. We wanted to know if beneficial anti-adhesive properties of bee mandibles reduce propolis adhesion. The anatomy of bee mandibles was studied in a (cryo-)scanning electron microscope. Adhesion experiments were performed with propolis on bee mandibles to find out if bee mandibles have anti-adhesive properties that enable bees to handle the sticky material. A scale-like pattern was found on the inside of the mandible. Fresh mandibles were covered with a seemingly fluid substance that was at least partially removed during the washing process. Propolis adhesion on bee mandibles was measured to be 1 J/m2 and was indeed significantly lower compared to five technical materials. Propolis adhesion was higher on mandibles that were washed compared to fresh, unwashed mandibles. Results indicate that the medial surface of the mandible is covered with a fluid substance that reduces propolis adhesion. First results suggested that the surface pattern does do not have a direct effect on propolis adhesion.In a biomimetic top-down process, challenging the problem of resin deposition on woodworking machine tools, an adequate biological model was sought, which hypothetically could have developed evolutionary anti-adhesive strategies. The honeybee ( Nature has solutions for many of the problems and challenges humans face, since different creatures have adapted to similar challenges during their evolution. A common problem category in technology is unwanted adhesion; whether it is cake sticking to the cake tray, smeared glasses, or industrial production processes where unwanted build-up occurs. Specifically, this problem also occurs in woodworking processes, where escaping resins contaminate the cutting edge of the tools and, subsequently, blunt the carbide cutting edges through abrasion. This was the starting point of a biomimetic top-down development process that led to the anatomical investigation of honeybee mandibles.Apis mellifera) was chosen as a possible model because it collects and processes propolis, a sticky and ductile material, that is created by mixing plant resins, wax, and other substances. Similar to tree resins, which contaminate the surfaces of processing tools, propolis also adheres well to different types of surfaces [The hypothesis is that animal species that regularly have close contact with resinous plants or even actively harvest resins may have developed counter-stickiness strategies. This is because animals that permanently stick to a plant are deprived of further reproduction, which exerts a significant evolutionary selection pressure. The honeybee   in whichOne might expect that highly adhesive substances such as resin and propolis stick to the bee’s mouthparts and legs rendering them unusable. But bees are able to process these sticky substances and apparently without being negatively affected by them. It is therefore possible that anti-adhesive properties minimize adhesion of resins and propolis to their body parts. Various anti-adhesive strategies have been found in nature. Different mechanisms can lead to low adhesion. Possible strategies to reduce adhesion on the insect cuticle, as suggested in , are speOne example for possibly anti-adhesive surfaces that is especially relevant to this work, are Bornean stingless bees . Similar to honeybees, stingless bees harvest resin to build their nests and defend it against intruders . Resin cThe main objective of this work was to understand the interaction between honeybee mandibles and propolis. The hypothesis is that beneficial anti-adhesive properties have evolved to handle propolis without being hindered by resin contamination. Possibly, these anti-adhesive properties could serve as an inspiration to help solve problems such as resin contaminating woodworking tools.The anatomy of bee mandibles has previously been described –819, tho8Raw propolis provided by private beekeeper Dr. Oliver Schwarz   was harvApis mellifera) were collected in gardens in Kiel (Germany) in July 2019 and immediately used for experiments. They are hereinafter referred to as „nectar collectors“. In October 2018, bees were taken directly from the hive . They are further named „hive bees“. They were kept in a cage and provided with water and honey until they were prepared for experiments. The remaining bees were frozen and stored at −20 °C for three months to be used in further experiments.Adult worker bees (Apis mellifera), returning to the hive with resin attached to their pollen baskets, were caught and subsequently frozen in September and October 2018. These resin-collecting bees will be henceforth referred to as “propolis bees”.Honeybees , and a confocal 3D laser scanning microscope in order to identify anatomy and surface structure.Mandibles of all collected bees were prepared under binoculars by carefully separating them from the insect’s head with a scalpel . GeneralSurface structures on mandibles were studied in the SEM as described above. Additionally, the medial surface profile of the mandible was studied on fresh, chloroform-washed specimens fixed to a glass slide using a drop of a melted mixture of 50% paraffin wax and 50% colophony. The mandibles were examined with a confocal 3D laser scanning microscope . The MultiFileAnalyser software  was used to measure profiles and the structural dimensions in different areas of the mandible.Freshly defrosted and prepared mandibles of propolis bees were studied in the cryo-SEM in the frozen state so the resin contaminations did not dry out. They were placed on holders , rapidly frozen on the table in the preparation chamber at −140 °C, sputter coated with gold palladium (3 nm), and studied at −120 °C with a cryoSEM  equipped with a cryopreparation system  at 3 kV accelerating voltage.SEM images were processed using Gimp, version 2.10.14. All adjustments were applied to the whole image. Color levels, contrast, and brightness were adjusted, and scale bars and other labels were added. Profiles measured with the confocal 3D laser scanning microscope were digitized using WebPlotDigitizer .After insects for experiments were caught, they were placed and stored in the freezer at −20 °C for a minimum of 15 min and up to many months. The mandibles were prepared as described above . WithoutAdhesion experiments were performed in a manner similar to that in . Just be/m and 539 N/m were used) and a fibre-optic sensor  was attached to the metal spring with cyanoacrylate glue . The freshly formed, cone-shaped sample of propolis was then mounted on the tip of the capillary without any additional glue. The propolis sample was brought into contact with the substrate and retracted from the surface as soon as the load force reached 5 mN. The load was chosen to resemble the force applied by bees when handling propolis. As no studies exist on mandibular forces and pressures of honeybees, pressures measured at the tip of mandibles of predacious coleoptera [The effective elastic modulus and pull-off force of propolis were measured with a microforce measurement device  –24. The c sensor . The pieFa is the applied force and A is the contact area obtained from the contact radius.where With each propolis sample, a set of ten single measurements was performed, each on a different spot on the spoon-shaped tip of the bee mandible, starting at the sharp edge and moving towards the hairy edge . AnotherN = 5–15 propolis samples and mandibles, n = 10 measurements on mandible per sample). After the adhesion experiments, the substrate material was examined under a binocular microscope (Leica M205 A) in order to find possible propolis residues/prints in the contact area. Adhesion tests on fresh mandibles were performed in July and November. As a reference, propolis adhesion on bee mandibles was compared to propolis adhesion measured on glass under dry and fluid conditions [Experiments were carried out at room temperature (24.00 ± 0.53 °C) and a relative humidity of 36.80% ± 9.0%. For every condition, 50–150 single tests were performed on different spots of the mandible . The samples were then fractured within the preparation chamber by cutting off a part of the mandible tip using a cold scalpel blade mounted on a user-controlled handle. Fractured samples were then sputter coated and examined in a frozen state as described above.Where possible, the contact angle of the substance coating the mandible surface was measured on SEM images of fresh fractures of untreated bee mandibles using Gimp, version 2.10.14. For this purpose, two measurement lines were drawn: one along the surface of the cuticle and one along the surface of the substance on top of the cuticle. The angle was then measured between the two lines.® HPLC stabilised with 1% ethanol, Carl Roth GmbH & Co. KG), or acetone  for 5 min followed by 5 min washing with distilled water. Distilled water was used as an agent for removing hydrophilic substances from the surface of bee mandibles. Chloroform and acetone are known to remove hydrophobic substances and the wax layer from mandibles [Furthermore, different solvents were used to wash fresh mandibles in order to try removing any potential surface coating. Mandibles were washed in an ultrasonic bath with distilled water for 10 min or with either chloroform . Mandibles washed with chloroform and water were studied in the cryo-SEM as described above for untreated mandibles.To test the effect of potential surface coatings on propolis, adhesion experiments with propolis were performed on mandibles washed with the different methods as described for untreated bee mandibles (section “Investigation of propolis adhesion on mandibles”).In order to test how shape and surface structures of the mandibles affect adhesion, replicas of bee mandibles were made using a two-step moulding method ,28. The N = 5–6 propolis samples and mandibles, n = 10 measurements on mandible per sample).Adhesion experiments with propolis were carried out as described for real bee mandibles (section “Investigation of propolis adhesion on mandibles”). For mandible replicas, reference measurements were performed on the smooth resin substrate. 50–60 single tests were performed on different spots of the mandible replica . An unpaired two-sample When bees handle propolis, the mandibles are the first surfaces that will come into contact with plant resin or propolis. They were thus analysed using various imaging methods.Apis mellifera) was studied using a binocular microscope and SEM on acetone-cleaned specimens. Mandibles have a strong stem at the proximal end and a concave, spoon-shaped tip at the distal end . For some measurements, near the hairy edge of the bee mandible the point of contact was not clearly distinguishable in the loading curve due to hair touching the sample before it contacted the cuticle. However, in the unloading curve the adhesive failure (pull-off) was easy to identify in most cases.The adhesion of propolis samples on several untreated bee mandibles (BM) was measured to determine whether mandibles of bees possess anti-adhesive properties. No statistical differences were found between work of adhesion values of single measurements that were conducted on different positions of the bee mandible  , Table 1/m2 (pull-off force: 0.71 ± 0.33 mN), which is significantly lower than adhesion on dry glass   [/m2, P = 1.0) [The mean work of adhesion on fresh, untreated mandibles was measured to be 1.01 ± 0.21 JP = 0.9999). Compared to fresh mandibles, propolis adhered significantly stronger to mandibles that had been stored in the freezer (−20 °C) for three months prior to the experiment .There were no significant differences between adhesion on fresh mandibles caught and tested in October and July (/m2 (P < 0.0001) when the contact time of the propolis sample was increased to 60 s.Compared to the work of adhesion on fresh bee mandibles where the propolis sample was retracted as soon as the maximum load was reached, the work of adhesion increased significantly to 3.45 ± 1.44 JTo figure out possible reasons for low propolis adhesion on bee mandibles, freeze fractures of freshly prepared mandibles were studied in the cryo-SEM . The difDifferent washing treatments were applied in order to remove the surface layer from the mandibles. Examination in the SEM showed that on mandibles washed with water the residues of the substance covering the surface were still present to a similar extent as on untreated mandibles studied with the same method –F. On ma/m2 (P = 1.0). Compared to fresh, untreated mandibles, a significantly higher work of adhesion of 1.87 ± 0.49 J/m2 (P = 0.0014) was measured on mandibles washed with chloroform and water and an even higher work of adhesion of 2.14 ± 1.20 J/m2 (P < 0.0001) on mandibles washed with acetone and water. The work of adhesion of propolis on washed bee mandibles was significantly higher, when the contact time was increased to 60 s and compared to values for 0 s . Comparing the work of adhesion obtained from tests with a 60 s contact time performed on untreated and washed mandibles shows a significantly higher propolis adhesion on washed mandibles (P < 0.0001).Adhesion of propolis was tested on washed bee mandibles and compared to adhesion on untreated specimens , Table 1/m2) and on a smooth surface of Spurr’s resin (P = 1.0). Adhesion on fresh bee mandibles was however significantly lower than on both substrates made from Spurr’s resin (P < 0.0001).In order to test the effect of the surface structures and topography of the mandible as well as material properties on propolis adhesion, further adhesion experiments were performed. The morphology of real mandibles including the microstructures were successfully replicated in mandible replica made from Spurr’s epoxy resin . PropoliA honeybee mandible is a highly specialised organ that is used for multiple purposes such as feeding, defence, and propolis foraging and processing –7. UsingMicrostructures were found on the medial surface of honeybee mandibles. These anisotropic structures looked like scales and their proportions and orientation differed depending on the location on the mandible. The structures faded and eventually disappeared towards the apex and hairless edge of the mandible. It is possible that the scales in these areas are worn out by intensive use. However, it can be assumed that this is the normal morphology of the mandibles, as all examined mandibles had this characteristic. Anisotropic structures are also present on other animals such as snakes and were proposed to support anisotropic properties, for example, anisotropic friction . The scaMandibles of propolis bees exhibited the same features as mandibles of regular worker bees. They were however frequently contaminated with varying amounts of resin. This shows that bees do not seem to be able to completely prevent resin adhesion. Nevertheless, resin residues were easy to remove from the mandibles and did not adhere strongly to the mandible surface. Worker bees that were not observed foraging resin did not show similar contaminations. This could indicate that bees are able to clean their mandibles and get rid of resin contaminations. But bees exhibit a division of labour and older bees are believed to be responsible for resin foraging . Therefo2 (2) occurred on fresh bee mandibles compared to technical surfaces such as glass, steel, PTFE, or Spurr resin (2 [It has been reported  that proresin (2 ). It wasresin  is structured with scales that were suggested to lower adhesion [Nepenthes alata and lizard skin have been found to enhance unidirectional liquid transportation [In some cases, it has been described that microstructures can reduce adhesion by decreasing the contact area –49. The adhesion . Resin aadhesion . As scaladhesion . For insortation . The conReduced propolis adhesion was observed on the surface of bee mandibles. The anti-adhesive strategy found in bees might also be helpful in solving technological adhesive problems, for instance, unwanted resin contamination of woodworking tools.The next step in the further course of the top-down development process is the attempt to further understand the principle of the presumed anti-adhesive strategy of the honeybee and to verify it in replicas. The abstraction of the principle or structures found and the transfer to the technosphere with an effectiveness test under technical production processes are the next steps in the top-down procedure.Studying the interaction between propolis and bee mandibles revealed that propolis adhesion is indeed reduced on bee mandibles. Propolis adhesion is four times reduced in the presence of a natural secretion layer on top of the mandibular epicuticle, while the scale-like micropattern on the mandible might help the spreading of the fluid. The lubricating layer could be removed with organic solvents. In addition, the hair on mandibles is likely to help in the cleaning process.


The precise segmentation of the optic cup (OC) and the optic disc (OD) is important for glaucoma screening. In recent years, medical image segmentation based on convolutional neural networks (CNN) has achieved remarkable results. However, many traditional CNN methods do not consider the cross-domain problem, i.e., generalization on datasets of different domains. In this paper, we propose a novel unsupervised domain-adaptive segmentation architecture called CAE-BMAL. Firstly, we enhance the source domain with a convolutional autoencoder to improve the generalization ability of the model. Then, we introduce an adversarial learning-based boundary discrimination branch to reduce the impact of the complex environment during segmentation. Finally, we evaluate the proposed method on three datasets, Drishti-GS, RIM-ONE-r3, and REFUGE. The experimental evaluations outperform most state-of-the-art methods in accuracy and generalization. We further evaluate the cup-to-disk ratio performance in OD and OC segmentation, which indicates the effectiveness of glaucoma discrimination. In the past few years, retinal fundus images have been used to diagnose retinal diseases. Glaucoma is a disease that causes damage to the optic nerve of the eye, resulting in decreased vision, but timely detection can further control the effect of glaucoma. Automated segmentation of the optic disc (OD) and optic cup (OC) in fundus images is helpful for the screening and diagnosis of glaucoma , to augment the data and improve OD/OC on the dataset segmentation accuracy. Our method is based on two main observations. First, as depicted in Our main contributions are summarized as follows: (1) We propose a new domain-adaptive segmentation framework that uses convolutional autoencoders to enhance source domain images to generate enhanced domains with the same semantics as the source domain images, but with different light and vessel shapes. Then they are put into the network to train together, in the segmentation process, the adaptive segmentation of the network under the influence of surrounding blood vessels is strengthened, and the generalization ability of the model is improved; (2) Our proposed network integrates the ideas of boundary prior and adversarial learning, and the two boundary subnetworks have their functions. Combined, the boundary discrimination can still show good segmentation performance in harsh conditions . (3) The proposed segmentation method is clinically meaningful in glaucoma screening based on extensive evaluations on three publicly available fundus image datasets.boundary discriminator for enhanced and source domains, mask discriminator for new source and target domains, boundary discriminator for new source and target domains, as shown in The network architecture is exhibits in How to generate images useful for fundus image segmentation has become a major challenge due to the time-consuming and labor-intensive manual labeling of fundus images and the rarity of labels. To solve this problem, we use a convolutional autoencoder to generate images similar to the original image but different in blood vessel structure and brightness. For the convenience of elaboration, we have added Q, and the decoder function is represented by G. Assuming that the encoder function gets the intermediate code z, the input image is represented by xs ∈ XS, and the output image is represented by Formally, we aim at solving the problem of domain adaptation: A model is trained on a source domain XS in vascular performance and brightness. In other words, we want to maximize the domain discrepancy between XS. Therefore, the reconstruction error LCAE for domain augmentation can be formulated as:Intuitively, we expect the augmented domain LCAE creates large domain transportation.The pre-trained CAE can better capture the distribution of the source domain and maximize The reason for choosing a convolutional autoencoder is that data slicing and data stacking can cause a large amount of information to be lost. The convolutional autoencoder abandons the stacked data, keeps its spatial information unchanged when the image data is input, and gently extracts the information in the convolutional layer. This process aims to preserve the spatial relationship in the data, but it does not generate the same data as GANs  in the augmentation domain. This method can further expand the source domain so that data from other domains can also perform well when training on the model, achieving the purpose of model generalization.W is the width of the picture and H is the height of the picture. And our enhanced domain generated by the convolutional autoencoder CAE is denoted as xs ∈ XS belonging to the source domain, an image ys ∈ YS. When the source domain image is generated by CAE and becomes the enhanced domain, it will be put into our model together with the source domain image and the target domain image for training. The difference is, as shown in The source domain is XS (denoted as 1) or belongs to the enhancement domain There are three discriminators with a different function for each. The first one is the discriminator LD is a common binary cross-entropy loss. At this time, in order to ensure that the boundary distribution of the enhancement domain will approximate the boundary distribution of the source domain, we need to further optimize the segmentation network:where N is the number of images in the source domain and the enhanced domain. Since the images in the enhanced domain and the source domain are generated with 1:1 (see Section 2.1), the number is N. And Db, input is judged to belong to the union XU of the source domain XS and the enhanced domain XT (marked as 0). Henceforth, this union XU is called the new source domain. This setting is to ensure that the boundary distribution of the target domain is close to the boundary distribution of the new source domain, and to improve the prediction accuracy of the target domain in a boundary-driven manner. Therefore, the training objectives of the boundary discriminators of the new source and target domains can be set as:The second one is the discriminator NU is the number of pictures in the new source domain, and NT is the number of pictures in the target domain. At this time, in order to ensure that the boundary distribution of the target domain will approximate the boundary distribution of the new source domain, we still need to further optimize the segmentation network:where Dm. If only boundary-driven confrontation is used, it is still easy to generate uncertain predictions inside the optic cup and optic disc, resulting in an only good performance at the boundary. However, the goal is to segment the entire optic cup and the optic disc. Therefore, we also need to perform adversarial learning on masks to narrow the distribution difference between the new source and target domains. Specifically, the objectives of our mask discriminator for new source and target domains can be set as:Finally, there is a discriminator At the same time, we still need to optimize the segmentation network and perform adversarial learning to fool the discriminator and make the target domain images generate prediction masks close to the new source domain. The detailed settings are as follows:Ladvm, Ladvb, and Combine As it is shown in Db and Dm have a similar structure, which contains five convolutional layers, and the number of channels is 64, 128, 256, 512, 1 and in that order. And the kernel size of these five layers is 4 × 4, and the stride size is 2 × 2. After each convolutional layer, there is a LeakyReLU activation function instead of the ReLU (α = 2). But the last layer is special, using the Sigmoid activation function. Through recursive iteration, the receptive field size of each discriminator is 94 × 94. Subsequently, each patch output size is 16 × 16 and is distinguished as true (1) or false (0) by Db and Dm.For the specific structure of the convolutional autoencoder part, see Section 2.1. The last part is the discriminator. The three discriminators Lm is the mask prediction loss, which takes the form of cross-entropy and performs multi-label classification processing , (5), and (7).where λWe evaluate the proposed method with three public available datasets REFUGE challenge dataset  and the Vertical Cup-to-Disk Ratio (CDR) to evaluate the segmentation performance. Among them, DSC and CDR are defined as:TP, TN, FP, and FN represent the number of true positives, true negatives, false positives, and false negatives at the pixel level, respectively. d(OC) and d(OD) represent the vertical diameters of OC and OD and are used to represent the error between the predicted cup-to-disk ratio and the cup-to-disk ratio given by the actual clinician. We want the bigger the DSC the better and the smaller the better. The reason why we choose these two evaluation indicators is that the dice coefficient is usually a commonly used evaluation indicator for segmentation tasks, especially for optic cup and optic disc segmentation tasks, and the CDR value is one of the important indicators used in glaucoma screening in clinical medicine.where We compare our method with state-of-the-art domain adaptation or domain generalization segmentation methods: BEAL  is defined as the area under the ROC curve. The larger the area, the better the effect. We plotted the ROC curve in We combine the optic cup and optic disc segmentation results of the MICCAI 2018 report REFUGE challenge and use the same dataset. The challenge is weighted according to the following formula, and the individual and total scores are ranked:A total of 12 teams were selected for this challengeDSCcup and DSCdisc, the BMAL has a higher improvement than the CAE module. However, for the δ, CAE improves more than the BMAL module; (2) After combining the two modules, there is a common improvement in the three indicators, especially δ is reduced by 1.59%; (3) At the same time, adding DSCcup and DSCdisc, the accurate segmentation of OC and OD does help to estimate the value of δ.In this section, we discuss the performance of each part on the REFUGE dataset and its impact. We use the backbone based on DeepLabv3+ and the architecture with Atrous Spatial Pyramid Pooling (ASPP) components as the baseline. We added CAE, BMAL, and By observing This paper compares the three backbone networks of ResNet-34  http://medimrg.webs.ull.es/research/downloads/; (REFUGE)  https://refuge.grand-challenge.org/.Publicly available datasets were analyzed in this study. This data can be found at: (Drishti-GS) XZ: conceptualization, supervision, writing, and review and editing. JS: conceptualization, methodology, writing, data acquisition, analysis, and interpretation. CW: validation and visualization. ZZ: data acquisition, data curation, validation, and visualization. All authors contributed to the article and approved the submitted version.This research was supported in part by the Natural Science Foundation of Chongqing, China (No. cstc2019jscx-mbdxX0021), in part by the Project of Key Laboratory of Tourism Multisource Data Perception and Decision (Ministry of Culture and Tourism) and in part by the Key Cooperation Project of Chongqing Municipal Education Commission (No. HZ2021008).Author CW was employed by Chongqing Telecom System Integration Co., Ltd., Chongqing, China. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.