The objective was to modify functional properties of breadfruit flours using twin-screw extrusion and test the physicochemical properties of the extruded flours. Extruded breadfruit flours were produced with twin-screw extrusion using different last barrel temperature (80 °C or 120 °C) and feed moisture content (17% or 30%). These conditions resulted in four extruded flours with different mechanical  and thermal (melt temperature) energies. At temperatures below the gelatinization of the native starch (<70 °C), swelling power was increased in all extruded treatments. Solubility was dramatically increased in high-SME extruded flours at all tested temperatures. Water holding capacity was dramatically increased in the low-SME extruded flours. A two-fold higher cold peak viscosity was obtained for low SME-high temperature extruded flour compared with the other extruded flours. Low SME-low temperature extruded flour still exhibited a hot peak viscosity, which occurred earlier than in native flour. Setback was decreased in all extruded flours, especially in high-SME treatments. The incorporation of extruded flours into soy protein gels did not affect cooking loss, while hardness and springiness decreased with the addition of extruded flours. Overall, extrusion of breadfruit flour altered functional flour properties, including water holding capacity and pasting properties, and modified the texture of soy protein gels. Breadfruit is a high-yielding tropical staple crop grown throughout Oceania, Central Africa, and Central America. Breadfruit is a relatively low-input crop since the tree does not need to be replanted each year and its production lifespan is 50 years or potentially more . TherefoChemical, physical, and enzymatic modification have been widely used to improve the functional and physicochemical properties of flours and starches. In recent years, physical modification has garnered attention because of several advantages, such as cost-saving and consumer acceptance (and demand) in terms of natural and clean-labeled ingredients ,19. The Proteins are able to form different gel structures and provide various textural properties in processed food products . The properties of the gels are affected by the protein source, concentration, pH, ionic environment, processing time, and interactions with other ingredients . Soy proThe goal of this study was to modify functional properties of breadfruit flour with twin-screw extrusion technology using different conditions during extrusion. Therefore, breadfruit flours were extruded at different moisture (17% or 30%) and temperature conditions (80 °C or 120 °C in the last barrel) in order to achieve different mechanical and thermal energies. It was hypothesized that extruded flours would have altered viscosity (more viscous at lower temperatures and less viscous at greater temperatures), greater solubility, and improved swelling power when compared with native flour. It was further hypothesized that the extruded flours would have great potential as a texture enhancer and stabilizer in hydrothermal conditions close to the gelatinization temperatures of the native breadfruit flour.Native breadfruit flour was provided by Natural Foods International . Breadfruit flour was extruded as detailed in Pico et al.  with sliExtruded products were dried overnight at room temperature and then ground with a rotor mill  equipped with a 500 μm mesh screen. Extruded breadfruit flours were stored in air-tight plastic bags at −20 °C until further analyses. v/v) to remove D-glucose and stirred with 2 M potassium hydroxide at approximately 4 °C (ice/water bath) to pre-dissolve resistant starch.Moisture, lipid, protein, and ash content were determined with the AOAC methods 925.10, 920.85, 992.15, 923.03, respectively ,49,50,51pH values were measured in a homogenate prepared with 2 g of flour sample (both native and extruded flour samples) and 10 mL of distilled water using a benchtop pH meter  following calibration with buffer solutions of pH 4.0 and pH 7.0.g on a benchtop centrifuge . The clear supernatant was carefully discarded following centrifugation. Water holding capacity was expressed as grams of water bound by 1 g of dried flour sample. Oil holding capacity was expressed as the weight in grams of oil bound to 1 g of dried flour sample.Water and oil holding capacity were measured using similar methodology that was previously reported by Huang et al. . The floPasting properties of the flours (both native and extruded flour samples) were analyzed using a Rapid Visco Analyser 4800  with the maximum holding temperature set at 95 °C. The moisture of flour samples was measured prior to analysis using a moisture analyzer  in order to obtain the correct flour sample weight and the amount of water required for the test. The flour suspension  was poured into the RVA canister. Subsequently, the samples were subjected to a heating-cooling cycle in which temperature was initially held at 25 °C for 6 min, increased to 95 °C with a heating rate set to 14.0 °C/min, maintained at 95 °C for 6.5 min, decreased to 25 °C with a cooling rate set to 14.0 °C/min, and finally maintained at 25 °C for 5 min. The following pasting parameters were calculated from the pasting curves—cold peak viscosity as the maximum viscosity of the flour slurry at room temperature (25 °C); hot peak viscosity as the maximum viscosity during the heating step; breakdown as the difference between minimum viscosity upon cooling and hot peak viscosity; final viscosity as the viscosity at the end of the test, which indicated the ability of the flour to form a viscous paste or gel after cooking and cooling; setback obtained as the difference between minimum viscosity upon cooling and final viscosity; and peak time was estimated as the time at which hot peak viscosity was reached.Scanning electron microscopy  was used for the determination of granule morphology. Flour samples were sprinkled onto double-sided adhesive tape attached to a circular specimen stub. A uniform layer of sparsely scattered particles was obtained after carefully removing excess powder with a brush. The specimen holder was then transferred to the sample chamber and the flour samples were observed and photographed at an accelerating potential of 10 kV.g for 10 min. The supernatant was poured into a weighed aluminum dish and evaporated at 100 °C in a drying oven  for 24 h. The weight of dry solids and the weight of wet sediments in the centrifuge tube were determined for the calculation of solubility and swelling power, respectively.Solubility and swelling power were measured using a similar methodology that was previously reported by Huang et al. . One wt%Soy protein isolate  and sodium chloride  were sourced from commercial vendors. Water, soy protein isolate, flour (both native and extruded flour samples), and sodium chloride were formulated at consistent levels of 74%, 20%, 4%, and 2%, respectively. The soy protein isolate and flour inclusion levels of gels was kept constant at inclusion levels of 20% and 4%, respectively, to ensure that a self-supporting gel was formed. The soy protein isolate inclusion level of 20% was similar to previous studies ,53. The Thirty grams of uncooked, mixed and prepared protein/flour batter was placed in 50 mL centrifuge tubes with constant agitation to prevent air voids. Samples were placed in a water bath  set to 87 °C and cooked to an internal temperature of 85 °C. The samples remained at 85 °C for 25 min. The cooking temperature was kept constant at 85 °C to ensure that a self-supporting gel was formed after cooking. This endpoint cooking temperature was determined based on preliminary experiments.After cooking, test tubes were removed from the water bath and inverted for 16 h at room temperature to release the exudate. The samples were weighed again to determine cooking loss after the release of the exudate.Texture profile analysis (TPA) of protein gel samples was conducted using a texture analyzer  in accordance with methodology described previously by Huang and Bohrer . Protein65, a 0° viewing angle, and an 8 mm aperture. The standard CIELAB Color System  was used for the evaluation of instrumental color. The cooked protein gel samples were freshly cut into cylindrical cores (10 mm length and 28 mm diameter) immediately prior to evaluation. Six cylindrical cores were obtained and evaluated from each gel sample.Instrumental color was measured using guidelines presented by the American Meat Science Association . Instrumn = 3 for each flour). Each replication was at the minimum conducted in duplicate for each laboratory procedure, and the coefficient of variation thresholds were used to ensure precision and repeatability. Statistical analyses were performed using Statistical Analysis System software . The data were analyzed with PROC GLIMMIX of SAS with the fixed effect of flour and the random effect of replication. Least square means were separated using the PDIFF option with a Tukey–Kramer adjustment. Differences were considered statistically different at p ≤ 0.05. Pearson correlation coefficients were calculated among parameters using PROC CORR of SAS.All laboratory procedures were conducted in three independent replications for each treatment  moisture content compared with the native flour. This caused starch, protein, lipid, and ash content to differ (p < 0.05) on an as-is basis (p = 0.20) starch content in extruded flours when expressed on a dry matter basis. Extrusion did not change (p > 0.05) the protein content (expressed on a dry-matter basis) in LS-LT, HS-LT, and HS-HT extruded flours compared with native flour (protein ranged from 5.41% to 5.53%), but the protein content of LS-HT extruded flour (5.26%) was slightly less than (p < 0.05) the native flour (5.54%). The lipid content (expressed on a dry matter basis) of the native flour was 3.51%, which was greater than (p < 0.05) all the extruded flours (ranging from 1.74% to 1.99%). This was expected, as detectable lipid content is often reduced following extrusion due to reduced extractability [p < 0.05) for all extruded flours compared with native flour, which may be due to a better extractability of minerals in the disrupted tissue matrix after extrusion.The extruded flours had less . In this sense, breadfruit pulp is rich in ascorbic acid , and this value varies with maturity state . Howeverp < 0.05) for LS-LT and LS-HT extruded flours, while the water holding capacity was only slightly increased (p < 0.05) for HS-LT extruded flour and did not significantly increase for HS-HT extruded flour compared with native flour. The differences in water holding capacity among extruded flours was likely due to the fact that extrusion in the lower SME treatments (LS-LT and LS-HT) was carried out at a greater moisture content (30%). Moisture can operate as a plasticizer during extrusion cooking, which can reduce the degradation of starch and lead to an increased water absorption ability [The water holding capacity of extruded flour in this study ranged from 2.66 g water/g flour to 5.01 g water/g flour. These values were similar to the values for the water absorption index (which ranged from 2.543 to 4.285 g water/g flour) of extruded and expandable breadfruit products reported by Ma et al. [ ability . On the  ability , as discp < 0.05) in all extruded flours when compared with the native flour. Among the extruded flours, oil holding capacity was slightly less (p < 0.05) in HS-LT extruded flour compared with the other three extruded flours. Oil holding capacity depends on hydrophobic groups in the material and the ability of the material to maintain oil within its structure [Oil holding capacity was dramatically lowered (tructure . The redtructure .p < 0.05) cold peak viscosity and HS-HT extruded flour had the lowest (p < 0.05) cold peak viscosity. Lower values of cold peak viscosity suggested more amylopectin was fragmented during extrusion [p < 0.05) final viscosity and setback values compared with the extruded flours. During cooling, the extent of the increase in paste viscosity is governed by the tendency of the starch to re-associate by hydrogen bonding, known as retrogradation [Pasting viscosity profile and pasting properties of native and extruded breadfruit flours are shown in xtrusion . These rxtrusion ,27. The radation . The redradation . This drradation ,27,56,65The typical structure of pre-gelatinized flour was observed in the extruded breadfruit flour samples . Starch p < 0.05) compared with native flour. At 30 °C, the solubility of extruded flours ranged from 25.76% to 41.84%, while the solubility of the native flour was 11.34%. Likewise, Ma et al. [p < 0.05) than that of high SME extruded flours (HS-LT and HS-HT), with significant differences between LS-HT, HS-LT, and HS-HT extruded flours at higher temperatures (70 °C and 90 °C). Low SME extruded flours (LS-LT and LS-HT) were extruded at a greater moisture content compared with high SME extruded flours (HS-LT and HS-HT), which suggests that starch was more degraded in high SME extruded flours due to a greater shear-fragmentation on starch molecules [The solubility of both native and extruded flours increased when samples were heated from 30 °C to 90 °C A. At eacolecules . Interesp < 0.05) swelling power at lower temperatures (30 °C and 50 °C) compared with native flour (The extruded flours were pre-gelatinized and had greater (ve flour B. Moreovp < 0.05) than the extruded flours at 90 °C . Regarding extrusion conditions, LS-HT extruded flour had greater (p < 0.05) swelling power at 30 °C and 50 °C compared with the other extruded flours. This was in agreement with the results seen for pasting profile, which showed that LS-HT had greater viscosity compared with the other extruded flour until approximately 65 °C to 70 °C, at which point LS-LT extruded flour reached similar viscosity values. This could imply that LS-HT was more capable of interacting with water compared with the other extruded flours at lower temperatures. However, swelling power was not different between LS-LT and LS-HT extruded flours at 70 °C and 90 °C, yet both were greater (p < 0.05) compared with HS-LT and HS-HT extruded flours . This was also supported by the RVA results, which showed that LS-LT extruded flour was not fully pre-gelatinized (or the granule structure fully disrupted), as the presence of a small peak swelling at approximately 82 °C suggested. However, this increment in viscosity was less significant, and occurred at an earlier temperature and time than in the native flour whose swelling power was only significantly higher at 90 °C.Conversely, the swelling power for native breadfruit flour increased with increasing temperature, especially from 70 °C to 90 °C, at which point starch gelatinization in breadfruit flours occurs . Interep = 0.29) for the cooking loss of protein gels between the flours (p < 0.10) between the cooking loss of protein gels and the water holding capacity of the flour.There were no differences (e flours . This inp < 0.01) for gels prepared with the native flour compared with gels prepared with the extruded flours. In addition, hardness was greater (p < 0.05) for protein gels prepared with low SME extruded flours (LS-LT and LS-HT) compared with gels prepared with high SME extruded flours (HS-LT and HS-HT). The gumminess of protein gels was similar to the values of hardness, as gumminess was lower (p < 0.01) in extruded flours compared with native flour. Among the extruded flours, the gumminess of protein gels was greater (p < 0.05) for gels prepared with LS-LT extruded flour compared with all other extruded flours, and was greater (p < 0.05) for gels prepared with LS-HT extruded flour compared with HS-LT and HS-HT extruded flour. There was no difference (p > 0.05) for the springiness of protein gels prepared with low SME extruded flours (LS-LT and LS-HT) compared with native flour. However, springiness was lower (p < 0.05) for protein gels prepared with high SME extruded flours (HS-LT and HS-HT) compared with the other flours. The ability of successful gel formation largely depends on the mechanisms of bonding and interactions, such as hydrogen and covalent bonds, and electrostatic and hydrophobic interactions [p < 0.01) with final viscosity and the setback of the flours. All textural attributes of protein gels were negatively correlated  with solubility at 90 °C and positively correlated (r ≥ 0.69 and p < 0.01) with swelling power at 90 °C. Moreover, adhesiveness of protein gels was negatively correlated  with flour water holding capacity and positively correlated  with setback, which would highlight greater adhesiveness  of protein gels prepared with extruded flours. In this sense, starch de-polymerization causes greater stickiness of flours and on the surface of protein gels.The hardness of protein gels was greater  for the L* value (lightness) of protein gels prepared with LS-LT, LS-HT, and HS-LT extruded flours compared with protein gels prepared with native flour. Yet, the L* value was greater (p < 0.05) for protein gels prepared with HS-HT extruded flour compared with all other treatments. Perhaps more meaningful was that protein gels prepared with extruded flours had a greater (p < 0.05) a* value (redness) compared with native flour. Extrusion treatment can cause several heat-sensitive chemical reactions to occur, including the formation of Maillard reaction compounds [Regarding the instrumental color of the gels, there was no difference  led to an increased capacity for water absorption and reduced solubility (compared to high SME conditions) as a result of the lower shear-fragmentation of starch molecules, which could be desirable in food applications and result in improvements in processing yields. In fact, a two-fold higher cold peak viscosity was obtained for LS-HT extruded flour compared with the other extruded flours. This flour may prove to be useful in situations where cooking at high temperatures is undesirable and/or in instant food applications. Conversely, low short-term retrogradation properties, as denoted by setback, were obtained for all extruded flours, especially for those subjected to higher SME (175 kJ/kg) during extrusion.Based on the results of cooking loss and texture in the protein gel modeling systems, it can be concluded that the incorporation of extruded flour can maintain adequate processing yields  while adjusting the texture and color of protein/starch food systems. This research is beneficial for the food industry as it provides a characterization of the extruded breadfruit flour and its potential as a gelling agent in protein/starch food systems. Research on the incorporation of extruded breadfruit flour in specific food applications is necessary to further these initiatives.


For thinner films, a minimum is reached for w before diverging to a monodomain. Although this behaviour is known to be qualitatively unaltered when the dielectric environment of the film is modified, we consider the quantitative changes in that behaviour induced on the ferroelectric film by different dielectric settings: as deposited on a dielectric substrate, sandwiched between dielectrics, and in a superlattice of alternating ferroelectric/dielectric films. The model assumes infinitely thin domain walls, and therefore is not expected to be reliable for film thickness in the nanometre scale. The polarization field P(r) does vary in space, deviating from ±PS, following the depolarizing field in linear response, but the model does not include a polarization-gradient term as would appear in a Ginzburg–Landau free energy. The model is, however, worth characterizing, both as paradigmatic reference, and as applicable to not-so-thin films. The correct renormalization of parameters is obtained for the thick-film square-root behaviour in the mentioned settings, and the sub-Kittel regime is fully characterized. New results are presented alongside well-known ones for a comprehensive description. Among the former, a natural separation between strong and weak ferroelectric coupling in superlattices is found, which depends exclusively on the dielectric anisotropy of the ferroelectric layer.The electrostatics arising in ferroelectric/dielectric two-dimensional heterostructures and superlattices is revisited within a Kittel model in order to define and complete a clear paradigmatic reference for domain formation. The screening of the depolarizing field in isolated ferroelectric or polar thin films via the formation of 180° domains is well understood, where the width of the domains  Within the same model but making no approximations on the electrostatics arising from an ideal, regular polydomain structure, for w reaches a minimum and grows again when decreasing d, until the monodomain is reached [The formation of ferromagnetic –4 and fe reached ,7. A sim reached –11. ThisIt is now possible to fabricate ferromagnetic and ferroelectric samples by growing alternating layers of different thin films, just a few unit cells in thickness, in a periodic array (superlattice) –14. AlteThe dependence of the domain structure on superlattice geometry cannot be described using the theory of a thin film in an infinite vacuum, however. Some generalizations have appeared in the literature which include the effects of surrounding materials ,22–28. FBy placing a ferroelectric thin film together with a paraelectric layer between two short-circuited capacitor plates, it was found that the domain structure could be controlled by tuning the properties of the paraelectric layer, and the stability of the ferroelectric film could be improved –28. This3/SrTiO3 (LAO/STO) [A number of experimental and computational advances have revived interest in this problem. Interesting effects can occur at interfaces between different materials such as the formation of a two-dimensional electron gas (2DEG) at polar/non-polar interfaces like LaAlOLAO/STO) ,32. It iLAO/STO) , and simLAO/STO) ,35. ThisLAO/STO) –38. ThisAlthough ferroelectric thin films have been frequently simulated from first principles in different settings and environments ,39–43, fThe continuum electrostatic description of a monodomain ferroelectric thin film is essentially unaffected by a dielectric environment of the film. This is because there is zero field outside the thin film and hence these regions make no contributions to the electrostatic energy. For a polydomain ferroelectric thin film, the domain structure introduces stray electric fields into the regions outside the film . We expeFirst, we review the continuum model of an isolated film (IF) with the full treatment of the electrostatics and a domain wall term. We then generalize the theory for three different systems: a thin film on an infinite substrate , a thin film sandwiched between two infinite dielectric media , and a FE/PE superlattice (SL). We keep the prevalent nomenclature in the literature of referring to a spacer material such as STO as paraelectric, but the description will be exclusively that of a dielectric material with a given isotropic dielectric permittivity.w ≪ d), for which analytic expressions are obtained for w(d), and also in the general situation. Previous studies of periodic superlattices have assumed ferroelectric and paraelectric layers of equal width. Here, we provide a more general study of domain structures as a function of superlattice geometry. We also present a detailed derivation of the electrostatic energies in appendix A.All of these systems except the OL have appeared in the literature in various contexts and with different levels of detail. We compare the different cases, first in the Kittel limit  is taken to deviate from the spontaneous polarization ±PS in linear response to the electric depolarizing field, according to the dielectric susceptibilities normal and parallel to the film, κc and κa, respectively. This model makes significant approximations about the form of P(r), such as neglecting domain-wall width and surface/interface effects. Ferroelectric domain walls tend to be much thinner than ferromagnetic domain walls, typically of order 1 nm. Realistic descriptions of nanometric films should rather resort to theories with proper consideration of those effects, such as explicit first-principles calculations or Ginzburg–Landau . ,55. P inau e.g. ,56), and, andP inPS, except for its modification in linear response to the depolarizing field implicit when using a dielectric permittivity for the material normal to the field, κc. This assumption is equivalent to replacing the form of Since we will be interested in the electrostatic effects due to a finite polarization, we will consider the polarization to be equation  by its hw (in appendix A different widths are considered). For an IF, the electrostatic energy for that structure is given by [κa, κc are the dielectric permittivities in the directions parallel and normal to the film and w/d ≪ 1, equation (ζ(n) is the Riemann zeta function. An analytic expression is obtained for the equilibrium domain widthAs mentioned above, we consider an ideal domain structure made by regular straight stripes, all of them of the same width given by 2.4Felecel limit , w/d ≪ 1n = 100 terms. We use PbTiO3 (PTO) and SrTiO3 (STO) as examples of ferroelectric and paraelectric materials, respectively, in all of the plots in this paper, using suitable parameters.1 PTO and STO are some of the most widely studied ferroelectric and paraelectric materials, respectively, both experimentally and theoretically, particularly in the context of FE/PE superlattices. The predictions of the model should be reliable in the Kittel regime, but other materials will better conform to the approximations of this model for single sub-Kittel thin films. The model is suitable, however, for strongly coupled PTO/STO superlattices, as for the situations described in [Beyond the Kittel regime, we can obtain the equilibrium domain width from the numerical solution to equation  for the ribed in ,56.Figud, but, for decreasing d, w reaches a minimum at dm and then diverges at d∞. We can understand this behaviour by studying the shape of the energy curves as a function of domain width and thickness, which is done in w, the energy is approximately linear in w, following Kittel’s Law  in In equation ). As w idm was reported2 of the formThe described deviation from Kittel’s Law is sensitive to the system’s parameters. In , an exprκc. Increasing κc decreases the curvature of the electrostatic energy and also decreases the monodomain energy (the asymptotic energy for large w). By increasing κc for a fixed value of d, the total energy minimum again becomes shallower and then disappears.In dm, below which the width begins to diverge, we havedm has the same dependence on the system’s parameters as equation , the coupling between them becomes weak, the SW system being realized for c, which is almost identical to figure 7b.For large spacings between the ferroelectric layers (dm/lk(κs) is independent of κs. At this ratio, the dielectric permittivity of the spacer has no influence on the equilibrium domain structure, relative to the length scale given by lk(κs). This is shown in figure 9b. In dm as a function of κs for different values of α. We see that when dm increases with κs, while it decreases for αc represents a natural boundary between the strong and weak coupling regimes of superlattices.Interestingly, when αc can be predicted from both the asymptotic and analytic approximations. Using the analytic approximation to the SL system (see appendix C), we haveκs vanishes.The critical ratio 4.We have extended the continuum electrostatic description of an isolated ferroelectric thin film within Kittel’s model to thin films surrounded by dielectric media and FE/PE superlattices. While some of the generalizations have previously appeared in the literature, a detailed comparison had not been done before. In doing so, we have understood how the surrounding dielectric materials influence the domain structure in the ferroelectric materials, both in the Kittel limit and beyond.κs. This provides a useful correction to measurements of domain width with film thickness, as Kittel’s Law for an IF typically underestimates domain widths. Beyond Kittel’s regime, we found that increasing κs decreases dm, that is, the thickness for which the domain width is minimal.In the Kittel limit, the square-root behaviour is only affected in scale, the domain width increasing with dielectric permittivity, κs can either decrease or increase dm, depending on the ratio of thicknesses, κs. When α is small, the ferroelectric layers are strongly coupled, and dm increases with κs. Remarkably, when dm/lk is unaffected by κs. In reality dm does change, since the Kittel length depends on κs, but the scaling is different above/below αc. The critical ratio αc serves as a clear boundary between the strong and weak coupling regimes from an electrostatic viewpoint.For FE/PE superlattices, we found that ussed in  for examPS , however, dm increases with κs, and we would have dm ≫ dc, and therefore the thin-limit behaviour presented above should be observable before the films becoming paraelectric.If A ≠ 0, ± 1, see appendix A). In addition, the polarization gradients expected close to surface, interfaces and domain walls are better described within a Ginzburg–Landau theory, which will give significantly different predictions for ultrathin films, where complex structures such as polar vortices and skyrmions have been observed [The model described in this paper makes use of a number of significant approximations. Domains are typically not straight or of infinite length, and the domain structure may not be an equilibrium one . While the domain width outside of Kittel regime may not be a realistic description for some materials, the values of


Lysine-specific demethylase 5B (KDM5B/PLU1/JARID1B) is found to be overexpressed in numerous malignancies, including breast, lung, skin, liver, and prostate cancer. Identification of molecules targeting the KDM5B enzyme could be a potential lead in cancer research. Although many KDM5B inhibitors with promising outcomes have been developed so far, its further application in clinical practice is limited due to toxicity and lack of target specificity. Here, we summarize the significance of targeting KDM5B in anticancer therapy and report the molecular docking studies of some known anti-viral agents, decitabine, entecavir, abacavir, penciclovir, and 3-deazaneplanocin A in the catalytic domain JmjC of KDM5B. These studies show the repurposing potential of identified anti-viral agents in cancer therapy. Studies over the past few decades and ongoing research reveal that, along with genetic alterations, epigenetic aberrations play a vital part in the evolution and progress of numerous diseases, including several types of cancers . EpigeneHistone, being the functional protein, not only actively takes part in gene expression, but also acts as a spool for DNA to wind around. Any modification on histone molecules via methylation, acetylation, phosphorylation, ubiquitylation, or sumoylation alters the compact chromatin architecture, leading to abnormal gene expression . HistoneLysine-specific demethylation is finely tuned by two enzyme categories, called lysine-specific demethylase (KDM1/LSD1) and the JumonjiC (JmjC) domain, encompassing histone demethylases (KDMs). JmjC, being the largest among two families, displays high substrate specificity, with demethylase activity targeting histones H3 and H4 . In mammThe KDM5/JARID1 subfamily consists of four interrelated histone demethylase enzymes—KDM5A, KDM5B, KDM5C and KDM5D—involved in several human biological processes, including hormonal response, stem cell regeneration, genomic stability, cellular proliferation, and differentiation ,9. KDM5BN-terminal Jumonji (JmjN) domain, a catalytic JmjC domain, an AT-rich domain (ARID), a C5HC2 zinc finger domain, a PLU1 motif and three plant homeobox domain . The domain architecture of KDM5B is highly conserved and homologous from yeast to humans. JmjN and JmjC are vital domains required for enzymatic activity, whereas AT-rich domain binds to GC rich DNA sequences. PHD domains contribute to substrate recognition in which it prevents reverse reaction ..2+ (20].These results also show that small molecules, which possess planar bicyclic heterocyclic rings, exhibit favorable binding toward the JmjC domain of human KDM5B. This is due to their ability to undergo interactions with the catalytic site metal ion, which is an important criterion in KDM5 inhibition and is consistent with previous studies ,77,88. OThe leading role of KDM5B oncogene in tumorigenesis and cancer progression is well established in various malignancies and is therefore, considered as a potential therapeutic target. Although many small molecule inhibitors of KDM5B have been identified so far, their possible clinical application in cancer treatment is a work in progress. This study provides a rationale on how repurposing antiviral drugs could contribute to the discovery and development of KDM5B inhibitors. We anticipate that these studies will stimulate further research in considering drug repurposing approaches to target KDM5B, and to discover novel therapies for various cancers.


Triticum dicoccum, T. monococcum, T. aestivum, T. durum and T. turgidum, corn, barley, rye and oat from a certified producer in Greece were used either as raw materials or for the production of bread, pasta and flakes. A small portion of the flour or the corresponding product was extracted with DMSO-d6. The liquid part was directly analyzed by NMR (400 MHz). The simplicity of the NMR spectrum of the total extract and the lack of overlapping peaks permitted the development of a high throughput quantitative method for the measurement of total bioactive alkylresorcinols in less than 15 min. Grains, whole grain flours and breads from old varieties of T. dicoccum and T.monococcum showed high contents of alkylresorcinols (455–1148 mg/Kg), while the same compounds were completely absent from white flour and the corresponding bread. The term high-phenolic flour is proposed to distinguish among flour types.The main objectives of the current work were to investigate differences among flours from traditionally preserved Greek varieties of cereals, and especially those of wheat, and in parallel, to correlate those potential differences with the presence of bioactive natural ingredients. In this context, we developed a new, fast, and simple method for the measurement of total 5-alkylresorcinols in cereals and related foods by qNMR. Several types of flour (white or whole-grain) coming from wheat, i.e.,  Triticum spp.), olive trees and grapevines are considered to be the three basic plants of the Mediterranean diet; in particular, wheat is the most important plant in the history of human nutrition in Europe and the Middle East. Although famous bioactive small molecules like resveratrol or oleocanthal have been discovered in grape and olive oil, wheat products are not similarly known, from the consumer’s perspective, as sources of health-protective ingredients. However, alkylresorcinols (ARS), an important class of phytochemicals found in wheat, have attracted the interest of scientists in the last thirty years /Msample, where Msample = 330 mg and mmol I.S. = 0.5 mg/MW syringaldehyde = 0.00270. To express the concentration in mg ARS/mg of dry material, we used the average molecular weight between C19 (m/z 376) and C21 (m/z 404) alkylresorcinols.A portion of the flour or the comminuted corresponding product (330 ± 0.1 mg) was extracted in an ultrasonic bath for 5 min with deuterated DMSO (1 mL) containing syringaldehyde (0.5 mg/mL) as an internal standard. The supernatant liquid part was obtained by centrifugation (5 min at 3000 rpm) and was directly transferred to a 5 mm NMR tube. Each sample was analyzed in triplicate using a standard 90 degree excitation pulse, with a pulse width of 10 μsec and a prescan delay of 6.5 μsec. All measurements were performed at 298 K. Typically, 16 scans were collected into 32 K data points over a spectral width of 0–13 ppm (5263.18 Hz) with a relaxation delay of 10 s, an acquisition time of 3.11 sec and a FID resolution of 0.32 Hz. The appropriate relaxation delay was determined by gradual increases , an exponential weighting factor corresponding to a line broadening of 0.3 Hz was applied. For the peaks of interest, accurate integration was performed manually. The concentration of ARS was measured by comparing the area of the selected signal at 6.00 ppm with that of the internal standard (IS) at 9.79 ppm, which was set as 1. The calculation of the concentration of total ARS in mmol/mg of dry material was performed using the following formula: C = [ and LOD = 10 μg or 30 mg/Kg (S/N > 3).6 were directly recorded by 1H-NMR without any other treatment. The spectra were compared and a singlet was observed at 6.0 ppm only in wheat and rye samples  contents (>450 mg/Kg), while the compounds were completely absent from white flour and all the commonly consumed corresponding types of bread.Grains, whole grain flours and breads, especially from old varieties of Secale cereale flour, while no 5-alkylresorcinols could be detected in Hordeum vulgare (barley) flour, Avena sativa (oat) whole grain flour and flakes, Zea mays whole grain flour, nor in the seeds of Lens culinaris, Lathyrus clymenum, Phaseolus vulgaris, Cicer arietinum, Linum usitatissimum, Fagopyrum esculentum and Zea mays. The quantitation results are presented in We also found a high total alkylresorcinol content only in samples of 1H-NMR for the qualitative and quantitative characterization of bioactive ingredients in olive oil, wine and beer [The main objectives of the current work were to investigate the possible differences among different types of flours from traditionally preserved Greek indigenous varieties of cereals, and especially those of wheat, and in parallel, to correlate those differences with the presence of bioactive natural ingredients. Based on the previous successful use of and beer ,22,23, w3, CD3OD, Pyridine-d5, DMSO-d6 and Acetone-d6, we found that DMSO was the most efficient solvent, presenting in parallel the clearest spectra concerning the aromatic area which showed the most significant differences. A comparison among the different spectra  only in rye and the wheat species. Targeted isolation of the molecules corresponding to this peak using liquid chromatography and subsequent characterization by NMR and GCMS led us to attribute it to a series of 5-alkylresorcinols. Interestingly, when the NMR spectrum of the isolated compound was recorded in CDCl3, Acetone-d6 or Pyridine-d5, it presented a doublet and triplet, as described in the literature for 5-alkylresorcinols [3 [After several trials of extraction and simultaneous spectra recording with CDCl spectra  of the forcinols , correspinols [3  were repinols [3  as a douARS) and comparisons with the integration of the internal standard at 9.79 ppm, which was set as 1. The calculation of the concentration of total ARS in mmol/mg of dry material was performed using the formula presented in Materials and Methods, which was based on the molar ratio between the target analyte and the internal standard, following the general guidelines presented by Pauli et al. [The simplicity of the NMR spectrum of the total extract in DMSO and the lack of overlapping peaks from other compounds allowed us to develop a high throughput quantitative analytical method for the measurement of total alkylresorcinols. The method was based on the integration of the peak at 6.0 ppm  of ARS. Longer extraction times or more than one cycle of extraction led to a negligible increase of the recovery, i.e., less than 5% .Advantages of the qNMR method in comparison to chromatographic methodsAlkylresorcinols (ARS) have been previously investigated and quantified in several wheat products, mainly using time-consuming chromatographic methods and long extraction or pretreatment procedures ,18,19,20Our new method is the first to make possible the selective and high throughput quantitative determination of ARS in grains, flours and foods in less than 15 min per sample, including the extraction and analysis time. In addition, the most important advantages are: (i) no need for standards for target compounds; (ii) no need for derivatization; (iii) short time of extraction in ultrasonic bath (5 min); (iv) very simple procedure for the separation of the solution for analysis by centrifugation; and (v) adequate limit of detection and precision. The only limitation is that the current method cannot determine the length of the side chain, which, in some cases, is useful to differentiate rye from wheat ,5.Comparison among the studied cereals and other common seeds and beansT. aestivum has significantly reduced the nutritional intake of ARS in Mediterranean populations. As previously reported, ARS were found only in wheat and rye, and not in other cereals. Indeed, analyses of the grains of the studied cereals, as well as several of other commonly used beans and seeds, confirmed that the main nutritional source of ARS is wheat and rye. Wheat should be considered as a primary nutritional source, especially for Mediterranean populations, since rye is consumed to a lesser extent. Until the mid-20th century, the main flour used for bread preparation was whole-grain wheat flour, usually coming from local varieties, and consequently, the traditional Mediterranean diet should be considered as a diet that is very rich in ARS. In modern days, the massive use of white (refined) flour from Comparison among wheat species and previously reported ARS content in wheat grains and flourT. monococcum (known as “Kaploutzas”) showed the highest content of ARS in whole grain flour, i.e., even higher than the corresponding rye flour. It showed a small difference in comparison with T. dicoccum and a big difference with T. durum, T. aestivum and T. turgidum. Interestingly, the analysis of the grains showed that T. dicoccum presented the highest content of ARS (>1000 mg/Kg), close with that of T. monococcum, but again, very different from those of T. aestivum and T. durum. The results obtained herein are in agreement with previous analyses [T. monococcum and T. dicoccum are richer than T. aestivum and T. durum, but herein, the concentrations in local varieties of T. monococcum (925 mg/Kg) and T. dicoccum (1148 mg/Kg) were found to be much higher than what was previously reported from Germany and Hungary (391–819 mg/Kg) [T. timophevi   and alsoimophevi . A compaARS content of bread, pasta, wafersT. dicoccum grains and the fact that this species is becoming popular again for baking prompted us to study the fate of ARS during the production of bread, flakes, wafers and pasta. Interestingly, all products contained significant amounts of ARS (The results obtained for s of ARS . The flaAlkylresorcinols are a class of major bioactive ingredients present in wheat flour and wheat-based products, and found in high amounts in old wheat varieties while being almost absent from the common white flours that are used for the mass production of bread nowadays. Their concentration can easily be measured by qNMR, permitting high-throughput analyses of flour and related products.


Asfarviridae family, was originally described in Africa almost 100 years ago and is now spreading uncontrolled across Europe and Asia and threatening to destroy the domestic pork industry. Neither effective antiviral drugs nor protective vaccines are currently available. Efforts to understand the basis for viral pathogenicity and the development of attenuated potential vaccine strains are complicated by the large and complex nature of the ASFV genome. We report here a novel alignment-free method of documenting viral diversity based on profile hidden Markov model domains on a genome scale. The method can be used to infer genomic relationships independent of genome alignments and also reveal ASFV genome sequence differences that determine the presence and characteristics of functional protein domains in the virus. We show that the method can quickly identify differences and shared patterns between virulent and attenuated ASFV strains and will be a useful tool for developing much-needed vaccines and antiviral agents to help control this virus. The tool is rapid to run and easy to implement, readily available as a simple Docker image.African swine fever virus (ASFV), belonging to the  Asfarviridae family, was first described in Kenya nearly 100 years ago (African swine fever virus (ASFV), belonging to the ears ago . The virears ago . Outsideears ago . Since tears ago , with cuears ago . Furtherears ago . These rThe ASFV virion is enveloped and spherical or pleomorphic in shape with a diameter of 175–215 nm. The virus has a linear, dsDNA genome of 170–195 kb with complementary terminal sequences. The ASFV genome encodes >150 open reading frames ORFs; . In addiThe p72 gene  is frequently used for PCR diagnosis of ASFV . AdditioCoronaviridae genome sequences  as well as any indication of virulence derived from the original literature. A summary of the 47 genomes used for the analysis is provided in Collection of the ASFV genomes. All ASFV full genomes were retrieved from GenBank (5 April 2019) using the query: txid137992[Organism] AND 170000[SLEN]:200000[SLEN] yielding forty-eight complete genomes. Two genomes were identical: genome 2.1The Pfam domains encoded by ASFV genomes were identified using the hmmsearch function of HMMER-3.2.1 , searchi2.2txid137992[Organism] AND 170000[SLEN]:200000[SLEN] NOT patent). From the ‘Send to’ menu, the option ‘all coding sequences’ was selected and these entries were retrieved to a fasta file. MGF entries were selected from the complete ASFV coding sequence file by sorting for the presence of the term “MGF” in the coding sequence ID with a simple python script. This yielded a set of 660 MGF entries.All ASFV encoded MGF protein coding sequences were retrieved from GenBank as follows. An initial query to the NCBI nucleotide database was made to retrieve complete or nearly complete ASFV genomes (MGF_100 (thirty-eight entries), MGF_110 (nine entries), MGF_300 (thirty-nine entries), and MGF_360 (forty-one entries). To increase resolution for ASFV genome comparisons, profile HMMs were prepared for these proteins as follows. The 660 MGF ORFs were clustered using Usearch . These were classified in GenBank as  Usearch  at an aa Usearch , and a p Usearch .These cuThe computational tools for performing this analysis are openly available as a platform independent Docker image of the tool and instructions for installing and using the tool have been made available (see Data availability section and Readme document in the 2.3NC_001659 for the BA71V strain and used in an online BLAST search (MEGABLAST default settings) to identify closely related sequences. Using the download menu, all hits  were retrieved to a fasta file, the UK domain coding sequence from the NC_001659 genome was added, and the set was translated into protein sequences using Geneious, aligned in Mafft , eighty-two domains were identified at least once per genome in the set of forty-seven genomes, and seventeen domains were found twice or more per genome in the set indicating repeat occurrences in some genomes see . The dom3.1NC_001659 UK protein as a model and the HMMER-3 score is correlated with the differences of query domains from this early ASFV sequence. Thus, an HMMER-3 search can be used both to find members of a domain family in a query genome and to provide a quantitative score (bit-score) of the distance of the query domain from the model domain.As an illustration of the domain classification approach, we examined the UK gene’s ORF encoding a ninety-six aa protein expressed early in ASFV infection . Althoug3.2We identified all profile HMM domains from the Pfam collection which were encoded in a set of forty-seven ASFV genomes. Using a domain_i-Evalue cutoff of 0.0001 (a measure of the probability of finding the domain by chance), eighty-two domains were identified at least once in the set of forty-seven genomes, and seventeen domains were found twice or more in the set indicating repeat occurrences in some genomes see . As descLS478113, identified from a wild boar in 2014; The forty-seven full ASFV genomes were ordered by hierarchical clustering based on the Pfam + MGF domain scores and compared with a p72 ML tree with the major genotypes in each analysis indicated by colored boxes . In vali3.3Five MGFs have been defined  with the naming based on the mean number of amino acids in the gene product.MGF_100: 38; MGF_110: 148; MGF_300: 46; MGF_360: 267; MGF_505: 160 entries) and examined for Pfam domains. Three MGFs consistently encoded at least one domain . These were MGF_110: domain v110, MGF_360: domain ASFV_360, MGF_505: domain DUF249. To capture the diversity in these MGFs, we prepared individual profile HMMs from a comprehensive set of MGF ORFs. Briefly, we grouped each MGF protein by aa sequence identity, identified forty-five MGF subfamilies and then constructed custom profile HMMs for each of these (see Section 2). We then analyzed the clustering pattern of all MGF ORFs based on their custom profile HMMs  virulent strain and the NH/P68 (NHV) nonvirulent strain, which have been described and compared for virulence differences . Domain 4.We have demonstrated the utility of a novel method of characterizing ASFV-encoded protein diversity on a genome scale based on profile HMM descriptions of conserved protein domains. The method exploits the Pfam collection of profile HMMs  as well We have identified greater diversity in the five MGFs than previously noted. We further reveal the presence of a set of unconventional MGFs  that appThe domain method described here also allows a rapid assessment in both the qualitative features of encoded domains and reports a bit-score for each identified domain, which is a protein distance from the model domain. Furthermore, the method also reports copy number changes in domains. For example, examining changes in domain instances showed that the GII ASFV strains, responsible for large global outbreak of ASF, encoded a substantial increase in several MGF gene families . These cThe added benefit of domain-based classification as described here is that there is no requirement to prepare an alignment of the query genomes. The resolution of any phylogenetic constructions relies heavily on accurate alignment of homologous regions of sequences. In the case of ASFV, there are differences in MGFs across different ASFV strains, either duplications or deletions, which are very difficult and time-consuming to reliably align. Furthermore, if certain genes are missing from some of the genomes for some of the alignment, this region of the alignment may be masked in the entire alignment and will not contribute to the phylogenetic signal. However, such deletions, duplications, or inversions of domains are captured by the domain scoring system used and may be an important component of the increased resolution of the domain method.In conclusion, hierarchical clustering based on profile HMM domain scores has provided a rapid method for comparing similar genomes to identify differences in the encoded proteins. It is not intended to replace genome-scale evolutionary analysis, rather it complements standard phylogenetic approaches by focusing on shared functional information in virus genomes. We applied the method to three sets of ASFV genomes from contemporary outbreaks with known phenotypic differences in their ability to replicate in and kill pigs . The novThe computational tools for performing this analysis can be downloaded as a platform independent Docker image using this command (docker pull matthewcotten/asfv_class_tool). Instructions for installing and using the tool are available in the This work was supported by a Marie Sklodowska-Curie Individual Fellowship, funded by European Union’s Horizon 2020 Research and innovation programme , by a Wellcome Trust Intermediate Fellowship , by the ASF-RESIST African Union Commission , and MRC .Conflict of interest: None declared.veaa044_Supplementary_DataClick here for additional data file.


Agbagoma and Balabi were evaluated. The samples Agbagoma and Balabi had 8.90%–9.00% moisture, 16.80%–17.40% protein, 47.80%–48.00% fat, 7.70%–7.80% ash, and 18.20%–18.40% carbohydrate. The mean values of functional properties revealed significant differences (p < .05) between okra seed flour samples studied. The samples’ bulk density range was 0.80–0.83 g/ml. Water absorption capacity and oil absorption capacity ranged from 511.65% to 504.32% and 88.38 to 160.67%, respectively. The solubility was 14.10% for Agbagoma and 10.97% for Balabi, whereas swelling power was 16.37% and 14.68% for Agbagoma and Balabi, respectively. All pasting properties except peak time and pasting temperature of Agbagoma seed flour were higher than those of Balabi seed flour. The study revealed that dried okra seed flour is rich in nutrients, which could be used for baking and fortification of foods.The physicochemical and functional properties of dried okra seed flour of two genotypes  p < .05) between the okra seed flour samples. 0kra seed flour from Balabi could be used as composite for baby foods due to its low viscosity whereas Agbagoma flour could be used as composite for high viscous food.The physicochemical and functional properties of dried okra seed flour of two genotypes Agbagoma and Balabi were evaluated. The mean values of physicochemical and functional properties revealed significant differences ( Economically, okra is an important vegetable crop grown in tropical and sub‐tropical regions of the world. Mostly, it is grown for its green leaves and pods as green vegetable according to Naveed, Khan, and Khan, , dried, cooked, frozen, fried, and pickled. Utilization of okra seeds is numerous. In Turkey, mature okra seeds are roasted, ground, and used as a coffee substitute  and packaged in zip‐lock bags and stored at −20°C in a freezer  for further analysis.Harvested 2.3Proximate composition of moisture, crude protein, ash, and crude fat of the dried okra seed flour samples was determined according to AOAC . Protein2.4The pH was determined by the method as described by AOAC . Two graThe color of the okra seed flour was measured using the CR‐400 Chroma Meter which is a handheld, portable measurement instrument designed to evaluate the color of objects.The color meter coordinates system L* a* b* values were recorded, and the Light index was calculated as (100/0). The white tile used for calibrating the Hunter L* a* b* color scale had L* = + 97.51, a* = + 0.29 and b* = 1.90 as standards.Bulk density was determined by modifications of the gravimetric method by Asoegwu, Ohanyere, Kanu, and Lwueke . Fifty g2.5‐1) was calculated as the difference between the initial volume of water added to the sample and the volume of the supernatant. The same procedure was carried out to determine the oil absorption capacity as below;Water and oil absorption capacity was determined by the method of Beuchat . One gra2.6Swelling power and water solubility indices were carried out based on a modification of the method of Leach, McCoven, and Scoch . One gra2.7Rapid Viscos Analyzer  was used in the analysis of the pasting properties of the dried okra seed flour samples. The calculated moisture content was inputted into the RVA which gives the mass of flour and water to be measured. The masses displayed by the instrument were measured into a dried empty canister. The dispersion was mixed thoroughly and then placed into the RVA. The slurry was heated from 50°Ϲ to 95°Ϲ.2.8p < .05).Two samples independent test was used for mean comparison to identify significant differences between the samples. Statistical significance was accepted at 95% confidence limit  falls within the acceptable range of 0%–13%  than Agbagoma (16.80%). These values were generally low when compared to other variety (iwo agborin) with protein content of 41.11%  as compared to Balabi which had the lowest (47.80%) (Table iwo agborin) with a fat content of 31.04%  Table . HoweverAgbagoma and lower for Balabi. In a study on okra pods by Gemede  for Balabi as compared to Agbagoma okra seed flour samples  than that of the flour from Agbagoma. The L* color brightness of both flours can be improved by preventing enzymatic browning by washing the okra seeds with ascorbic acid solution or 1% sodium metabiosulfite solution before sun drying.Color value was between the ranges of 60.29 to 65.37 in all okra seed flour samples. The color (L* a* b*) values were significantly higher  compared with Agbagoma seed flour (0.80 g/ml). In previous work done by Adebowale, Adeyemi, and Osohodi  between the two varieties. A lower WAC was recorded for irradiated and nonirradiated cowpea flours (110%–113%), full‐fat, and defatted mucuna flour (120%–220%) by Elkhalifa, Schiffler, and Bernhardt (Water absorption capacity (WAC) and oil absorption capacity (OAC) of ernhardt  and Adejernhardt . Accordiernhardt , proteinThe oil absorption capacity (OAC) of food determines the mouth‐feel, flavor retention as well as shelf‐stability of baked or fried foods and especially meat products. According to Adebowale and Lawal, , OAC of The swelling power of the okra seed flour ranged from 14.68 to 16.37 g/g. The swelling capacity values were not in accordance with  between okra seed flour samples in terms of solubility index. Often, the solubility and swelling power are influenced by the water‐binding capacity of the flour sample, which is a function of proteins and carbohydrates present in the flour  revealed a high content of fat and proteins. The high protein content could be a valuable protein supplement for cereals based foods. The pH values of the okra seed flour were within the recommended range which is ideal for individuals with ulcers‐related problems. The functional properties of okra seed flour were appreciable that could be exploited in food formulations such as cereals and wheat flour. All pasting properties except peak time and pasting temperature of Agbagoma seed flour were higher than that of Balabi seed flour suggestion that Balabi seed flour could be used as composite for baby foods because of its low viscosity whereas flour from Agbagoma could be used as composite for high viscous food.The dried okra seed flours (All the authors hereby state that there is no conflict of interest regarding this publication.


TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein. An abundance of cellular functions is controlled by the attachment of a 76-residue ubiquitin (Ub) molecule to a protein substrate. Ubiquitination occurs via the combined action of three enzymes: an ATP-dependent ubiquitin-activating enzyme (E1) conveys the ubiquitin molecule onto a conjugating enzyme (E2), which in turn transfers it, in the case of RBR or HECT ligase enzymes (E3), first to the E3 or, in the case of ‘Really Interesting New Gene’ RING ligases, directly onto the substrate . RING-tyThe TRIM ligase family contains over 70 members, which, with a few exceptions, are characterized by a common architecture of an N-terminal tripartite motif  comprising a RING domain followed by one or two zinc-coordinating domains called B-box1 and B-box2 and a long coiled-coil (CC) region  6–10]. . 6–10]. The RING core is a small domain of 40–70 residues found in proteins from plants to humans. A cross-braced pattern of conserved cysteine and histidine residues coordinating two zinc ions maintain the native fold  16,17].,17.16,17In all TRIM family RINGs that have been shown to self-associate, but TRIM19, RING dimerization is achieved via hydrophobic interactions between aliphatic side chains of residues present in short α-helical segments at the N- and C-termini of the zinc-coordinating core RING domain that form a 4-helix bundle. Additional contacts contributed by the core RING domain, mainly from residues in the loop connecting ß-sheets ß1 and ß2 stabilise the dimer interface  and 2A–CAt present only two RING domains, from TRIM32 and TRIM69, have been shown by biophysical techniques to form a constitutive dimer in solution ,31. AnalIn contrast to TRIM32 and TRIM69, other TRIM RING domains analysed thus far exist in a monomer-dimer equilibrium in solution, which in the case of TRIM25 is strongly shifted towards the monomer, with dimerization only observable by NMR as protein concentration increases . The RINThe anti-parallel nature of the CC domain places the two RINGs at opposite ends of a given TRIM homodimer implying that RING dimerization and catalytic activity will require association of two or more TRIM homodimers into a higher-order assembly, or alternatively occur within one homodimer. In fact, the ability to form tailored higher-order assemblies is key to the activity of some TRIMs, best understood in the case of TRIM5α, a retroviral restriction factor that targets the viral capsid core and synthesizes K63-linked polyubiquitin chains to activate innate immune signalling pathways. A range of biochemical and biophysical studies have analysed the self-association properties of individual domains of TRIM5α, and combined with cellular studies, shed light on the mechanism behind higher-order self-assembly and dimerization of the RING domains . These sin vitro. However, in a physiological setting, activity might possibly be regulated by recognition of substrate and cognate E2s, or through additional stimuli such as post-translational modifications. The first example of such regulation has recently been identified in TRIM21, a cytosolic antibody receptor .,52.32,52Dimerization of the RING domain, whether entirely self-promoted or supported by other elements within the full-length protein, appears to be a common strategy for conferring E3 ligase catalytic activity to TRIM family proteins. The ability to form helical segments N- and C-terminal to the core RING is imprinted in many TRIM primary sequences  and appears to be a prerequisite for the formation of dimers. Nevertheless, while some TRIMs form constitutive RING dimers, in others these N- and C-terminal segments, which contain the aliphatic residues that stabilize the core of the four-helix bundle, might be in dynamic exchange in solution between helical and random-coil secondary structures. A shift of the equilibrium towards a stable dimer may only occur as the local RING concentration increases - for instance, following the binding of the E2∼Ub conjugate, which could be a regulated event. In the context of the full-length protein, where the RING domains are located at either end of a rigid anti-parallel coiled-coil, RING dimerization likely requires additional protein interactions mediated by other structural elements or the formation of higher-order assemblies, potentially driven by substrate-binding.The functional requirements underlying these differences in behaviour are not clear at this time and we speculate that the strength of RING self-association and need for other supporting elements might be a means to regulate overall catalytic activity. Detailed studies on the full-length proteins are urgently required to understand the key factors dictating the relationship between the overall oligomeric state and the formation of catalytically active RING dimers in TRIM ligases, and cryo-electron microscopy will likely play an important role in providing a molecular description of these factors.Importance of the field: TRIM E3 ubiquitin ligases are crucial regulators of many cellular processes and their dysfunction is associated with a variety of pathological conditions. Insight into the molecular features driving catalytic activity is crucial to understanding their physiological function.Summary of current thinking: Self-association of TRIM ligases is known to be important for their function yet the precise details of the link between ligase activity and oligomeric state remain largely unknown. Dimerization of the catalytic RING domain is important for ubiquitination activity in most TRIM proteins studied so far.Comment on future directions: The future availability of structures of full-length TRIM proteins, bound to E2-Ub conjugates and substrates, will have a major impact on our understanding of the function, catalytic activity and regulation of this protein family.


The stress–strain curve showed that the hot deformation behavior of the Cu–0.55Sn–0.08La (wt.%) alloy was significantly affected by work hardening, dynamic recovery, and dynamic recrystallization. The activation energy Q was 261.649 kJ·mol Copper alloys are structural and functional materials with excellent electrical and mechanical properties. These features make them suitable for applications in designing the frame of large-scale integrated circuits, contact wires of electrified railways , lining 2O3 composites  c, respecEquation (8) represents the apparent activation energy Q of plastic deformation.−1. However, the apparent activation energy of the thermal deformation of pure copper with different impurity contents is around 208–245 k·J·mol−1  was fitted linearly as a function of lnZ and the results are provided in Based on the above-mentioned analyses, the constitutive equation of Cu–0.55Sn–0.08La at high temperatures was determined as: As a result, Equation (9) can be deduced as:Hot deformation of Cu–0.55Sn–0.08La (wt.%) alloy was successfully studied using a Gleeble-3180 testing machine at deformation temperatures (400–700 °C) and various strain rates. The following conclusions were drawn:−1), the stress–strain curve increased with deformation. At high deformation temperature (700 °C) or low strain rates (0.01 and 0.05 s−1), the deformation process was accompanied by the formation and growth of recrystallized nuclei. Furthermore, the softening rate of the alloy was equal to the deformation hardening rate, and the stress–strain curve tended to stabilize.1. The flow stress of the Cu–0.55Sn–0.08La alloy decreased with the deformation temperature and increased with strain rate. At low temperature (400 °C) or high strain rates (1 and 10 s−1, the relationship between peak flow stress of the Cu–0.55Sn–0.08La alloy and strain rate was determined as: −1. Thus, the constitutive equation can be expressed as: 2. At 500–700 °C and 0.01–10 s3. The microstructure of Cu–0.55Sn–0.08La alloy showed the presence of slip bands and shear bands in the grains at the deformation temperature of 400 °C. Recrystallization grains were noticed near the shear band at the grain boundary as the deformation temperature increased. At 700 °C, the dynamic recrystallization appeared to be relatively complete with the growth of recrystallization grains.


Streptococcuspneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles, it was assumed for a long time that these pneumococcal surface proteins represent a protein family comprised of eleven subgroups. Recently, however, the evaluation of more proteins revealed a greater diversity of individual proteins. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are also present in other virulence factors encoded by pneumococci and other bacterial pathogens. This knowledge improved pattern evaluation at the level of full-length proteins, allowed a sequence comparison at the domain level and identified domains with a modular composition. This novel strategy increased understanding of individual proteins variability and modular domain composition, enabled a structural and functional characterization at the domain level and furthermore revealed substantial structural differences between PspC and Hic proteins. Given the exceptional genomic diversity of the multifunctional PspC and Hic proteins a detailed structural and functional evaluation need to be performed at the strain level. Such knowledge will also be useful for molecular strain typing and characterizing PspC and Hic proteins from new clinical S. pneumoniae strains.The PspC and Hic proteins of  S. pneumoniae (the pneumococcus) is the leading cause of community-acquired pneumonia. In addition, this Gram-positive pathogen can cause otitis media and may also cause acute life-threatening invasive infections such as sepsis and meningitis4. Malnutrition and S. pneumoniae infections are the major cause of childhood mortality worldwide. Pneumonia accounts for approximately 16 percent of the 5.6 millions of deaths among children under five years of age, killing around 808,000 children in 2016 according to the United Nations Children’s Fund (UNICEF) and the World Health Organization (WHO)7. At any point in time pneumococci can reside asymptomatically in the upper respiratory tract of about 50% of children, from where they can spread to other sites and cause disease or be transmitted to other individuals8. Based on the differences in the polysaccharide capsule 100 S. pneumoniae serotypes have been identified so far10.12.Pneumococcal diseases are widespread and antibiotic resistant strains are constantly emerging resulting in a need for new therapeutics. In addition, currently available vaccines are based on the capsular polysaccharide and only provide protection against the limited number of serotypes included. Vaccines protecting against a higher number of serotypes or a serotype-independent vaccine is needed to combat the pathogen efficiently. These limitations make it important to identify new virulence determinants that may serve as novel vaccine or therapeutic targets, to understand the diversity of these determinants and also to define the immune escape strategies of this pathogenic bacteriumS. pneumoniae. Common mechanisms of complement evasion are emerging as a large list of pathogenic microbes bind and exploit the same human complement regulators17. Thus, it is important to understand the exact role of individual pneumococcal virulence determinants in complement and immune evasion. Furthermore, it is important to establish whether the virulence determinants are localized to the surface and, if so, the specific regions of the protein exposed21.Immune and in particular complement evasion is critical for all pathogenic microbes, including 22. The majority of virulent S. pneumoniae strains express at least one PspC or Hic variant, and strains that have the pspC/hic genes deleted show significant amelioration of lung infection, nasopharyngeal colonization, and bacteremia in mice23.The PspC and Hic proteins are important pneumococcal immune evasion proteins and adhesins that represent promising vaccine candidates25. Ianelli et al. identified several domains within the 43 PspC and Hic proteins evaluated including, the leader peptide, α-helical regions with a seven-amino acid periodicity, repeat domains and a proline-rich stretch followed by either a choline-binding or sortase-dependent anchor26. At that time, the cell wall anchors were used as the criterion to differentiate between PspC and Hic family members and based on sequence differences six PspC-type and five Hic-type clusters were defined. However, today there are still no precise criteria regarding cluster specific domain composition or domain characteristics. Because the patterns of domains are not exactly known and the borders of individual domains are not well-defined, a straightforward system of variant designation is at present difficult to achieve. This makes assignment of existing and newly identified pspCandhic genes, including those from novel clinical pneumococcal isolates, difficult or even impossible27.Based on overall sequence similarities PspC and Hic variants are considered to represent one group of pneumococcal immune evasion proteins. Initial analyses by Brooks Walter in 1999 and Iannelli et al. in 2001 revealed both sequence similarity and diversity among PspC and Hic proteins28. Because pspC and hic genes were identified independently by multiple groups, different names were given, including CbpA (choline-binding protein A), SpsA (secretory IgA binding protein), PbcA (C3-binding protein A), or Hic (Factor H binding inhibitor of complement)  and the polymeric IgA receptor (pIgR)t) Table 29–39. OvPspC and Hic proteins are are attached to the bacterial cell wall. PspC proteins attach non-covalently to the phosphorylcholine (PCho) moiety of teichoic acids (TAs) via their C-terminal choline binding domains and Hic proteins, are covalently linked to the peptidoglycan via an LPsTG motif. The fact that both proteins are anchored via their C-terminal regions suggests that the N-terminal region of the protein spans the capsular polysaccharides and extends beyond the capsule into the external environment. However, the different mechanisms of localization suggest that there might be differences between PspC and Hic in the strengths of interaction with the bacterial surface. Furthermore, attachment of Hic to the peptidoglycan will result in the protein being attached closer to the cell membrane.41. These multifunctional proteins represent one of the most diverse group of immune evasion proteins41. PspC and Hic proteins have a mosaic structure, comprised of distinct regions that consist of multiple domains. Furthermore, a substantial overlap of domains exists between PspC and Hic variants. Standard domain or sequence-based comparisons between members of this protein family are complex due to structural differences and variable domain composition. Currently, the protein NCBI databank lists 54,852 entries for PspC or Hic and 12,193 entries for CbpA, including both full-length proteins and partial protein sequences . The individual entries show homology, but also exhibit considerable variation in structure and sequence. Examination of several PspC and Hic proteins revealed proteins composed with variable domain patterns, different combinations of domains, and novel domains.PspC and Hic proteins bind several human plasma proteins including Factor H, C3, C4BP, Plasminogen, thrombospondin-1, and vitronectinOur understanding of these important pneumococcal immune evasion proteins is currently incomplete. Thus, our ability to understand the function of single domains, know the binding sites for host ligands, determine how the proteins of different strains vary in structure, and correlate these properties with disease states is limited. To achieve these goals it is essential to define the exact domain composition of individual PspC and Hic variants.40. We further aimed to define domain composition and position. Our studies illustrate structural and compositional differences between the full-length PspC and Hic proteins, within the PspC or Hic group and between the N and C-terminal regions. Furthermore, this comparison also identified nine new domains and several subvariants.Thus far, the domain organization of individual PspC and Hic variants, whether each domain is likely within or extending beyond the cell wall and precise borders of the domains is unclear. Furthermore we do not know exactly which domain(s) are integrated into the bacterial cell wall, which domain(s) span the capsule and which domains are externally positioned. Given these limitations, and the heterogeneity among these important immune evasion proteins, we aimed to evaluate the structure and domain composition of six PspC and five Hic variants, each representing one of the clusters defined by Ianelli et al.40. These are the six PspC variant clusters, i.e. PspC1.1, PspC2.2, PspC3.1, PspC4.2, PspC5.1, PspC6.1, and the five Hic variant clusters, Hic/PspC7.1, Hic/PspC8.1, Hic/PspC9.1, Hic/PspC10.1, Hic/PspC11.1. At the date of the cluster designation Ianelli et al. considered the PspC and Hic variants as one protein family and used a PspC nomenclature for both protein groups37. To preserve the differentiation between Hic and PspC families and at the same time follow the nomenclature suggested by Ianelli et al. we combined the Hic and PspC designations , which is considered a cell wall-spanning and flexible domain and the β-sheet region represented the Choline-Binding Domain (aa 483–701) which mediates attachment to the cell wall . In contrast, the C-terminal region contains a lower percentage of charged residues (15.0%), an increased percentage of polar and amphipathic amino acids (9.5%) and a high level of Tyr residues (8.9%)  Fig. C. Thus, Next we evaluated if the structural composition, as outlined for PspC3.1, is conserved in the other PspC and Hic variants. The N-terminal region of all analyzed PspC and Hic variants consists mainly of α-helices, and the C-terminal Proline-Rich Domains are predominantly coiled-coil structures. The Choline-Binding Domains within the C-terminal PspC variants consist mainly of β-sheets, while the Hic specific LPsTG anchors consist of a coiled-coil stretch followed by an α-helical segment , and were rich in Tyr residues (8.3–9.8%)  to 633 (PspC5.1) residues. A homology alignment of the N-terminal regions showed two distinct clusters. One N-terminal cluster included five PspC variants  and the Hic/PspC11.1 variant, while the second N-terminal panel included PspC4.2 and four Hic variants   to 113 aa (PspC2.2). As their name suggests, they were highly variable in sequence with each PspC and Hic variant examined encoding a distinct variant  present in all variants; although, additional residues are conserved in several variants. Factor H binding by PspC3.1 is mediated by a 12 amino acid region28, we identified diversity in this region of different variants and whether these domains all bind Factor H remains to be determined  is present in PspC3.1, PspC5.1, and Hic/PspC11.1, HVD-B is present in PspC2.2, PspC1.1, and PspC4.2, and HVD-C is present in PspC6.1, Hic/PspC7.1, Hic/PspC10.1, Hic/PspC9.1, and Hic/PspC8.1 . Five PspC variants  contain a second Repeat Domain. These Repeat Domains are rich in charged residues, and include conserved RNYPT motifs, which are binding sites for sIgA/pIgR Fig. . RelatedThe Random Coil Domains are typically positioned downstream of the first Repeat Domain. They are approximately 30 aas in length, have a coiled-coil structure and are relatively conserved in sequence. No homologous sequences were identified in the sequence database  or the unique Hic/PspC11.1 domain have the consensus SnD/GS2 and are up to 24 aa in length. The serine-rich elements following the Random Coil Domain  are comprised of S2DS2, units and can be up to 18 aa long. The domain of Hic/PspC10.1 shows a variation to these common features .Serine-Rich Elements with the overall motif S2DS2 combination of domains.Two new domains were identified downstream of the Random Coil Domain-SStreptococcus agalactiae. These domains are predicted to be involved in bacterial adherence or cell wall binding44.Two proteins, PspC1.1 and PspC5.1, contain an almost identical new 83 aa domain. This domain includes several charged residues, and shares homology with RICH type domains in other proteins, including PspC Q9KK19, SpsA O33742 and IgA Fc receptor binding protein P27951 from PspC4.2 and Hic/PspC10.1 have 114 and 126 aa domains that follow the Random Coil Domain and which share moderate sequence identity. The N-terminal domain of Hic/PspC10.1 has a 37 aa extension, with the remainder of the domain being sequence similarity with the PspC4.2 domain. The biological role of this unique segment is unclear. In PspC4.2 this domain includes a long α-helical stretch that is followed by a 30 aa coiled-coil region.46, suggesting that the newly identified domains in PspC1.1 and PspC5.1 bind lactoferrin48.PspC1.1 and PspC5.1 have related, new domains following Repeat Domain II. These 130 or 131 aa domains are rich in charged residues, and share 84.5% sequence identity with the A*/B element of PspA from pneumococcal strain DBL6A. The A*/B element includes a lactoferrin-binding regionDomain pattern analysis identified an element in PspC4.2 which is positioned between the Hypervariable Domain and the Random Coil Domain. This 33 aa α-helical structured element, lacks homology to other proteins in the databank, thus its role remains unclear.PspC4.2, Hic/PspC7.1, and Hic/PspC10.1 contain related 92, 82, and 68 aa domains, respectively. These mostly α-helical domains are distantly related (41.6% aa identity) to the Repeat Domains, but lack the sIgA/pIgR binding motif (RNYPT) binding motif and seem to be specific to PspC and Hic proteins.A new two-domain segment was identified in PspC4.2 and the three Hic proteins, Hic/PspC7.1, Hic/PspC10.1, Hic/PspC9.1.Extracellular Proline Rich Segments. The high Proline content may suggest a function as linker separating domains49. These External Proline Rich Segments lack homology to other bacterial proteins, and thus seem unique to PspC proteins.The 24–40 aa upstream segments of this domain are rich in proline residues, have a predicted coiled-coil structure, and due to their location in the N-terminal region of PspC are termed Streptococcus agalactiae (strain P27951/Uniprot). This protein binds the Fc region of human IgA, likely via two putative binding sequences50 which are also found in several other bacterial immune evasion proteins including SpsA from S. pneumoniae. Based on the many charged residues this IgA binding domain (pfam05062) is also named RICH (Rich In Charged residues) the proposed function of which is bacterial adherence or cell wall binding.The 78 or 89 aa elements are rich in charged residues, lack proline residues, and have an α-helical structure. A blast search revealed 51.1% aa identity with an IgA binding domain within the trypsin sensitive beta-antigen of Following the Hypervariable Domain, Hic/PspC11.1 contains a unique 102 aa α-helical domain. Related domains were identified in most Hic/PspC11 variants, but not in other bacterial proteins Thus far, the function of this domain is unknown.49. The C-terminal regions of the PspC and Hic proteins analyzed are relatively conserved in length (ranging from 237 aa (PspC5.1) to 348 aa (Hic/PspC8.1)). A general pattern is emerging: PspC proteins link shorter Proline-Rich Domains (57 to 77 aa) to longer Choline-Binding Domains (179 to 219 aa), while Hic proteins combine longer, Proline-Rich Domains (186 to 286 aa) with shorter LPsTG anchors (50 to 62 aa).The C-terminal region of each variant contains a modular Proline-Rich Domain with a Choline-Binding Domain for PspC variants or an LPsTG anchor for His variants51. The proposed role of these domains as spanning the bacterial cell wall-spanning is consistent with the position proximal to the anchor52. Our in-silico analysis identified a modular composition and further distinct proline-rich domains, which differ in length (57 to 286 aa), modular composition, and sequence.Proline-Rich Domains have a modular structure and connect the N-terminal region to the cell wall anchor54.Five PspC variants have highly related 59 to 77 aa domains, termed Proline Rich domain I. This modular domain can consist of two  or three  segments  MQPSPQPEGKKPSVPAQPGTED). Each repeat contains nine proline residues and two KKPS(A/V)P motifs. The repeats are followed by a truncated 24 aa repeat element ETPKPEVKPE repeats (variant residues are displayed as white letters on a black background). In each case, these repeats are followed by one shortened repeat and a nearly identical 16 aa-long C-terminal module, which varies only at position 15 (T/P variation)  to 248 aa (PspC1.1) and consist of modules most of which are 20 aa in length  and a penicillin binding protein20. Three domains, the Signal peptides, the Hypervariable Domains and Proline-Rich Domains are found in all analyzed variants  to 610 aa containing eight domains (PspC4.2). These regions share structural features, including long α-helical structures, and a high proportion of charged residues. The Hypervariable Regions are most likely located most distant from the cell surface and show the highest degree of sequence variation. This diversity can reflect differences in antigenic variability, which is relevant for evading immune recognition by antibodies. Six of the N-terminal domains are unique to PspC and Hic variants, others like the PspA Related Domain and the region with homology to the IgA binding β antigen are found in other bacterial immune evasion proteins  a hic-variant, 19 isolates (5.4%) had pspC and hic gene and only 10 isolates (2.9%) did not possess either gene64. In addition, invasive, PspC expressing strains bound more Factor H, and Factor H binding and immune control was more effective in encapsulated as compared to unencapsulated strains. Similarly, the PspC variants PspC2 and PspC6 were more efficient in Factor H binding and complement inhibition on the bacterial surface as compared to the Hic variants, Hic/Pspc9 and Hic/PspC1166.When comparing prevalence and distribution of PspC and Hic variants among 349 pneumococcal isolates from adult patients with invasive pneumococcal disease, 298 isolates (85.4%) had a single Evaluating the domain composition of selected PspC and Hic variants and an in-depth characterization of the domain composition advanced our understanding of the structure of these virulence determinants. Our approach identified differences between PspC and Hic proteins beyond their distinct membrane anchors. Such knowledge allows a comparison of full-length proteins based on domain patterns, numbers and can result in a better comparison of different PspC and Hic/Hic variants. Similarly, individual domains can be compared based on structure, modular composition and sequence.Analyzing the additional > 60,000 PspC and Hic proteins deposited in the NCBI protein database or gene products from additional clinical isolates, will likely identify additional variants due to the discovery of new domains and subdomains, and novel domain combinations. Defining the diversity within these pneumococcal virulence factors will increase understanding of their role in immune evasion and provide important information for molecular strain typing and vaccine design. Finally, this may also allow a correlation between PspC or Hic type variants with invasive pneumococcal infections and with clinical outcome.40. The sequences were derived from the NCBI database (status: Feb 2018). The PspC/Hic designation is based on Iannelli et al.40. The protein names, corresponding bacterial strain, protein size, GenBank Accession number and protein ID are shown in . PspC3.1 most predicted structural similarity 2vyuA . Analysis of the other ten PspC / Hic variants revealed a similar secondary structure  of each selected PspC and Hic protein was evaluated using RaptorX (www.megasoftware.net). There was a total of 976 positions in the final dataset67. The CLUSTALW program and the BLOSUM amino acid matrix were used to compare the allelic variants of PspC, following which phylograms were generated using the Neighbor-Joining method . The phylogram for each domain was generated using the same method. Phylogenetic trees are modified in MEGA7.The PspC and Hic amino acid sequences and composition were evaluated using MEGA7 (http://www.ncbi.nlm.nih.gov/). Furthermore, BLAST targeting database UnipRotKB reference proteomes plus Swiss-Prot was used to find regions of local similarity between sequences (https://www.uniprot.org/blast/). All the domains in this work have been done a blast.BLASTp was used to identify related proteins or protein segmetns within the GenBank database available at the National Center for Biotechnology Information (Supplementary Figures.Supplementary Tables.


The effects of the package materials on the hot workability and stress-strain characteristics of high-Nb TiAl alloy with a nominal composition of Ti-46Al-8Nb (in at.%) were systematically studied via “sandwich structure” hot compression. TiAl sheet fabrication was conducted by hot pack rolling, and the microstructural characteristics and deformation mechanisms were investigated. Based on the analysis of compressed samples and stress-strain curves, the stainless steel/TiAl structure showed better deformation compatibility with homogeneous deformation and decreasing resistance. However, severe interfacial reactions were inevitable. Meanwhile, for the titanium alloy/TiAl structure, few interfacial reactions happened, but wavy deformation and high resistance complicated the compression process. Finally, a package structure with an outer stainless steel isolation layer and inner titanium alloy was determined for the pack rolling process. A TiAl sheet with no crack defects was obtained with 80% reduction. The pack-rolled TiAl sheet took on alternate microstructure of the grain-boundary Al-enriched ribbons and elongated lamellar colonies ribbons. The grain-boundary recrystallized α2 phase, lumpy γ phase, and massive α2/γ lamellae could be observed, which led to the scatter microstructure. The microstructural characteristics mainly resulted from the solute segregations of as-cast Ti-46Al-8Nb alloys, which triggered the local flow softening and deformation incompatibility during hot pack rolling. TiAl Nevertheless, the pack rolling process is not feasible enough for a sound TiAl sheet, especially for the thin-plate fabrication of the TiAl ingot with the intense heat-loss and strain sensitivity. In general, the pack rolling mainly depends on the package structure’s design and the materials, which directly determine the stress-stain conditions, heat protection, and sheet surface quality. The “black box” mode for the package structure always confuses the design for the pack rolling process . SemiatiAdditionally, the softening behavior and heat-deformed microstructure of TiAl alloy during the hot pack rolling process are closely connected with the original microstructure and rolling conditions ,16. For In this study, the pack rolling process and microstructural characteristics for Ti-46Al-8Nb sheet fabrication were investigated. Firstly, different packaging materials  were selected for the “sandwich structure” hot compression experiments. Then, the deformation compatibility, stress-strain conditions, and surface reaction were studied between the packaging layer and TiAl matrix. The optimal package structure design for Ti-46Al-8Nb sheet fabrication is presented. Finally, the microstructural characteristics and flow behavior were investigated for the pack-rolled Ti-46Al-8Nb alloy.−1. Three trials were performed for each reduction to reduce errors. The temperature was measured by W/Re thermocouples in the TiAl matrix material.The as-cast cylindrical Ti-46Al-8Nb ingot with an actual composition of Ti-46.3Al-7.8Nb (at.%) was fabricated by induction skull melting (ISM). Chemical analysis results were analyzed by inductively coupled plasma mass spectrometry (ICPMS) for both the alloys. Subsequently the homogenization heat treatment was performed at 1200 °C for 4 h. The microstructure is comprised of the α2/γ lamellar colony, B2 phase, and grain-boundary γ phase . The sam2O3 polishing solution, and etched with a solution of 10 mL HF + 10 mL HNO3 + 180 mL H2O. The different phases present in the microstructure were discriminated based on their contrast on back-scattered electron  images, and the component distribution was determined by EDS. Specimens for transmission electron microscope  were prepared by twin jet polishing. This was carried out in a solution consisting of 5% perchloric acid, 35% butanol, and 60% methanol (in volume fraction) at −25 °C at a voltage of 25–30 V.The packaging samples for Ti-46Al-8Nb alloy are also prepared from the as-cast ingot by ISM after the homogenized heat treatment at 1200 °C for 4 h, and the hot-rolling process was conducted on a mill with a roller 120 mm in diameter and 300 mm in width, which was preheated to 1250 °C for 30 min before rolling. Each sample was rolled at a speed of 40–50 mm/s with a reduction of 8% per pass, and the re-heating was conducted between each rolling pass. After the hot compression and the package rolling experiments, the specimens were cut for microstructural analysis performed by optical microscopy  and scanning electron microscopy  with an energy dispersive spectrometer . Samples for OM and SEM observations were rubbed by #100, #400, #1000, and #1500 sandpaper, mechanically polished by a CrThe respective deformations of the packaging layers and TiAl matrix for the hot-compressed sandwich samples are shown in Furthermore, the calculated stress-strain curves were obtained for the different packaging materials and deformation degrees during the compression process in Additionally, the high chemical affinity of the TiAl alloy should be always considered during the high-temperature conditions. Through the results above, the selected packaging materials are critical to the hot deformation processing for the TiAl workpiece, which should own the characteristics of deformation compatibility, heat protection, and low interfacial reactivity. For the stainless steel package, excellent deformation compatibility and heat protection can be obtained owning to its high-temperature strength, but the serious interfacial reactivity may lead to inhomogeneous deformation and crack defects due to the eutectic reaction of Ti-Fe interface at the temperatures of nearly 1100 °C. For TC4 alloy package, the interfacial reaction can be neglected, but the wavy large-reduction and the heat loss always occur due to its low strength during the hot deformation process. Therefore, the deformation compatibility and interfacial reaction should be considered for the TiAl sheet fabrication during the package rolling process, which is mainly centered on the design of the packing materials and packing structure.Based on the analysis above, the complex design with the outer stainless steel/isolation layer and inner TC4 alloy/coating structure was selected for the pack-rolled Ti-46Al-8Nb alloy, and the sheet macrograph, interface conditions, and macrostructure are shown in Furthermore, the heat-deformed microstructure of the pack-rolled Ti-46Al-8Nb alloy was investigated with increasing degrees of deformation, as shown in The results above are mainly attributed to the anisotropic plastic flow of lamellar colonies during rolling process. According to the calculation of polysynthetically twinned crystal (PST) TiAl at 1100 °C , the yieThrough the results above, the local flow softening exists in heat-deformed TiAl alloy, which may lead to complex microstructure under the pack rolling conditions, especially for the selected Ti-46Al-8Nb alloy with the highly solute segregation. Generally, the streamline structure, multiple- recrystallized grains, and retained lamellar with rolling reduction of 80% are observed in pack-rolled Ti-46Al-8Nb alloy, as shown in Additionally, the partial lamellar microstructures cannot be broken down completely by hot deformation processing, especially for the Ti-46Al-8Nb alloy with a local flow softening process, and massive retained lamellar colonies with nearly 60 μm are obtained in the pack-rolled TiAl sheet, as shown in Through the results above, the typical deformed-ribbons structure, lumpy γ phase, and retained lamellar structure are always obtained in pack-rolled Ti-46Al-8Nb alloy, leading to the scatter microstructure and mechanical properties. The multistructure above is mainly from the local flow softening process due to the original microstructural characteristics and solute segregation, and it can be seen that the design of TiAl alloy with the full and fine α2/γ lamellar colony, low Al segregation, and the improved hot-mechanical treatment should be required for the homogeneous, heat-deformed microstructure for TiAl sheet fabrication.Based on the above observation, the schematic illustration of the deformation processes of Al-enriched ribbons (Al segregation regions) and the elongated lamellar colonies ribbons (regions with net-like Nb segregations) can be deduced, as shown in (1)The sandwich structure of stainless steel/TiAl matrix has good deformation compatibility due to the approximate high-temperature strength during the hot compression process, showing a less deformation-resistant and homogeneous deformation process, but a severe interfacial reaction is observed leading the aggravation of the contact interface. For the TC4/TiAl structure, the interfacial reaction can be neglected, but wavy reduction and high deformation resistance occur due to the low high-temperature strength of the titanium alloy package.(2)Considering the deformation compatibility and the interface protection, the pack-rolled Ti-46Al-8Nb alloy is complexly designed with an outer stainless steel/isolation layer; an inner TC4 alloy/coating structure for the sheet fabrication; and finally, the sound TiAl sheet with the intact interface. No crack defects can be observed with 85% reduction under the rolling conditions, showing the proper packing structure design and rolling conditions.(3)The pack-rolled TiAl sheet takes on the alternate structure of the dark grain-boundary Al-enriched ribbons and the elongated lamellar colonies ribbons, in which the multistructure of the deformation ribbons’ structure, lumpy γ phase, and retained lamellar colony is obtained mainly from the local flow softening process due to the original microstructure and solute segregation in pack-rolled Ti-46Al-8Nb alloy.In this study, the pack rolling process and the microstructural evolution for Ti-46Al-8Nb sheet fabrication were investigated. The results and conclusions can be summarized as follows: