Triticum aestivum) on its rheological and baking properties. Raw materials were analyzed chemically  and physically . Selected rheological analyses (Farinograph® and Extensograph®) were carried out on wheat blends, including up to 30% alternative grains. The baking properties of the blends were evaluated on standard bread and sweet milk bread recipes. Results showed that low amounts (5%) of sorghum and millet improved the dough stability of the high-gluten wheat flour. For optimum dough extensibility, additions of 30% sorghum, 15% millet, or 20% amaranth were needed. The use of gluten-free grains increased bread volume and decreased crumb firmness of the sweet milk breads when added at lower levels . In conclusion, cereal blending is a supportive tool to mitigate the effects of ongoing climate change and can enhance biodiversity and nutrition.Due to the effect of climate change, wheat flour qualities with extremely high dough extensibility or dough strength are becoming more common, which impairs the production of selected wheat products such as pastries. The aim of this study was to investigate the effect of sorghum, millet, amaranth, or buckwheat addition to such a strong gluten common wheat flour ( In recent years the interest in bakery products made from cereal blends has increased for various reasons. Cereal blending can offer health-promoting benefits as it can diversify, complement, or even enhance the nutritional properties  of flour. This allows the development of end-products with higher nutritional quality, as was shown for, e.g., in maize tortillas fortified with amaranth, which then possessed significantly higher antioxidative activities , or for In Europe, wheat is the most important cereal in the food industry. Its uses are most evident within the baking industry, in a broad variety of products, ranging from bread, cakes, and pastries to waffles or cookies.Wheat is characterized by its excellent baking properties, resulting from the unique properties of the gluten protein fraction, which gives the dough a firm but extensible and elastic character. However, the ongoing climate change has become a challenging factor for the wheat cereal industry in recent years. On the one hand, intensely hot and dry summers have led to a decline in wheat yields. On the other hand, the wheat protein quality and amounts (in particular gluten) have been observed to vary greatly depending on the climatic conditions, as reported by Gagliardi et al. .At this point, it has to be stressed that high protein quantities have principally been valued by the baking industry and, thus, the wheat breeding industry. Harvested Austrian wheat can generally be considered to be of superior quality; they are also sought after and exported to many countries. Yet for some products, such superior wheat qualities are less desirable, as excessive dough extensibilities or dough strengths are not suitable for all products. Some require lower wheat qualities, in particular fine bakery products such as cakes and waffles and other fine bakery products that are made from batter rather than from dough. So far, only a minimal amount of data is available that describes in detail such climatic effects on wheat quality. Wrigley et al.  pointed Possible solutions may be the use of food additives to adjust the wheat dough/bread quality or to import wheat from other countries to balance out wheat crop losses and qualities. However, these solutions offer rapid but not sustainable options and do not meet the growing consumers’ trend towards regional, nutritional, and clean-label foods. Another feasible approach to compensate for wheat crop losses and the altered wheat qualities may be offered by using flour blends of wheat and non-wheat grains for Western-style bakery products.A considerable amount of literature has been published on the rheological and baking properties of wheat blends with alternative cereals. However, the existing body of this research has primarily focused on the nutritional enrichment of final products from such cereal blends, thus aiming to introduce the highest possible amounts of non-wheat  grains into the flour blends. Until now, inadequate attention has been paid to the substitution of wheat in smaller amounts with the aim of optimizing or improving the rheological properties of doughs made from flour blends and their final product quality. While it is common knowledge that the lack of gluten affects baking properties negatively, less consideration has been given to the idea of whether low amounts of (gluten-free) cereals are advantageous in the baking of products from strong gluten wheat flours . Promisi® and Extensograph®, Brabender & Co. KG, Duisburg, Germany) and up to 40% in bakery products in 5% steps (2.5% steps for some specific conditions). The bakery products selected were a standardized yeast bread and a standardized sweet milk bread, the latter representing the fine bakery product range.Therefore, the general aim of this study was to compare how the addition of different non-wheat, gluten-free grains  affects the dough and baking quality of bakery products when blended with wheat flour. Emphasis was put on the use of the same methods and recipes for all flour blends, in order to allow a reliable comparison of eventual differences between the selected specialty grains, which so far has not been considered in previous publications. The decision criteria for selecting the non-wheat grains were their (existing or known) ability to be cultivated in Austria/middle Europe and their suitability to hot and dry climatic conditions. This was true for sorghum, millet, and amaranth; buckwheat, though, is minimally drought tolerant, but as it is more known in Austria than the beforementioned grains, it was included as well. All raw materials were thoroughly characterized for their chemical , physical (WAI and WSI), and pasting properties (RVA). For the experiments, wheat was blended with the respective grain to up to 30% for dough rheological determinations  and water-soluble index (WSI). These results are summarized in A noticeable lower ash content was found for wheat, which was expected as wheat was the only refined flour. Both red and white sorghum showed similar ash contents; the highest ash content was found in amaranth, followed by buckwheat. Decortication of the seeds only partially explains the difference in ash content, as amaranth was the only seed that was milled un-decorticated. Low protein contents were observed for red sorghum and millet. In white sorghum, it was higher, similar to buckwheat, and amaranth possessed the highest protein content. Among all alternative cereals, buckwheat showed the lowest fat content and was again highest in amaranth. The highest starch values were evaluated for wheat and buckwheat, while amaranth was characterized by the lowest amount, which was even lower than reported results from the literature . All detThe water absorption index  is known for determining the hydration properties of a grain material. It is mostly influenced by the starch properties but also by fat and dietary fiber content. Both sorghum varieties had the highest WAI, which is most likely related to the fact that sorghum shows a huge bandwidth regarding starch granule sizes of 4–35 µm as reported by Zhu  in diffeThe RVA pasting properties of the flours are presented in Overall, pasting properties are mainly influenced by the starch content and its properties, such as amylose/amylopectin ratio, amylopectin chain lengths, and starch granule sizes.The pasting temperatures (°C) in descending order for red sorghum, white sorghum, wheat, millet, amaranth, and buckwheat were 91.77 ± 0.5, 89.05 ± 0.5, 88.55 ± 0.5, 78.75 ± 0.5, 77.52 ± 0.1 and 73.17 ± 0.5, respectively. It is known that amylose is able to form complexes with lipids during thermal treatments, which can result in higher pasting temperatures . This miPeak viscosity was highest in wheat (216.39 ± 12.11), followed by white sorghum (197.89 ± 13.88), millet (164.47 ± 0.82), red sorghum (127.58 ± 0.29), and amaranth (97.58 ± 6.22). For buckwheat, no defined peak viscosity was determinable. The grains showed significant differences with respect to breakdown, which was highest in wheat (87.33 ± 4.07), followed by millet (66.67 ± 3.18) and white sorghum (41.33 ± 9.06). Interestingly, the two sorghum varieties were significantly different from each other as only red sorghum showed almost no breakdown (6.83 ± 0.42), similar to the two pseudocereals. The breakdown of hot paste viscosity is an indicator of the stability of starch during heat treatment and is highly influenced by the inherent starch granules properties such as granule stability, crystallinity, or number of amorphous regions. A lower breakdown might also indicate that starch granule swelling and starch granule disruption occur at the same time . LookingIn In Dough development time b decreasRegarding the Extensograph data e–h, the Selected results of the baking trials can be seen in In the standard wheat bread (SWB) baking trials, differences between the grains and also between varieties (within sorghum varieties) could be observed. Those results were already discussed in the previous publication of Rumler et al. . While wAs hypothesized for this study, the addition of alternative grains could positively influence the MWB final quality. Most successful in this respect was the blending with millet. At 5 and 10% millet addition, a significant increase in the MWB volume and a remarkable decrease in crumb firmness was observed, and no detrimental effects, even at 40% addition, were detected. Similar results were obtained by Maktouf et al. , who fouThe crumb structure of a bread is an essential quality parameter. In In SBW, sorghum addition and their variety significantly affected crumb pore sizes to a different extent a,c. Red In MBW, the porosity was generally less influenced by alternative grain addition compared with SBW. Sorghum addition slightly increased the pore area, mainly on account of the number of pores, and was more pronounced for white than red sorghum. In the case of millet addition, no clear changes were observed for both average pore size and total pore area b,d. An aThe color of the bread crumbs was measured and is shown in Triticum aestivum) without food additives was obtained from GoodMills Österreich GmbH . The wet gluten content of the wheat flour was 33.3%, which characterized the flour as a high gluten flour. Wholegrain sorghum flours , wholegrain millet flour  and wholegrain buckwheat flour (Fagopyrum esculentum MOENCH) were purchased from Strobl Naturmühle . Amaranth  was obtained from a farmer in lower Austria (harvested in 2020) and milled to wholegrain flour using a pin mill . Sorghum varieties Armorik and Ggolden, millet, and buckwheat were harvested in the year 2019 and decorticated by abrasive milling prior to flour milling.Refined common wheat flour  according to ICC standard methods 115/1 and 114/1, respectively. All analyses were carried out in triplicate, except for the Farinograph and Extensograph trials, which were performed in duplicate.Ash, protein, starch, and fat contents were analyzed according to ICC or AACC standard methods as described by Rumler et al. . The pasStandard wheat bread (SWB) preparationFor the standard wheat bread (SWB) preparation, a dough weight of 1000 g was produced based on the following recipe (% based on flour): 100% flour, 58% water, 3% dry yeast, 1.8% salt, and 1% sugar. All dry ingredients were homogenized at step 1 using a laboratory kneader . Afterward, the yeast (dissolved in 50 mL of the water) and the remaining water were added while kneading at step 2 for 30 s. Then, the dough was kneaded for 6 min and afterward molded by hand for 1 min. The obtained dough was put into a fermentation chamber  set at 30 °C and 85% relative humidity (RH) for 30 min. After dough fermentation, the dough was separated into three 300 g portions, molded, folded, and rolled into a screw, which was placed into an oiled baking tin and submitted to a second fermentation period  for 50 min. Finally, the breads were baked at 220 °C in a conventional deck oven  for 25 min. After baking, breads were cooled at room temperature for 2 h and then stored at 20 °C, 50% RH in a climate chamber overnight until further analyses.Sweet milk wheat bread (MWB) preparationFor the sweet milk wheat bread (MWB) preparation, a dough weight of 1000 g was produced based on the following recipe (% based on flour): 100% flour, 50% milk, 6% yeast, 12% butter, 12% sugar, 6% fresh egg yolk and 1.5% salt. First, the milk was warmed up to 30 °C and homogenized with the yeast. Afterward, sugar, egg yolk, and salt were added, then homogenized with the flour by using a laboratory kneader  at step 2 for 2 min. Subsequently, the butter was added, and the dough was kneaded for a further 4 min, then molded by hand for 1 min. The obtained dough was placed into a fermentation chamber  at 30 °C and 85% RH for 30 min, divided into 300g dough pieces, and fermented for a second period for 20 min. Finally, the doughs were baked at 170 °C in a conventional deck oven  for 25 min. The cooled breads (2 h at room temperature) were stored at 20 °C and 50% RH relative humidity in a climate chamber overnight until further analyses. All SBW and MBW baking trials were carried out in triplicate, resulting in a total number of 9 bread loaves per formula.3) of the bread was evaluated by using the bread volume analyzer BVM 6600 . Bread weight (g) was measured after the bread was fully cooled. Every loaf was measured in duplicate, resulting in a total number of 18 values per formula. The specific volume was calculated as displayed in Equation (1):Specific volume: The volume (cm3/100 g flour) defines the loaf volume related to 100 g flour and is typically used by bakers. The calculation can be seen in Equation (2).Volume yield: The volume yield , which was equipped with a 50 kg load cell and a cylindrical compression probe (SMS P/100). The settings were chosen according to Waziiroh et al. : Test spBaking loss describes the amount of water lost during baking and was calculated as ratio of dough weight to loaf weight (Equation (4)).Crumb and crust color analysis were carried out as described by Bender et al. . BrieflyFor the determination of the crumb porosity, the procedure of Waziiroh et al. (2021) was applied using the Image J software. Values measured were the average pores size (mm) and the total pore area [%].All results were statistically analyzed by using Statgraphics Centurion 19.4.01 3 . Applying the ANOVA procedure and Fisher’s least significant test (α = 0.05). The correlation of data was carried out using the Pearson correlation test (α = 0.05).Results of this study demonstrated that blending wheat flour with specialty gluten-free grains offers a new perspective for the use of (too) strong gluten wheat flour as affected by climate change. Rather than a decline in quality, in this study, it was seen that low levels of selected gluten-free cereals indeed could improve the rheology and (fine) baking properties of wheat flour, which was characterized by a high/strong gluten content, as determined by high dough extensibility and high dough strength. The addition of gluten-free grains  led to a relaxation of the dough; ideal dough extensibility values were observed when 25% white or red sorghum and 20% millet or amaranth were added to wheat. Baking trials demonstrated that the used gluten-free grains were able to improve final product quality  when added at lower levels . While the used cereals, sorghum, and millet, could often be added at higher amounts of up to 40% without deteriorating the product quality significantly, the pseudocereals amaranth and buckwheat had a lower optimum level of addition (2.5–5%) without detrimental quality effects. This implies, in consequence, that if the aim is to replace wheat in higher amounts with alternative grains, e.g., to improve the nutritional properties of the final product, or to increase the biodiversity of grain production and consumption, the use of cereals such as millet or sorghum is recommendable. If the aim is to simply balance the overall flour blend quality, then gluten-free cereals and also pseudocereals  can be incorporated, all at low levels of approx. 2.5–10%. Overall, wheat blending offers beneficial effects for the future in terms of increased biodiversity and improved nutrition and is thus a supportive tool to mitigate the effects of the ongoing climate change.


These improvementsenabled the demonstration that this chemotype engages PI5P4Kγin intact cells and that compounds from this series do not inhibitPI5P4Kα or PI5P4Kβ. Furthermore, the first X-ray structureof PI5P4Kγ bound to an inhibitor has been determined with thischemotype, confirming an allosteric binding mode. An exemplar fromthis chemical series adopted two distinct modes of inhibition, includingthrough binding to a putative lipid interaction site which is 18 Åfrom the ATP pocket.Phosphatidylinositol 5-phosphate4-kinases (PI5P4Ks) are emergingas attractive therapeutic targets in diseases, such as cancer, immunologicaldisorders, and neurodegeneration, owing to their central role in regulatingcell signaling pathways that are either dysfunctional or can be modulatedto promote cell survival. Different modes of binding may enhance inhibitorselectivity and reduce off-target effects in cells. Here, we describeefforts to improve the physicochemical properties of the selectivePI5P4Kγ inhibitor, NIH-12848 ( Despite the lack of structuralsimilarity between lipid and protein kinases, a comprehensive studyof the selectivity profiles of protein kinase inhibitors previouslyshowed that some exhibit weak activity toward the lipid PI5P 4-kinases,27 while other publications have shown that someprotein kinase inhibitors display activity against PI5P4Kγ,for example, tyrphostin28 and palbociclib29 (see Supporting Information Table S1 for more details from our own screening of selected PI5P4Kγinhibitors). Most significantly, NIH-12848 (1)30 and NCT-504 (2)23 have been disclosed as selective PI5P4Kγ inhibitors(1 was reported to have an IC50 of 2–3 μMin a radiometric 32P-ATP/PI5P incorporation assay (see Supporting Information Table S2). Compound 2 was identified following a high-throughput screen and, whentested against a panel of 442 kinases, was found to have activityagainst only PI5P4Kγ (IC50 = 16 μM in a 32P-ATP/PI5P incorporation assay).The role of PI5P4Kγin disease makes it a potentially importanttherapeutic target; however, development has been hampered by thelack of potent and specific inhibitors. Furthermore, in contrast tothe protein kinase family, which has a relatively well-conserved activesite,hibitors1. Compou3), “compound 30” (4),32 and noncovalent “compound 13” (5)33 (Table S1). Compound 4 displayedan IC50 of 1.3 μM against PI5P4Kα in a bioluminescentassay and 9.9 μM against PI5P4Kβ in a fluorescence polarizationassay, but inhibited PI5P4Kγ by only 22% when tested in a KINOMEscanassay at 1 μM. In the same assays, compound 3 showedsimilar activity against PI5P4Kα and β but with a moresignificant PI5P4Kγ inhibition of 91% and a PI5P4Kγ KD of 4.8 nM.34 Compound 5 has demonstrated similar levels of activity in these PI5P4Kαand β assays and 100% inhibition of PI5P4Kγ in the KINOMEscan assay whentested at 1 μM and a PI5P4Kγ KD of 3.4 nM.34In 2020, pan PI5P4K inhibitorswere reported, including covalentinhibitors THZ-P1-2  with poorsolubility (<5 μM). Furthermore, 1 has beenproposed to interact with an alternative region of the PI5P4Kγcatalytic site, potentially acting as a competitor for the PI5P substraterather than for ATP binding30 offeringa unique opportunity to study lipid kinase structural biology.Herein, we describeefforts to improve the physicochemical propertiesof 1, either through purchasing or synthesizingclose analogues with subtle changes to the trifluoromethylphenyl groupor thiophene (25 and has enabled the development of an inhibition assay with a useablewindow. The mutations  correspondto the PI5P4Kα residues and the resulting PI5P4Kγ constructcontaining these has been referred to as PI5P4Kγ+.25 To mitigate concerns about being misled by theuse of the PI5P4Kγ+ construct and the possible introductionof PI5P4Kα activity into the molecules, our screening cascadeincluded the measurement of ADP-Glo activity at PI5P4Kα-WT (andPI5P4Kβ-WT to fully understand emerging selectivity profiles).Moreover, cellular target engagement was periodically determined forexemplars; this determined compound binding to overexpressed PI5P4Kγ-WTin HEK293 cells through changes in target protein thermal stability,monitored using a DiscoverX’s InCell Pulse assay. Furthermore,we established a cell-free thermal shift assay with PI5P4Kγ-WTand determined that this chemical series showed a good correlationfor measured ΔTm with PI5P4Kγ-WTand the ADP-Glo pIC50 with PI5P4Kγ+ .In general, for this chemical series, we observed a good correlationbetween PI5P4Kγ+ and PI5P4Kγ-WT binding and the undetectablelevels of PI5P4Kα and PI5P4Kβ inhibition.The optimization campaign began with simple modifications of theparent molecule hiophene 1. To ena35 enabled us to pinpoint theATP-binding pocket for PI5P4Kγ. However, 1 couldnot be docked into this pocket in a convincing binding pose, a findingthat is consistent with Clarke et al.’s evidence from hydrogen–deuteriumexchange-mass spectrometry (HDX-MS)30 thatthis ligand does not bind in the ATP-binding pocket. No other bindingpocket for 1 could be identified in the 2GK9 structure, so weproceeded with our attempts to optimize 1 through classicalmedicinal chemistry approaches without structural guidance.Structuralinformation for each of the PI5P4K isoforms was publiclyavailable at the start of this program in the form of X-ray crystalstructures deposited in the Protein Data Bank, but there were no publishedstructures with drug-like ligands bound. The only crystal structureof PI5P4Kγ publicly available at that time (pdb: 2GK9) does not have aligand bound, but the crystal structures of PI5P4Kβ bound tonucleotides1 didnot reveal any advantages  nor was N-methylation(data not shown). On balance, from this initial set of thiophene replacements,pyridine 14 was preferred as it showed a reduced lipophilicitycompared to 1 (c log P 5.5) and a modest improvement in solubility to 9 μM, whichresulted in a significant improvement in measured Caco-2 permeabilityfrom 6.4 to 85 × 10–6 cm/s (14 showed a ΔTm of 6.3 °C versus 4.7 °C for 1 and ΔTm < 1 °Cagainst both PI5P4Kα and PI5P4Kβ (Table S4).During initial structure–activity relationship (SAR) scopingwith PI5P4Kγ+, a brief scan of a variety of small substituentswith different electronic properties at position R e.g. 6–91, and th0–6 cm/s 2. In the1 modificationwas then conductedusing the 2-pyridyl analogue 14 as a template. The additionof a fluoro substituent was beneficial for potency at position 4 butnot 5 (16 and 17). The movement of the trifluoromethylgroup from the 2-position to either the 3- or 4-position of the phenylring was not tolerated ,whereas 3- or 4-methoxy was tolerated to some degree . Small lipophilic substituentsgave the greatest PI5P4Kγ+ inhibition when used at position2, with i-Pr > c-Pr > Et > Ph .Indeed, 22 was one of the most ligand efficient (LE)analogues identifiedduring this campaign (LE = 0.34) and showed submicromolar activityin the cellular target engagement assay with PI5P4Kγ-WT , which appears to limit its aqueous solubility and microsomalstability  and electron-donatinggroups (27).A further iteration of R5P4Kγ-WT 3. Howevetability 2.Polar 28 and 29, respectively) was not toleratednor was the addition of a 5-trifluoromethyl substituent (30). The addition of a 3-hydroxy group (31) was also well-tolerated.This modification introduced some inhibition of PI5P4Kα andsome polarity that may be of use for further development, but theseaspects were not pursued.Having identified2-isopropylphenyl as the preferred substituentat position 2 of the quinazoline for potency gains, we next lookedat adding further substituents to the 2-pyridyl ring and modifyingthe quinazoline core. The deletion or branching of the methylene linker gave a small increase in potency at the cost ofsignificantly increased molecular weight (MW) and c log P, whereas an amino group at the same position(33) maintained activity with a smaller MW penalty anda positive impact on c log P.The quinazoline core presented a numberof opportunities for investigation,including adding substituents, introducing heteroatoms to modulatelog ometries 3. We genet al.,36 and the structural similarity of ML323 , we performed a truncation of thequinazoline core to afford compound 34. In our assays,ML323 was inactive at both PI5P4Kα and PI5P4Kγ+ (datanot shown), whereas 34 showed a pIC50 of 5.4against PI5P4Kγ+ and no detectable inhibition of the αor β isoforms. A similar profile was seen with analogue 35, possessing a trifluoromethyl instead of the isopropyl and particularly at position8 (37 and 38). Pyridopyrimidine 37 showed an improved potency, a reduced c log P and good selectivity versus PI5P4Kα and PI5P4Kβ.Furthermore, 37 showed submicromolar activity in thecellular target engagement assay with PI5P4Kγ-WT.In order to maximize the efficiency of the core, we sought to deletelipophilic atoms or replace them with heteroatoms. Intrigued by theparallels with the work by Dexheimer sopropyl3. The LE39 and 40 were also evaluated.Although compound 39 was inactive,compound 40 showed good potency and LE in the primaryassay, and submicromolar activity in the cellular target engagementassay. It also maintained selectivity versus PI5P4Kα and PI5P4Kβ,showed high levels of permeability, and moderate microsomal stability was not well-tolerated.Regioisomericpyrrolopyrimidines tability2. The alKDs) were determinedfor compound 40 using commercially available assays forPI5P4Kγ-WT (68 nM) and PI5P4Kβ  and comparedversus compound 1 (Table S6). This assay was not available for PI5P4Kα but a commercialIC50 determination was possible via Thermo Fisher to corroborateour own data .Compound 40 was also tested for selectivity against apanel of 140protein kinases and 15 lipid kinases . Only one of these targets (PAK2) showed<50% residual activity when tested at 10 μM.Binding constants . The other threemonomer chains  show the ligand located 18 Å awayfrom the ATP site in a lipid-binding pocket.38 In this protein conformation, residues Gln378, Tyr379, and Asp380of the activation loop occupy an inhibitory position in the ATP-bindingsite .The allosteric binding site in the PI5P4Kγ protein is formedby residues Asp161, Met162, Asn165, Leu166, Tyr169, Leu182, Phe185,Phe272, Leu273, Leu276, Asp374, and Thr377 with the isopropylphenylmoiety of c pocket3. The pyf Asn165 3c. Finalf Asn165 3d. The i40 in the allosteric pocket is 4.4 kcals/mol more favorablethan in the ATP pocket. There is evidence from the electron densitythat there is some occupancy for both ligand-binding modes in allthe chains.In chain B, wherethe ligand occupies the ATP site, the allostericbinding site is occupied by residues Ile375 and Leu376, which formthe start of a loop that is mostly missing in PI5P4K crystal structures4. Ile37540-PI5P4Kγcomplex shows that one side ofthe ATP-binding pocket is formed by residues 134–141 when 40 is bound. In the apo structure 2GK9, residues 136 to 139 of this loop aremissing, but they are clearly visible in chain B of the complex crystalstructure, although some side chain density is poor , except forF-atoms (16 and 17). It is not clear whycompounds 20 and 21 show weak activity,but from the obtained crystal structure, binding to the ATP pocketcannot be ruled out. The ortho isopropyl group is important both forfilling the lipophilic pocket formed by Leu182, Pro183, Phe185, Phe272,and Leu273 and for stabilizing the binding conformation through internalhydrophobic contact. A trifluoromethyl group at the same positionis also able to fill the pocket and stabilize the conformation, butall other groups tested were, in retrospect, too small  or too large (25) to fill the binding pocket or likely forced the ligand into a nonbindingconformation (26 and 27). The SAR aroundthe R2 substituent in 14) is slightlyfavored over a phenyl substituent (11), and the crystalstructure suggests that this is due to the interaction with Thr377.The changes shown in 39 is inactive, and this is likelydue to the N-methyl group clashing with the N165side chain.The optimization of 40 found in chainsA, C, and Dof the complex fits well with the findings from mutational studiesand HDX-MS experiments by Clarke et al. that suggestedthe putative PI5P-binding site of PI5P4Kγ, not the ATP-bindingsite, to be the likely region of interaction30 of 1. The HDX-MS method measures the rate of exchangeof solvent-accessible protons from protein amides with deuterium.Changes in exchange accessibility generated by the binding of an enzymewith an inhibitor are detected by peptide MS. The regions with reducedHDX accessibility may indicate ligand-binding sites or structuralchanges in the protein. The main sites showing an HDX-MS effect inClarke et al.’s study are shown in 40 complex, where site 1 comprises residues 158–162 and site2 comprises residues 373–407. Both sites are in close proximityto 40 in the allosteric binding mode. Residues 378–380in particular change their environment on binding with 40, as they move from a flexible loop conformation without electrondensity in the apo structure and chain B to a buried location in thealternative protein conformation of chains A, C, and D  that is highly similar to the one shown in 40 was observed to bindmainly to the allosteric pocket in this structure, with some densityfor 40 in the ATP pocket (occupancy of 0.35). However,we did not observe simultaneous binding of 40 to theallosteric pocket and AMP-PNP to the ATP pocket. Overall, the combineddata set is most consistent with the A/C/D chain-binding mode, withsupport from the HDX data and with the SAR developed around this series.Previousstudies have shown that basal ATP turnover in the absenceof PI5P is not inhibited by 40 are able to engage the PI5P4Kγ target in cells andthat, in X-ray costructures with PI5P4Kγ, 40 canadopt two distinct modes of inhibition, with binding to a distal lipid-bindingsite that is 18 Å from the ATP pocket preferred. The nature ofthis novel mode of inhibition remains to be further elucidated andmay involve PI5P competition or conformational changes in the proteinthat manifest through changes in the interactions with distinct proteincomplexes. Further studies to understand the cell biology of theseinhibitors and to identify novel chemotypes with further improvedpotencies and physicochemical properties are underway and will bethe topic of future publications.In conclusion, we have optimized a series of selective small moleculePI5P4Kγ inhibitors to improve both potency and physicochemicalproperties and enable structural biology studies. We have presentedbinding constants, thermal shift data, and biochemical data, whichshow that, in general, this chemotype does not inhibit PI5P4Kαor β. In addition, we have shown that exemplars such as 9 was purchased fromAmbinter (Amb16536894) and was determined by ultra-performance liquidchromatography (UPLC) to have a purity of >95%. All the other compoundswere synthesized as described below, and all the tested compoundshad a purity of >95% by UPLC analysis. Compounds were synthesizedaccording to Schemes S1–S3 as described in the Supporting Information section.Compound Escherichia coli BL21(DE3)Gold using a pET28b vector. Expression was induced using 0.1 mM IPTGand the cells were cultured at 18 °C for 16 h before harvestingby centrifugation. The protein is composed of residues His32 to Ala421with the region between and inclusive of residues 300–341 deleted.The purification of TEV-cleaved protein was by both affinity and size-exclusion(Superdex 75) chromatography. The structure of the ligand complexwas generated by co-crystallization of human PI5P4Kγ in thepresence of 40. The purified protein  overnight at 4°C. Crystals were grown from 22% w/v Peg3350, 0.3 M ammoniumtartrate, and 100 mM PCPT  pH 7.5 at 20 °C. Where AMP-PNP wasused, the protein was first incubated with 4 mM AMP-PNP (in buffer)for 2 h and then overnight with 10 mM 40 before settingcrystal trays. For X-ray data collection, they were flash-frozen andX-ray diffraction data was collected at 100 K. Diamond Light SourceSynchrotron Facility, Oxford, UK, Beamlines .Data were processed using XDS and Aimless software. The phase informationnecessary to determine and analyze the structure was obtained by molecularreplacement  using the previously solved structure ofhuman PI5P4Kγ (PDB code: 2GK9) as the search model. Subsequent modelbuilding and refinement were performed according to standard protocolswith software packages CCP4 and COOT. TLS refinement has been carried out, which resulted in lower R-factorsand higher quality of the electron density map. The ligand parameterizationand the generation of the corresponding library files were carriedout with ACEDRG (CCP4). The Ramachandran plots of the final modelsshow 91.3 and 92.3% of all the residues in the most favored regionsand 7.0 and 5.0% in the additionally allowed regions, for 7QIE and 7QPN, respectively. Statisticsof the final structure and the refinement process are listed in Table S5.Crystallographywas performed by Peak Proteins Ltd. Truncated human PI5P4Kγwas expressed in https://www.schrodinger.com/) was used to predict the 5 lowest energy conformers of 40. The predictor generates conformers using macromodel, and then performsdensity functional theory geometry optimizations on the structures,using the B3LYP-D3/LACVP** level of theory. The structures were thenranked using optimization energies at the M06-2X/cc-pVTZ(-f) theorylevel in solution (water/PBF) calculated using the geometries fromthe previous step. The root mean square deviation (RMSD) of the lowestenergy conformer with the ligand from chain A of the minimized crystalstructure  was thencalculated using the maximum common structure superposition algorithmin Maestro.The Maestro QM tautomer andconformer predictor , using thecrystal structure placement, with induced fit refinement guided bythe GBVI/WSA dG scoring function. E_refine for the two binding modesof 40 was compared.The free energy of binding was calculated usingthe Dock panel in MOE .Marvin was used for 25 The protein from PIP4K2C (UniGene 6280511),genetically modified to have a specific activity close to that ofthe active PI5P4Kα isoform25 andcloned into the expression vector pGEX6P (Cytiva), was expressed andpurified from E. coli BL21(DE3). Cultureswere induced with 0.4 mM IPTG and probe sonicated in the presenceof protease inhibitors. The GST fusion protein of PI5P4Kγ+ washarvested by binding to glutathione sepharose beads (Cytiva) and cleaved in situ with 50U of PreScission protease (Cytiva) for 4h at 4 °C. The cleaved protein was further purified by size-exclusionchromatography . The protein purity was confirmedby sodium dodecyl sulphate–polyacrylamide gel electrophoresis,and the concentration was determined by colorimetric assay (Bio-Rad).Recombinant mutant PI5P4Kγ+was prepared essentially as described previously.2, and 16.7 μM (ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid) (EGTA)).The plate was incubated for 60 min at room temperature after the additionof purified PI5P4Kγ+ (150 ng/well) and prior to the transferof 4 μL of the reaction mix into a second plate (Greiner 784904)containing a 4 μL of ADP-Glo reagent for a further 40 min ofincubation. After incubation with 8 μL of the kinase detectionreagent for 30 min, plate luminescence was read .PI5P4Kγ+ activity in the presence of inhibitor compoundswas determined by the ADP-Glo assay (Promega). The assay was performedin a 384-well plate (Greiner 784201) with serial dilutions of thetest compound in 18 μL of the reaction mix dimethylammonio]-1-propanesulfonate(CHAPS), 20 mM MgClThe binding of compounds to PI5P4Kγ in intactcells was assessedusing an InCell Pulse thermal stabilisation assay (DiscoverX). Hek293cells stably expressing ePL-tagged PI5P4Kγ were incubated with25 nL of the test compound in 100% DMSO in a black skirted polymerasechain reaction (PCR) plate for 60 min at 38 °C. After incubationfor 3 min at 46 °C, followed by cooling for 3 min at room temperature,12 μL of the EA-3 reagent (prepared as per the manufacture’sguidelines) was added to each well. The plate was then incubated for60 min in the dark prior to luminescence reading using a PherastarFSX plate reader (BMG Labtech).50 values and in vivo binding pEC50 valueswere estimated using a 4-parameter fit (Dotmatics).Statistical analysis was performed usingnonparametric testing in Prism 8 (GraphPad). Activity pIC2, and 100 mM NaCl. The plateswere heated from 25 to 90 °C at a rate of 0.5 °C/min. Thethermal melting point was calculated for each well by monitoring theminimum point of the negative derivative of the fluorescence unfoldingcurves. Thermal shifts were calculated by comparison to control wellswith no compound (5% DMSO). At least two chemical replicates of eachligand were performed, with control compounds used to check for consistencybetween plates.The thermal shift assay was performedwith an Applied Biosystem StepOne Real-Time PCR System in 96-wellplates sealed with optically clear lids. The fluorescent dye SyproOrange was used to report on protein unfolding. The final concentrationof the PI5P4K protein was 4 μM, the ligand was at 63 μM,in a final volume of 20 μL. A 5000× stock solution of SyproOrange was used, at a final concentration of 5×. The buffer was50 mM HEPES pH 7.4, 5 mM MgCl


Sandwich panel structures (SPSs) with lattice cores can considerably lower material consumption while simultaneously maintaining adequate mechanical properties. Compared with extruded lattice types, triply periodic minimal surface (TPMS) lattices have light weight but better controllable mechanical properties. In this study, the different types of TPMS lattices inside an SPS were analysed comprehensively. Each SPS comprised two face sheets and a core filled with  Triply periodic minimal surfaces (TPMSs) were originally discovered and described by Schwarz in 1865 ,3,4. A wSPSs are commonly observed in nature, such as in human skulls, butterflies, and dragonflies . SPSs prAn SPS contains a lattice core. Several struct-based cellular geometries are possible, such as the cubic primitive, body-centred cubic, face-centred cubic, and octet truss ,9,10. AnThe most significant structural characteristic of a lattice pattern is the relative density (ρc*/ρs) , where ρn et al. , increasn et al.  tested tn et al.  and Lee n et al.  indicaten et al. ,25.Simsek et al.  numericaTPMSs under compressive loading and sandwich beams with a 3D truss cell/foam core under three-point bending have been studied extensively ,30,31,32A minimal surface is defined as the surface of the minimal area between any given boundaries. When a minimal surface repeats itself by rotating, mirroring or replicating in three dimensions, it is defined as a TPMS . TPMSs hSP:S2:I-WP:F-RD:Various studies have performed intensive experiments on sandwich beams with extruded lattices or foam inside the core under three-point bending loads ,37,38. TDeformation occurs when the SPS is loaded and reaches equilibrium. The total deformation includes the bending component δ, can be calculated as follows:The total deformation, fE is the Young’s modulus of the solid material making up the face sheets, and The total weight of the SPS Previous studies ,13,39 haIn this study, Grasshopper and computer codes developed in house were used to generate single cells of TPMSs. The single cells were generated in an 8 mm × 8 mm× 8 mm cubic box and patternised to generate Many studies have demonstrated that discretising the core surface as a shell rather than a solid mesh produces superior results. This is because the thickness aspect ratio is large, and the shell model can provide more accurate results ,27,28. TAcrylonitrile butadiene styrene (ABS) is a common thermoplastic polymer with nearly constant elastic modulus and other mechanical properties under the operation temperature. It has loads of advantages, such as great heat resistance, high tensile strength, and low cost. Hence, ABS was selected as the material of the SPS specimens, and In this study, the mechanical performances of SPSs with four different TPMS lattice cores were evaluated and compared. In addition, the calculation results based on the equations presented in the ‘Theoretical background’ section were compared with the numerical results obtained using the FEA approach.To compare the mechanical performance of SPS with different TPMS lattices, the total weight of each SPS must be considered first. The weight calculated from Equation (11) and the weight acquired from the FEA models are listed in The total deformations of the SPSs with different TPMS lattices cores under a three-point bending load calculated using Equation (5) were compared against the numerical predictions obtained by FEA. 90 in the followings. 90 at relative densities of 10% and 20% were extremely large compared to the calculated results. This clearly indicates that the cell direction causes this extremely huge difference. Other TPMS lattices considered in this study  do not demonstrate this difference because their cell rotation does not affect the array direction –(15) can reliably predict the deformations of SPSs with different TPMS lattices.Because the numerical results were based on a three-point bending load, the deformation in the loading direction was also considered. 90 lattice did not increase when the relative density was increased from 20% to 30% but instead declined. In addition, the maximum allowable load was generally lower for S290 than for S2. These results demonstrate that the geometry and relative density of the lattice core are significant to the structural performance. The maximum allowable load increased with the relative density. The array direction of the lattice had a significant effect on the performance of S2, which was the only TPMS in this research with different cell direction because of its two openings in the geometry.The SPSs were made from ABS, and its maximum yield strength is about 45 MPa. Thus, the maximum allowable load at different relative densities can be calculated by numerical analysis. An SPS beam may fail according to several failure modes. All failure modes must be considered to determine which is dominant. With regard to face yielding, plastic deformation occurred once the stress on the face sheet was greater than the yield strength of the face material. The face normal stress The shear stress has the largest effect on the core tearing during the three-point bending test ,29,30. TIn conclusion, the normal failure modes of the SPSs were face yielding and bond failure because of the thinness of the face sheets. Between these two failure modes, face yielding was the most severe because the stress greatly surpassed the yield strength of the material. Other failure modes, such as face wrinkling and core failure, can be prevented by increasing the relative density. These results agree with those of other studies ,37 showiFace stress:Bond stress:Strain energy release rate:Moment:In this study, the mechanical performances of SPSs with four types of TPMS lattice cores at different relative densities were evaluated thoroughly. FEA results for the deformation under the three-point bending test showed good agreement with the calculated values, with a difference of less than 21%. The lattice geometry and relative density were the primary factors affecting the mechanical performance. For a fixed relative density, the SPS with the SP lattice core had the smallest deformation, and the F-RD lattice core had the maximum allowable load before yielding. The cell direction had a significant effect on the mechanical performance of the SPS with the S2 lattice core. The most dominant failure mode was face yielding because the stress greatly surpassed the yield strength of the material used in this study. Other failure modes such as face wrinkling and core failure can be prevented by increasing the relative density. Increasing the total amount of the lattice core or using different materials for the face sheets can improve the mechanical performance of SPSs, and the SPS can be optimised to meet the requirements for various engineering applications.


With food insecurity rising dramatically in Sub-Saharan Africa, promoting the use of sorghum, cowpea and cassava flours in staple food such as bread may reduce wheat imports and stimulate the local economy through new value chains. However, studies addressing the technological functionality of blends of these crops and the sensory properties of the obtained breads are scarce. In this study, cowpea varieties , dry-heating of cowpea flour and cowpea to sorghum ratio were studied for their effects on the physical and sensory properties of breads made from flour blends. Increasing cowpea Glenda flour addition from 9 to 27% (in place of sorghum) significantly improved bread specific volume and crumb texture in terms of instrumental hardness and cohesiveness. These improvements were explained by higher water binding, starch gelatinization temperatures and starch granule integrity during pasting of cowpea compared to sorghum and cassava. Differences in physicochemical properties among cowpea flours did not significantly affect bread properties and texture sensory attributes. However, cowpea variety and dry-heating significantly affected flavour attributes . Consumer tests indicated that composite breads could be significantly distinguished for most of the sensory attributes compared to commercial wholemeal wheat bread. Nevertheless, the majority of consumers scored the composite breads from neutral to positive with regard to liking. Using these composite doughs, chapati were produced in Uganda by street vendors and tin breads by local bakeries, demonstrating the practical relevance of the study and the potential impact for the local situation. Overall, this study shows that sorghum, cowpea and cassava flour blends can be used for commercial bread-type applications instead of wheat in Sub-Saharan Africa. Currently, the food system in Sub-Saharan Africa (SSA) is severely challenged because of the growing urban population and climate change ,2. MassiIndigenous African crops such as sorghum, cassava and cowpea are climate-resilient crops (CRCs) as they have enhanced tolerance to biotic and abiotic stresses ,7. CowpeFrom a nutritional perspective, the relative overconsumption of starchy crops in SSA is a concern for protein as well as micronutrient security . CassavaSorghum flour has been studied for partial wheat replacement in pan breads , showingDespite the relevance for human nutrition and food security, studies on composite breads based on sorghum, cowpea and cassava are scarce. However, their combination offers opportunities to optimize the nutritional composition and technological properties of bread-type products , which wAgainst this background, this study evaluated the effect of variations in cowpea flour properties, namely variety and dry-heating treatment, and the level of addition  on the physical and sensory properties of breads made from sorghum, cowpea and cassava mixtures. Two cowpea varieties were used for this purpose. Relevant physicochemical properties of the flours were analysed in order to relate to the bread properties. Descriptive sensory analysis was performed to understand the effect of variations in flours and bread properties on the intensity of the sensory attributes. Additionally, consumer studies were performed to gain insights into the perception and liking of a selected CRCs-based bread formulation as compared to commercial wheat bread. Implementation potential of the developed doughs was tested in Uganda with small bakeries making tin breads and with street vendors making chapati.Red, non-tannin, King Korn superfine sorghum  meal was obtained from a local supermarket in Pretoria (South Africa). Cowpea seeds from a white (Bechuana white) and a red (Glenda) variety were sourced from Agrinawar Agricol Pty  and milled as whole grains at ProCorn Mills . Sorghum contained 10.2% protein, 11.5% dietary fibres (of which 2.3% was soluble) and 73.1% starch. Bechuana white cowpea flour (BECH) contained 23.9% protein (of which 18.6% was soluble at native pH), 20.6% dietary fibres (of which 7.5% was soluble) and 40.7% starch. Red Glenda cowpea flour (GL) contained 23.9% protein (of which 18.4% was soluble at native pH), 24.1% dietary fibres (of which 7.7% was soluble) and 39.7% starch. Dry-heated GL (GL-DH) was also tested based on the result of a recent study on the Glenda variety . One treCassava starch  was supplied by DADTCO . Psyllium husk powder  was from Unilecithin . Dry yeast , salt and sucrose (both sourced in The Netherlands for lab tests and in Uganda for implementation tests) were used for the breadmaking trials.The water binding capacity (WBC) of the native and dry-heated GL (GL-DH) samples was determined in triplicate, according to a modified version of . Flours The moisture content of the pellet was measured by drying overnight in aluminium dishes in an oven at 105 °C. The filled dishes were cooled for 1 h in a desiccator before weight determination.onset), peak temperature (Tmin) and gelatinization/denaturation enthalpy were determined using the analysis tool available in the Universal Analysis software . Experiments were performed in triplicate.Flour concentrations of 20% (on dry matter basis) in distilled water were used to measure starch gelatinization and protein denaturation with a TA Instruments type Q200 Differential Scanning Calorimeter (DSC) as earlier described . SamplesPasting behaviour was investigated using a Rapid Visco Analyser (RVA) Super 4  as earlier described . Flour oVariations in CRCs-based bread formulations were built upon a product concept recently developed containing sorghum, cassava and cowpea , as show2 production of 90 mL. The CO2 production was determined using a Risograph . After proofing, the doughs were put in a swing oven at 180 °C for 70 min. During the first minute, steam was injected twice to regulate the moisture content. After baking, the breads were cooled at room temperature for 40 min, sealed in plastic low-density polyethylene bags and stored at room temperature until further analysis one day after baking. Baking tests were performed in triplicate. The described procedure was used for bread intended for instrumental characterization and descriptive sensory analysis.In total, about 1300 g of dry ingredients were added to a Sinmag spiral mixer  and pre-mixed at low speed (speed I) for 1 min. Then, water was slowly added during mixing, which was performed for 5 min at speed I and for another 4 min at speed II. After mixing, the dough was divided and shaped manually, and put into three greased baking tins, each containing 700 g of dough. These tins were put in a fermentation chamber at 30 °C and 85% RH. The proofing time was defined as the time needed by 50 g of dough to reach a COFor the naïve consumers’ study, sorghum and BECH flours were first hydrated in excess water for 1.5 h in order to minimize sandiness in breadcrumbs, based on preliminary trials. After hydration, the flour and the water were combined with the rest of the ingredients in the Sinmag spiral mixer and the same breadmaking protocol was applied.Loaf volume was determined on 2 loaves from each baking test, with a rapeseed displacement according to . In totaCrumb texture was measured by means of Texture Profile Analysis (TPA), using a TA-XT2i Texture Analyser from Stable Micro Systems  with a 30 kg load cell and a 75 mm compression plate and performed as previously described . In totaThe moisture content of the bread crumb (5 g sample) was measured according to  by dryinSensory profiling of the pan breads was conducted as generic descriptive analysis with 8 trained panellists who were regularly trained. Written informed consents were obtained from the participants prior to the evaluation. The base attribute list was developed by five trained assessors in a consensus session using previous studies as a basis ,36,37. TFor serving, a slice of each sample was packed in closed plastic bag and presented with a 3-digit code. Water and a piece of wheat-based cream cracker were used as palate cleansers. The serving order was randomized with a Latin squares design. The samples were evaluated in duplicate in a complete block design. The attribute intensities were evaluated with a 0–10 continuous line (0 = non-perceivable and 10 = very intense). The data were collected with EyeQuestion version 5.3 .n = 51, 37 female, age: 19–28 years) were recruited from Wageningen University & Research campus using flyers, posters and social media. Subjects were regular consumers of bread. Other inclusion criteria (self-reported) were no allergies or intolerances to gluten, good dental health and non-smoking habits. Participants were mainly of Dutch nationality (n = 37), followed by Italian (n = 5), and the remaining 9 were each from a different nationality. A consent form was signed by all participants. Subjects received reimbursement for their participation and were naive concerning the experimental conditions and purposes.Fifty-one participants  of the two different formulations were presented to the panellists with randomized three-digit codes. The panellists received one slice of bread per formulation. Panellists were instructed to first taste the bread crumb of the two different bread samples before scoring them. Subjects were instructed to cleanse their mouth with water and have a break of at least 2 min between evaluating the samples.Participants were asked to evaluate commercial wholemeal bread  and CRCs-based bread prepared following the recipe of GL9 , but usip < 0.05) for flour properties and physical properties of breads were performed with SPSS . A two-way mixed model ANOVA with samples as fixed factor and assessors as random factor was used for sensory profiling. Principal component analysis (PCA) of sensory profiling data was performed with Rstudio  using the PCA function of the FactoMineR package [Statistical evaluations  were treated as continuous data ,39. A pa2 = 0.968, p < 0.05).Relevant physicochemical properties of the flours were studied to elucidate their contribution to the baking behaviour of the composite CRCs doughs . GL showonset), while sorghum and BECH had the highest (p < 0.05). The Tpeak of starch was the highest for BECH, followed by GL and GL-DH, while cassava was the lowest (p < 0.05). The enthalpies for starch and protein in cowpea flours could not be distinguished due to partial overlapping between the two peaks. Nevertheless, the gelatinization enthalpies of cassava and sorghum flour were significantly higher than the enthalpy for all the cowpea flours.The DSC analysis of the flours revealed the main differences as being in their thermal behaviour . Cassavap < 0.05). Samples GL and GL-DH showed pasting temperatures that were significantly higher than cassava but lower than BECH (p < 0.05). Dry heating significantly increased the pasting temperature compared to the untreated GL. Paste viscosities  were the highest for cassava starch, followed by sorghum flour, GL, BECH and GL-DH (p < 0.05). The paste viscosities of BECH were significantly lower than those of GL. Furthermore, dry heating of GL resulted in significant reductions in PV, HV and FV. BD was the largest for cassava starch and the lowest for sorghum, BECH and GL-DH. Sorghum flour showed the highest SB, followed by cassava starch, BECH and GL (p < 0.05). Dry heating significantly reduced SB compared to the untreated GL, which was in agreement with recent work on GL with similar treatments [With regards to pasting behaviour, cassava starch showed the lowest pasting temperature while sorghum flour had the highest, followed by BECH , as observed in cake batters . From thDuring the baking stage with CRCs dough, fixation of the expanding foam structure is mainly related to the starch gelatinization process, due to the absence of the gluten thermo-setting mechanism. Starch gelatinization increases the viscosity and shear modulus of the batter dramatically and the swollen starch granules further support against normal forces in order to withstand collapse . In bake1 and PC2) accounted for over 78% of the variance, thus providing a good representation of the differences among samples. Bread samples were differentiated in terms of texture  and yeasty flavour/fermented odour along the PC1, mainly dominated by samples GL27 and GL9-DH. PC2 was dominated by samples GL9 and BECH27, which were separated based on beany flavour, beany odour and darkness of the crumb (p < 0.05). Dry heating of cowpea provided bread GL9-DH with the most intense yeasty flavour. In a recent study, flatbreads made with the Bechuana variety of cowpea also showed a more intense beany odour and beany flavour compared to the Glenda variety [Significant differences in appearance, odour and flavour attributes and in texture were observed among breads . PCA anahe crumb A. The us variety . Differe variety ,48. Off- variety . By bind variety ,51. Flav variety  and bean variety ,54. In t variety . The lim variety  and shouBECH27 had the lightest crumb and GL27 had the darkest. In a recent study on flatbreads from composite flours, breads prepared with the Glenda variety were also perceived as darker than those with the Bechuana variety. Differences were attributed to the higher concentration of anthocyanins in the seed coats of the red variety . A dark p < 0.05). Perception of crumbliness and moistness were significantly correlated , with lower crumbliness scores observed with increasing moistness perception . For wet soft-solids, such as protein/polysaccharide gels, crumbliness perception increases with increasing recoverable energy [Crumbliness is an important quality aspect of bread. Crumbliness is defined as the degree to which a sample fractures into pieces during mastication . In our rception B. Crumblrception B. Howevee energy , with ree energy . Crumblye energy . Whethere energy . Drynesse energy . On the e energy  and casse energy . OverallSandiness was perceived in all samples and changes in the composition of the flour blends and dry heating did not have significant effects. In preliminary trials, it was observed that both sorghum and cowpea flours contributed to sandiness. Pre-trials with sieving flours to below 200 µm or re-milling did not result in noticeable improvements (data not shown). For legumes, the hard-to-cook phenomenon is widely reported and attributed to structural and compositional changes induced by storage under hot and humid conditions, as encountered in many subtropical and tropical African countries . Lignifip < 0.05).The overall liking and sensory perception of CRCs-based bread and commercial wholemeal wheat bread were compared in the consumers’ test. The wholemeal wheat bread was chosen since the sorghum and cowpea flours were not refined. The CRCs-bread formulation for this study was formulation BENCH9 ; BECH waWith regards to the RATA tests, many significant differences were found for appearance, texture and taste attributes B–D. CRCsThe differences in flavour were related to salty, beany, tangy, sweet and sour attributes. However, the largest differences in intensity scores were limited to beany flavour and sour taste. Saltiness was more pronounced in the CRCs bread, likely due to a higher salt content than in commercial wheat bread. Even though differences in beany flavour were clearly perceived by consumers, this attribute received a low intensity score, suggesting that the chosen level of cowpea flour addition may also be suitable for white varieties such as Bechuana. The slight sourness and tanginess perceived in the CRCs bread may be considered a positive attribute, when not excessive, as they are naturally associated with sourdough breads .One of the CRCs-based doughs in this study was evaluated for its implementation potential in SSA, as a means to reduce Africa’s dependency on imported wheat and valorise traditional and underutilised CRCs. Chapati-type flatbreads, the most popular convenience foods produced by street vendors , and tinKikomando , by preparing popular dishes such as komando’ D–F. The  (Rolex’ . VendorsBaking experiments for CRCs-based tin breads were also performed in Uganda in a BBROOD formal bakery in Tororo. The bakery produces and sells Western-type breads, including tin breads, which are becoming popular among the middle class in the evolving and rapidly growing urban market . The douTo date, wheat replacement initiatives have not been very successful, as consumers in many SSA countries perceive composite breads as a lower quality product and therefore prefer bread made from 100% wheat flour ,74. On tCowpea flour proved to be functional in modulating the baking properties of the CRCs breads. High cowpea to sorghum ratio improved specific volume and instrumental texture by enhancing softness and cohesiveness. Substantial differences in WBC and pasting properties of cowpea flour compared to sorghum flour and cassava starch could be related to these improvements. Among varieties, the red Glenda cowpea was the most functional in reducing perceived crumbliness and increasing perceived moistness, probably due to its high WBC. Additionally, the Glenda variety scored better with regards to taste, as beany flavour was most pronounced in breads with high inclusion levels of the Bechuana variety. Dry-heating of cowpea flour substantially affected its functionality, but had limited effects on bread texture. The most significant effects were observed for the perceived yeasty and fermented flavour of the bread. Sensory tests with naive consumers indicated that the CRCs bread was the most distinct from commercial wheat bread for its crumbly texture and for the beany, sour and yeasty taste. Despite that, the CRCs bread was neither liked nor disliked, while the commercial bread was only moderately liked. These blends of CRCs could be successfully used in Uganda to produce chapati and tin breads with street vendors and local bakeries, respectively. Overall, the result of this study showed that blends of African local crops can be used to fully replace wheat in bread-type products. The selection of the cowpea to sorghum ratio and cowpea variety are important for modulating their texture and taste. These results are relevant to alleviate African dependency on imported wheat, thus potentially contributing to food and nutrition security in SSA in the current conditions of climate change and geopolitical crisis.


A composite thin shell with a high fiber volume fraction prepared by resin transfer molding (RTM) may have void defects, which create deformations in the final curing and lead to the final product being unable to meet the actual assembly requirements. Taking a helmet shell as an example, a multi-directional compression RTM (M-CRTM) method with an adjustable injection gap is proposed according to the shape of the thin shell. This method can increase the injection gap to reduce the fiber volume fraction during the injection process, making it easier for the resin to penetrate the reinforcement and for air bubbles to exit the mold. X-ray CT detection shows that the porosity of the helmet shell prepared by the newly developed technology is 36.6% lower than that of the RTM-molded sample. The void’s distribution is more uniform, and its size is decreased, as is the number of voids, especially large voids. The results show that the maximum curing deformation of the M-CRTM-molded helmet shell is reduced by 13.7% compared to the RTM molded sample. This paper then further studies the deformation types of the shell and analyzes the causes of such results, which plays an important role in promoting the application of composite thin shells. Carbon-fiber-reinforced polymer (CFRP) has an excellent specific strength and specific modulus, and the composite thin shell is applied in the aerospace field for applications such as solid rocket motors and pilot helmets. These fields require a high precision and a small dimensional tolerance for composite parts due to the limited assembly tolerance. If curing deformations occur after shell demolding, this may exceed the assembly tolerance and affect the assembly of the composite shell. The forced assembly causes internal stress in the parts, resulting in the scrapping of parts and a huge amount of manpower and material resources being wasted. Therefore, in the manufacturing process, it is necessary to control the molding process to reduce curing deformations in the composite shell.In the curing process of thermosetting composites, the curing process parameters will affect the residual stress ,2 and thIn the meantime, resin transfer molding (RTM) can not only produce parts quickly and reduce labor costs, but it can also obtain a class A surface on both surfaces, which makes it a prime candidate for use in the high-volume manufacturing industry ,21. ThisTo date, the force applied to the mold during the CRTM process has been concentrated in a single direction on a flat surface, which is generally used to produce composite parts with simple shapes. Although plate shells have a certain effectiveness in practical applications, most applications are non-planar geometric components. Therefore, single-direction compression RTM cannot meet the practical production needs of some fields. Therefore, according to the shape and structure of the composite shell, this paper puts forward a multi-directional compression RTM (M-CRTM) method with an adjustable resin injection gap, which means that the resin injection gap can be adjusted and the fiber volume fraction can be reduced during the resin-filling process. In this paper, the RTM and M-CRTM methods were used to study the effect of porosity on curing deformations, and the deformation types of the composite shells produced by the two methods were compared. By analyzing the porosity, void size, and number of voids, the issues behind the differences in curing deformations are also discussed.The helmet shell reinforcement was made of 300 gsm carbon fiber satin fabric, which was purchased from Tianniao High Technology Co., Ltd. . The thickness of the helmet shell was 1.2 mm, and it was arranged in a symmetrical stacking order with the layering order of [90°/0°/0°/90°]. The resin used was TDE86# epoxy resin with a viscosity of 200 mpa·s, provided by Tianjin Jingdong Chemical Composites Co., Ltd. . The curing agent was HK-021, purchased from Wenzhou Qingming Chemical Co., Ltd. . The RTM injection machine used was a two-component precision epoxy RTM injection machine from Beijing Hengjixing Technology Co., Ltd. . The resin injection process adopted the equal flow resin injection method, and the resin volume flow rate was 200 mL/min. Finally, the sample was heated at a rate of 2 °C/min and kept at 90 °C for 120 min, followed by heating at a rate of 2 °C/min and kept at 140 °C for 120 min, and finally cooled down with cooling rate of 2 °C/min.The thickness of the composite helmet prepared in this paper was only 1.2 mm. Therefore, in order to achieve the stiffness and strength required and to reduce the curing deformation for the helmet’s shell, the fiber volume fraction of the thin shell was as high as 58.8%. In this paper, the RTM resin injection process was used to produce the helmet because the surfaces of products produced by this method are smooth. The results showed that the overall deformation was large after demolding, which increases the difficulty of helmet assembly. The reason for this is that the fiber volume fraction of the product was relatively high, and due to the closed mold forming, dry spots and tiny bubbles were formed in the thin shell. To minimize these defects, a M-CRTM injection method with an adjustable injection gap was proposed in this paper. By increasing the injection gap during resin infusion, the permeability of the fabric can be improved, thus improving the overall forming quality. The schematic diagram of the forming method is shown in The structure of the helmet shell is shown in The high-performance Linac CT -Diondo was used to detect the internal void distribution of the produced helmet shells. Scanning was performed using a high-resolution, small-focus CT detection system. The X-ray voltage was 150 KV, and the scanning mode was a standard cone-beam CT scan. CT detection can intuitively observe the product’s internal defects, such as defect type, location, size, etc. Current limitations of micro-CT technology mean that only small samples can be scanned. Therefore, it was necessary to cut the sample to a scannable size to estimate the void content. Three areas (areas a–c in ® software (3D Systems®) in bin format for filtering and noise reduction, followed by optimization and model reconstruction. Then, the reconstructed model was compared with the helmet design model to obtain the curing deformation of the helmet shell. The specific measurement steps are shown in As previously mentioned, the helmet shell is a special-shaped, curved structure unlike the flat plate or L-shaped models, which can be used to directly compare the warpage or spring-in. Therefore, a method is needed to quantify the curing deformation of complex-shaped structures. Reverse engineering can be used to obtain a surface point cloud of the model through 3D scanning technology, and then reverse engineering software can be used to process this point cloud to obtain the surface shape of the tested sample. Therefore, reverse engineering is often used to detect the deformation between the manufactured sample and the original model. For example, Xiang  used a 3According to the measurement method shown in To further characterize the curing deformation, the maximum positive and negative deformations of the shell were statistically analyzed, and the results are shown in According to the above analysis, although the preparation process has a partial impact on curing deformation, the overall comparison shows that the use of the newly proposed M-CRTM technology can reduce the generation of curing deformation, indicating that the influence of the manufacturing technique is the main factor influence curing deformation under the studied conditions. Therefore, in this study, other influencing factors can be regarded as recurrent overall. In order to explain the influence of forming quality caused by the manufactured technique on the curing deformation of the helmet shell, the first group of helmet shells was selected as the representative to study and analyze the curing deformation types.3 and a maximum void volume of 6.6mm3. The volume of most voids was below 2 mm3, and the number of voids above 2 mm3 was 22, accounting for 17.75% of the total void volume. 3, and a maximum void volume of 4.574 mm3. The volume of most of the voids in the M-CRTM molded helmet was below 1 mm3, and the number of voids with a volume size greater than 2mm3 was 11, accounting for 11.54% of the total void volume. Compared with the RTM-molded helmet, the porosity of the M-CRTM-molded helmet was reduced by 36.6%, and the number of voids was significantly reduced. In addition, in the area  system was used to capture the strain field of the sample in the tensile test. The results are shown in The helmet platform  method with an adjustable resin injection gap was proposed. By comparing the curing deformation of helmet shells manufactured by the RTM and M-CRTM methods, it is found that although the manufacturing techniques are the same, the curing deformations were slightly different. The reason for this result is that the helmet structure has complex characteristics and the processing technique used also has a partial influence on the curing deformation. The study found that the use of the newly proposed M-CRTM technology can reduce the overall curing deformation. Therefore, this paper selects a group of helmet shells as representatives to study the influence of forming quality on the type of curing deformation. The X-ray CT scan test found that there were large voids in the composite helmet prepared by RTM, even reaching or exceeding half of the shell’s thickness in some areas. In comparison, the M-CRTM-molded helmet shell not only reduced the size and number of large voids, but it also significantly reduced the internal porosity of the shell. By studying the curing deformation in the X, Y, and Z directions of three representative sections and the overall curing deformation, it was found that the warpage and torsional deformation occurred in the RTM-modeled helmet. Furthermore, the maximum positive deformation and maximum negative deformation of the M-CRTM helmet shell were reduced by 13.7% and 33.6%, respectively. The reason for this result is that when the forming quality of the shell is improved, the internal stress and stiffness of the shell are uniformly distributed, and the curing deformation is thus significantly reduced. Although the change in the degree of curing deformation takes place at a millimeter level, such a difference plays an important role in aerospace parts that require high assembly accuracy. Our proposed M-CRTM method thus has a positive effect on further improving the application of composite thin shells in industry.In this paper, the influence of the forming quality of helmet shells on curing deformation was studied. However, this study has two shortcomings. On the one hand, void distribution models of the helmet shells were not established in this manuscript. On the other hand, due to the influence of the material model and void model, the curing deformation of the composite helmet shell was not simulated and compared with the experiment.


Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the main infectious diseases affecting swine herds in the U.S. Control and elimination are facing challenges, including the absence of a vaccine that confers sterilizing immunity. Elimination through herd closure can also be a long, uncertain, and costly process, with herds often taking around 41 weeks to start consistently weaning virus-free piglets after an outbreak is detected. Thus, producers and practitioners may hesitate in pursuing elimination if there is a perception that the herd will soon face another PRRS outbreak, opting instead to maintain some level of immunity in the herd indefinitely. Of all the breeding herds monitored over 12 years, only about 1/6 eliminated PRRS from their herds and remained, on average, PRRS-free for two years. After eliminating PRRS, the average percentage of new outbreaks per year was 23%, similar to the national average of 20–25% (regardless of the previous PRRS status). Additional factors might contribute to the decision to pursue elimination, and further studies are warranted.Background: Hesitation on eliminating Porcine Reproductive and Respiratory Syndrome virus (PRRSV) from breeding herds exists since it is difficult to predict how long the herd will remain virus-free. We aimed to estimate the time that breeding herds remained virus-free (naïve) after PRRSV elimination was achieved. Methods: Production systems voluntarily shared their breeding herds’ health status weekly between July 2009 and October 2021. PRRSV incidence rate and the total number of days a breeding herd remained virus-free were estimated. Results: A total of 221 (17%) herds reached the naïve status 273 times. The median time sites remained in this status was approximately two years. The overall PRRS incidence rate after sites achieved a naïve status was 23.43 PRRS outbreaks per 100 farm years. Conclusion: Estimates obtained here provide insights on how frequently and for how long sites remain naïve, which contribute to informing management practices for PRRS control. Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine infectious diseases due to its high economic burden ,2. It waSeveral methods for herd level control  and elimination  have been proposed ,6. ProduIn efforts toward system-wide, regional, or national PRRS elimination, a common hesitation for producers to implement an elimination strategy is the uncertainty of how long the herd will remain free of the virus. Here, we aimed to assess for how long farms that chose to eliminate PRRS and rollover their herds remained at a naïve status to ultimately help in informing decisions toward PRRS management.Data for this study originated from the Morrison Swine Health Monitoring Project (MSHMP), which aims at developing tools for foreign animal disease preparedness while monitoring swine diseases such as PRRS in approximately 50% of the U.S. sow population . Particin = 658) were from the Midwest, 38.4% (n = 479) from the South, 6.4% (n = 80) from the Northeast, and 2.4% (n = 30) were from the West according to the U.S. Census [The MSHMP’s weekly breeding herd PRRS health status data from July 2009 up to October 2021 was used. For this period, 1316 breeding herds from 40 pig production systems were included. From the enrolled herds, location data was available for 1247 herds, of which 52.8% (. Census .n = 184), followed by the South , Northeast , and West , while 14 had no information on location. Filtration status was available for 139 of the sites that reached S4 status, of which 38, 5, and 18 were not filtered; partially filtered during the fall, winter, and spring seasons; or filtered year-round, respectively.The follow-up time of the included breeding herds ranged between 1 and 12 years. During this period, 221 herds achieved S4 273 times . The number of sites reaching S4 each year ranged from 3 (2009) to 35 (2014), with an average of 21 herds reaching S4 each year throughout the studied period. Most sites that achieved S4 were from the Midwest  days, ranging between 7 and 3962 before changing to a different status. Three breeding herds remained in S4 for a period of 7 days. Among those, two switched to an S1 due to a wild-type virus introduction and one to status S2vx. The probability of an S4 farm remaining as such and not having a PRRS outbreak according to the year when S4 was achieved is shown through the Kaplan–Meier curve A.In 91 out of the 273 times herds achieved the S4 classification, the status was successfully maintained until the end of the study period, whereas in 163 (59.71%) cases, herds eventually lost such status due to wild-type virus introduction. On 15 occasions, herds switched from S4 to S2vx as a result of herds being vaccinated with a modified-live vaccine. Information on the new status was not available for four S4 events that had ended during the study period. For sites that moved from S4 to S1, the median time between achieving status 4 to the new introduction was 602 days (inter-quartile range: 259–1050).n = 57) occurred in the winter, whereas 27.61% (n = 45) occurred in the fall, 23.93% (n = 39) occurred in the spring, and 13.50% (n = 22) in the summer.The PRRS incidence rate after sites reached a naïve status ranged from 10.65 to 38.83 outbreaks per 100 farm years, depending on the year the site reached S4 B. The ovBivariate survival models adjusted for multiple events in the same site and for the production system showed that air filtration and the year in which the site reached a naïve status did not differ in terms of time to PRRS introduction . The yeaS4 was achieved by 17% of the breeding herds during the study period. Those that reached S4 remained as such for a median of two years (or 738 days). However, it is important to note that events were censored at the end of the study period. Censoring might have particularly affected those that reached S4 more recently, possibly underestimating this average since about a third of status four events were still ongoing. Overall, for the correspondent of 100 S4 farms followed for one year, on average, 23 of them would have experienced a PRRS outbreak throughout this period. Although this is similar to the overall cumulative annual PRRS incidence observed in U.S. breeding herds through recent years, ranging approximately 20–25% ,16.Although the PRRS incidence rate fluctuated by the year in which sites reached S4, we found that the year reaching S4 was not statistically significant for the PRRS rate of introduction when accounting for sites and systems. This might be explained by the application of standardized PRRS management strategies consistently every year or by a lack of power in the study since, on average, only 21 sites reached a naïve status each year, ranging from three to 35. Additionally, the monitored population was not consistent throughout the years, as there was an open enrollment of production systems to the monitoring project during the study period. The yearly number of participating systems ranged from 19 to 40.While the South and the Midwest are within the highest swine-dense regions in the U.S. with a similar number of sites participating in the MSHMP, the Midwest has an over three times higher number of sites reaching a naïve status than the South. We found that sites in the Northeast and South that reached S4 had a lower risk of PRRS introduction over time compared to sites that reached S4 in the Midwest. A possible explanation is that while over 20 systems participate in this monitoring project in the Midwest, the South and the Northeast are mostly dominated by less than five production systems. While the production system was accounted for in our model, the inherent heterogeneity of other factors such as PRRS prevention, control, and elimination philosophy affecting transmission and heterogeneity of production systems distribution might not have been captured. Moreover, previous studies showed that pig-dense areas (as is the case for most Midwest and Southern participants) were associated with higher PRRS incidence ,17. The No difference in time to a PRRS introduction according to air filtration status was found in this study, even though lower PRRS incidence rates were reported in sites with year-round air filtration . HoweverOther important potential benefits of maintaining S4 were not assessed in this study, such as the impact on their breeding and growing herd production performance since they are not facing the same health challenges as sites that do not seek elimination. This can be of particular importance and needs to be further investigated since a frequent alternative to elimination is the continuous exposure of gilts entering the herd to the modified-live vaccine (S2vx), which can lead to viremia, viral shedding and potential crossing of the placental barrier in pregnant sows ,18,19. AAlthough we were unable to study other factors, such as the decision process to continue pursuing S4 and additional biosecurity practices implemented, among others that might impact days as S4 and the PRRS incidence rate, our study provides insights on how frequently sites are striving to eliminate and how long can they maintain their naïve status.


New variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high effective reproduction numbers are continuously being selected by natural selection. To establish effective control measures for new variants, it is crucial to know their transmissibility and replacement trajectory in advance. In this paper, we conduct retrospective prediction tests for the variant replacement from Alpha to Delta in England, using the relative reproduction numbers of Delta with respect to Alpha estimated from partial observations. We found that once Delta’s relative frequency reached 0.15, the date when the relative frequency of Delta would reach 0.90 was predicted with maximum absolute prediction errors of three days. This means that the time course of the variant replacement could be accurately predicted from early observations. Together with the estimated relative reproduction number of a new variant with respect to old variants, the predicted replacement timing will be crucial information for planning control strategies against the new variant. Since its first emergence in the human population in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been generating new variants. Natural selection selects new variants that have higher effective reproduction numbers than other circulating variants. As a result, the average transmissibility in the viral population increases over time [It is important to know the transmissibility of new variants in comparison with previously circulating variants because the average reproduction number of the circulating virus changes when new variants become dominant. Several studies have analyzed the reproduction numbers of new variants that have replaced old ones. Volz et al., estimated the effective reproduction number of Alpha in England to be 1.5–2.0 times higher than that of non-VOCs using a logistic growth model for relative variant frequencies . Leung eIn order to prepare control measures against new variants, it is crucial to predict the trajectory of the variant replacements in advance. The prediction of variant selection has been widely studied in seasonal influenza viruses . Łuksza The transmissibility of an infectious agent can be measured by its reproduction number. The effective reproduction number at time nchanged . When monchanged ,13. Howenchanged . This meNucleotide sequences of SARS-CoV-2 variants have been collected worldwide and accumulated in the GISAID database . It is kIn this study, we conduct retrospective prediction tests using the nucleotide sequences collected in England during the Alpha–Delta replacement. For each given time point, we use partial sequence data observed only up to that time point to estimate the relative reproduction number of Delta with respect to (w.r.t.) Alpha. The estimated relative reproduction number is then used to predict the future trajectory of variant replacement. The estimated relative reproduction numbers and the predicted trajectories are evaluated by being compared to those estimated using the entire dataset.Nucleotide sequences of SARS-CoV-2 viruses collected from England from 1 January 2021 to 31 July 2021 were downloaded from the GISAID database on 16 November 2021. Of these, 411,123 sequences had complete information about the date of sample collection in the metadata. The PANGO lineage names [We estimated the relative reproduction number of a variant w.r.t. a baseline variant using an approximated version of the renewal-equation-based model . Let X a of 1.36 . We discSince the effective reproduction number of variant Assuming that the viral population at time We assume that the numbers of new infections do not vary greatly for 20 days, i.e.,For erved It . Using tThe average reproduction number of circulating viruses can be determined by the expected value of the reproduction numbers of circulating variants. Since the relative reproduction number of Let We consider Alpha and Delta as variants kelihood ,20. The 4 July 20. We used4 July 20. We usedp = 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, and 0.95 we determined the calendar times Relative frequencies of Delta and average relative reproduction numbers of circulating viruses w.r.t. Alpha in the future were predicted using the maximum likelihood estimates of parameters calculated from early observations. For each proportion was 1.88 . The finil 2021) . TherefoWe conducted retrospective prediction tests on the future relative frequency of Delta and the average relative reproduction number of circulating viruses w.r.t. Alpha using model parameters in n = 7), 0.023 (n = 11), and 0.004 (n = 15) with maximum absolute errors of 0.092 (n = 7), 0.060 (n = 11), and 0.034 (n = 15), respectively (n = 7), 1 (n = 11), and 1 (n = 15) days with maximum absolute errors of 2 (n = 7), 2 (n = 11), 3 (n = 15) days, respectively  . The rel–406.26) . Using t–406.26) .Several studies have estimated the relative reproduction of Delta w.r.t. Alpha in different countries. Ito et al., estimated the relative reproduction number of Delta w.r.t. Alpha in Japan to be 1.35 . In thisOur model assumes that the sequences on the GISAID database were sampled following a beta-binomial distribution. We can use the binomial distribution in the model instead of the beta-binomial distribution. The model using beta-binomial distribution resulted in lower Akaike information criteria (AIC) compared to the model using binomial distribution . This meIt is possible to use a logistic regression model to fit the trajectory of the relative frequency of variants to achieve the same purpose as in this study. The renewal-equation-based model described in this study resulted in a lower AIC than using a multinomial logistic regression model  as shownThe lockdown restrictions in the UK were relaxed on 17 May 2021. The relative reproduction number estimated using data up to 28 May 2021 was slightly lower than the final estimation using entire observations . This isPrediction tests conducted in this study used the date of sample collection and did not consider the delay in sequence submissions. Nucleotide sequences collected during the period from 18 March 2021, the introduction of Delta, to 13 May 2021, the date when Delta reached a relative frequency of 0.95, were submitted after 10.78 days on average with a standard deviation of 3.76 days. Indeed, 95% of sequences during this period were submitted to the GISAID database within 16 days after sample collection. This means real-time prediction may need additional 10–16 days to achieve a prediction accuracy similar to the results shown in this study.Our model assumes that there was no difference between the generation times of both variants with a mean value of 4.64 days . HoweverDuring the period of analysis, the percentage of people receiving two-dose vaccinations in England was increasing from 7% on 1 April to 39% on 1 June 2021 . Our modIn this study, we showed that the relative reproduction number of Delta w.r.t. Alpha in England and its future trajectory of replacement could be predicted one month before it reached a relative frequency of 0.90. Public health policy-makers would have had only one month to prepare control measures for the increase in viral transmissibility. This implies that a quick decision-making process is needed to take advantage of the prediction. This period can be extended if accurate predictions are available using data earlier than one month. Further research is needed to investigate how much additional information was required for obtaining accurate predictions using data earlier than one month.