Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo  in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from  Summary: The PDZ domain of Drosophila Dishevelled functions in noncanonical Wnt signalling but is dispensable for signalling through Armadillo, the fly homologue of β-catenin. Wnt signalling pathways are conserved ancient cell communication networks that regulate cell fate determination and differentiation during development . All Wnt1dsh mutant flies, which bear a point mutation in the Frizzled-binding loop of the DEP domain, show reduced recruitment of Dishevelled to the apicolateral membrane  gene, to investigate the function of the Dishevelled PDZ domain in multiple developmental contexts following its deletion using CRISPR/Cas9.There have been numerous attempts to pin down the function of the Dishevelled PDZ domain in the transduction of Wnt signals, without reaching a clear consensus. Some have argued that this domain is required for both canonical and noncanonical signalling , whereasDrosophila Dsh is dispensable for canonical Wingless (Wg) signalling but is essential in multiple cellular contexts controlled by noncanonical signalling. Focussing on PCP in the pupal wing, we found that the PDZ domain is required for the asymmetric accumulation of Dsh itself, and of Flamingo , a cadherin-like transmembrane protein required for PCP signalling  targeting either side of the PDZ domain to generate a frame-shift deletion (PDZfsdsh) or a clean excision (ΔPDZdsh) (1dsh mutants (To delete the PDZ-encoding sequences from dshΔPDZ) A. Both mdshΔPDZ) . However mutants   and reduced association of Dsh with Frizzled at the apicolateral plasma membrane  controls, PS-Dsh was clearly visible , similar to those in V26dsh heterozygotes . This is in contrast to wt dorsal and ventral axons, which invariably projected (20/20 larvae) to the dorsal and ventral lamina, respectively (V26/+dsh larvae (20/20 larvae), confirming that the defect in the ΔPDZdsh larvae is not due to reduced Dsh protein levels. 1dsh larvae also showed normal axons (10/10 larvae)  A, sugges larvae) , whereas larvae)  and acts larvae) .Fig. 2.ΔPDZdsh homozygous flies (7/18 flies), as also seen in 1dsh flies (7/14 flies)  B. Howeveies)  were unsuccessful, we chose the PDZ domain of human DVL2 (DVL2PDZ), which shares 91.8% sequence similarity with DshPDZ, including complete conservation of the ligand-binding cleft residues . After bacterial expression and purification, we acquired 1H–15N correlation spectra of 100 µM 15N-labelled DVL2PDZ alone or after incubation with 300 µM individual purified PBMs tagged with lipoyl (Lip).Several PCP components contain putative PBMs in their C termini , indicating that Fz, but not Fz2, contains a functional PBM. Fz is the predominant Frizzled protein implicated in Drosophila PCP signalling (PDZ (PDB:2REY), whose resonances have been assigned previously  in PDZ backbone N–H resonances . F11stan flies were homozygous viable and failed to show any of the above-described mutant phenotypes (C1dsh) or N612 (C9dsh), both of which result in a unique class II PBM that is, however, unable to bind DVL2 PDZ . Both of these mutants were viable and aphenotypic (F1fz) and 568STOP . Intriguingly, we found that Fmi polarity is actually increased in double  and triple mutants  compared to the wt . These polarity alterations of Fmi suggest that the C-terminal PBMs in these polarity proteins have a regulatory role in modulating proximodistal polarity; however, these PBMs are clearly not essential for polarity per se.To test the functional relevance of these PBMs during l thorax   that showed misoriented thoracic bristles and wing hairs . Therefore, B4fz and 1dsh both compromise the interaction between the two proteins, which could explain the polarity phenotypes caused by each of these lesions.Recall that this recruitment step is mediated by direct binding of the Dishevelled DEP domain to the intracellular face of Frizzled . HoweverxW motif . TherefoB4fz and 1dsh also highlight the importance of the DEP domain-dependent interaction between Frizzled and Dishevelled in planar cell polarity.In summary, our study demonstrates that the Dishevelled PDZ domain is required exclusively for noncanonical Wnt signalling responses in multiple developmental contexts. However, the PCP phenotypes of Dsh-DEP–GFP was generated by exchanging Dsh-DEP with DVL2-DEP from DEP–GFP . SNAP–FzThe following antibodies were used for immunolabelling: 1:1000 rat anti-Dsh , 1:300 aDrosophila melanogaster were used throughout. Our wt strain and the background of our mutants was w1118. The following mutant and transgenic strains were used : y w Ubx-FLP; arm-lacZ FRT80 and Ubi-GFP FRT19A; Ubx-FLP; w1, dsh1; Wnt4C1; Wnt4EMS23; dshv26; omb.lacZ. Deletion and truncation mutants were generated essentially as described previously (https://crispr.mit.edu was used to design gRNAs targeting Dsh (PDZ: 5′-GGAGACGGTAATGATATTTA-3′ and 5′-GCTGGTGGTGGCCAAGTGCT-3′) or C-terminal PBMs . Multiple alleles were obtained, and their lesions were determined by genomic sequencing.Standard strains of eviously . The CRIomb-lacZ  and regularly tested for mycoplasma infection. DEP recruitment assays in HEK293T cells were performed as previously described  and imagProteins were purified essentially as described previously , NMR spe


C) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhAC and promote the cyclic open-close domain motions of FlhAC. The temperature-sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS.Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAIMPORTANCE Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly. Salmonella enterica serovar Typhimurium  is divided into three structural parts: the basal body, which acts as a bi-directional rotary motor, the filament, which functions as a helical propeller to produce thrust, and the hook, which is a universal joint connecting the basal body and filament and transmits torque produced by the motor to the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. To construct the flagellum on the cell surface, the flagellar type III secretion system (fT3SS) transports flagellar structural subunits from the cytoplasm to the distal end of the growing flagellar structure  as the energy source to unfold and translocate export substrates across the cytoplasmic membrane . In addition, FlgN, FliS, and FliT act as export chaperones that escort their cognate substrates from the cytoplasm to the fT3SS ocation 68. The fT. The fT+properly .Salmonella FlhA is composed of an N-terminal transmembrane domain with eight transmembrane helices , a compactly folded cytoplasmic domain , and a flexible linker  connecting these two domains  and adopts open and closed conformations , the chaperone-substrate complexes can bind to the conserved hydrophobic dimple in the open form but not in the closed form , allowing the chaperone-substrate complexes to efficiently bind to the FlhAC ring  mutation inhibits the protein transport activity of the fT3SS at a restrictive temperature of 42°C but not at a permissive temperature of 30°C (–flhA(G368C) mutation. Molecular dynamics (MD) simulation has shown that the flhA(G368C) mutation restricts cyclic open-close domain motions of FlhAC at 42°C and stabilizes a completely closed conformation of FlhAC (C coupled with flagellar protein export.The  of 30°C –29. The of FlhAC . Gly-368of FlhAC . This leC combined with MD simulation. We provide evidence that the flhA(G368C) mutation stabilizes hydrophobic side chain interactions between domains D1 and D3 and those between domains D2 and D4, thereby suppressing dynamic open-close domain motions of FlhAC, and that its intragenic suppressor mutations induce the remodeling of the hydrophobic interaction networks in FlhAC, allowing FlhAC with the G368C mutation (FlhAC-G368C) to restore the dynamic open-close domain motions.To clarify this hypothesis, we carried out mutational analysis of FlhAflhA(G368C) mutation stabilizes a completely closed form of FlhAC at 42°C, we first compared the interfaces between domains D1 and D3 and those between domains D2 and D4 in the open and closed forms of FlhAC-G368C obtained in the previous MD simulation (C-G368C (right panel), we propose that the flhA(G368C) mutation may stabilize these two hydrophobic interactions at 42°C, thereby suppressing cyclic open-close motions of FlhAC.To address why the mulation   mutation in domain D3 does not affect FlhA function at all. However, the combination of the flhA(G368C) and flhA(K548C) mutations results in a loss-of-function phenotype even at 30°C (flhA(G368C/K548C) mutation inhibits flagellar protein export at 30°C, we analyzed the structure and dynamics of FlhAC-G368C/K548C by MD simulation at 27°C for 1.5 μs  between domains D2 and D4 during MD simulation .The highly conserved Lys-548 residue of FlhA is located at the interface between domains D1 and D3 and contributes to D1-D3 interactions  Fig. 1BFig. 1B.  at 30°C . To clarr 1.5 μs . DomainsC-G368C, which changes its conformation back and forth between the closed and open forms at 27°C  mutation restores motility of the flhA(G368C/K548C) mutant to a considerable degree . Gly-368 forms the highly conserved GYXLI motif along with Tyr-369, Arg-370, Leu-371, and Ile-372  mutation, which has been isolated as an intragenic suppressor mutation of the flhA(G368C) mutant grown at 42°C  and Y369G/R370G/L371G/I372G (here referred to as GGGG) mutants and analyzed their motility in 0.35% soft agar. Immunoblotting with polyclonal anti-FlhAC antibody revealed that the AAAA and GGGG mutations did not affect the steady expression level of FlhA (Hydrophobic parts of the side chain of Tyr-369 and Arg-370 make hydrophobic contacts with Pro-415 and Ala-416 in the open form of wild-type FlhA Pro-434 . To clar of FlhA . The AAA of FlhA . In agre of FlhA . The GGG of FlhA . Consist of FlhA . These oflhA(M365I), flhA(A446E), and flhA(P550S) mutations have been reported to restore motility defects in the flhA(G368C) mutant at 42°C (flhA(G368C) mutation stabilizes the completely closed form of FlhAC at 42°C through the strong hydrophobic interactions between Gln-498 and Pro-667 and those between Phe-459 and Pro-646, we next analyzed the effect of the M365I, A446E, and P550S suppressor mutations on the conformational changes of FlhAC-G368C.The intragenic  at 42°C , 28. To C-G368C but not in its open form  mutation induces a conformational change of domain D2 as judged by far-UV circular dichroism (CD) measurements of purified FlhAC with the G368C substitution  mutation may induce hydrophobic side chain interaction networks in FlhAC to stabilize a complete closed form at 42°C and that Leu-413, Pro-415, Pro-434, Phe-459, Gln-477, Leu-492, Gln-498, Lys-548, Pro-646, and Pro-667 may be involved in dynamic open-close domain motions of FlhAC (flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, we constructed flhA(G368C) mutants with either L413A, P415A, P434A, F459A, Q477A, L492A, Q498A, K548A, P646A, or P667A substitution and analyzed the motility of these double mutants at both 30°C and 42°C (C antibody (MD simulation of FlhAof FlhAC . To testand 42°C . These dantibody .flhA(G368C) mutant even at 30°C (flhA(G368C) mutant at 42°C (flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, thereby inducing conformational changes of domains D1 and D2.The P415A, P434A, Q477A, Q498A, and K548A substitutions reduced the motility of the  at 30°C . Consist at 30°C . Further at 42°C . The P41 at 42°C . TherefoflhA(G368C) mutant at both 30°C and 42°C (flhA(G368C) mutant grown at 30°C  mutant at both 30°C and 42°C (flhA(G368C) mutant grown at 30°C but not the level of FlgD secretion  mutant was much worse than that of the flhA(F459A) mutant (flhA(F459A) mutant  mutant  and 6, w) mutant , thereby) mutant . TherefoflhA(G368C) mutant at both 30°C and 42°C  mutation may limit the hinge movement between domains D1 and D3 through a remodeling of the hydrophobic side chain interaction networks of domain D1, thereby allowing Pro-646 and Pro-667 to make the strong hydrophobic contacts with Phe-459 and Gln-498, respectively  mutation limits the cyclic open-close domain motions of FlhAC at 42°C, thereby reducing the flagellar protein export by the fT3SS and therefore the cell motility  mutation induced a remodeling of hydrophobic side chain interaction networks in FlhAC at 42°C, allowing Gln-498 of domain D1 and Phe-459 of domain D2 to hydrophobically interact with Pro-667 and Pro-646 of domain D4, respectively (flhA(G368C) mutation not only suppresses the dynamic open-close domain motions of FlhAC but also stabilizes the completely closed conformation at 42°C. Intragenic M365I, R370S, A446E, and P550S suppressor mutations, which restore motility defects in the flhA(G368C) mutant to near wild-type levels  mutation  affinity chromatography have revealed that the flhA(G368C) mutation reduces the binding affinity of FlhAC for the chaperone-substrate complex   (C subunits stably when FlhAC adopts the open form in the ring structure (right panel) because FliJ can contact both FlhAC subunits. However, the FliJ binding to FlhAC in the closed form appears to be unstable (left panel) because the distance between the neighboring D4 domains is longer in the closed form (ca. 80.8 Å) than that in the open form (ca. 36.9 Å), restraining the proper contacts of FliJ with two FlhAC subunits (middle panel). Because the open and closed forms of FlhAC represent the active and inactive states of the fT3SS, respectively (C may be important for an efficient and robust energy coupling mechanism of the fT3SS.FliJ binds to FlhAig. S1B) . FliJ alaffinity . It has tructure . This suith FliD . This raD: 6WA9) . FliJ caectively , we propSalmonella strains used in this study are listed in int (flhA mutations were confirmed by DNA sequencing (Eurofins Genomics).The d in int . DNA manFresh colonies were inoculated onto soft agar plates  containing 50 μg/mL ampicillin, and then the plates were incubated at 30°C. At least six measurements were performed.Salmonella cells were grown at 30°C in 5 mL of L-broth  containing 100 μg/mL ampicillin. The cells were attached to a coverslip , and unattached cells were washed away with motility buffer . A 1-μL aliquot of polyclonal anti-FliC serum was mixed with 50 μL of motility buffer, and then 50 μL of the mixture was applied to the cells attached to the cover slip. After washing with the motility buffer, 1 μL of anti-rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen) was added to 50 μL of motility medium, and then the mixture was applied. After washing with the motility buffer, the cells were observed by fluorescence microscopy  of ca. 1.2 to 1.4. Cultures were centrifuged to obtain cell pellets and culture supernatants. The cell pellets were resuspended in sodium dodecyl sulfate (SDS)-loading buffer solution  containing 1 μL of 2-mercaptoethanol. Proteins in the culture supernatants were precipitated by 10% trichloroacetic acid and suspended in a Tris/SDS loading buffer  containing 1 μL of 2-mercaptoethanol  using a transblotting apparatus (Hoefer). Then, immunoblotting with polyclonal anti-FlgD, anti-FlgK, or anti-FlhAC antibody was carried out using an iBind Flex Western device (Thermo Fisher Scientific) as described in the manufacturer’s instructions. Detection was performed with Amersham ECL Prime Western blotting detection reagent (Cytiva). Chemiluminescence signals were captured by a Luminoimage LAS-3000 analyzer (FujiFilm). At least three measurements were performed.oethanol . Both whhttp://www.ebi.ac.uk/Tools/msa/clustalo/).Multiple sequence alignment was carried out using Clustal Omega (C with either G368C/K548C or G368C/F459C/K548C substitution were conducted as described previously (C (b water molecules (MD simulations of FlhAeviously . The mutously (C . The Ambously (C  was usedolecules  and 0.17olecules  with a molecules  of AMBER


With increasing prior cold strain, the grain size, after a two-stage deformation, decreases continuously, while the BLDΣ3n increases firstly and then decreases. The mechanisms for improving the mixed grain boundary network via two-stage deformation were uncovered. The sub-grain boundaries formed in prior cold deformation stimulate the nucleation of DRX grains and twins; meanwhile, the driving force for grain boundary migration is enhanced due to prior stored energy. Then, DRX is activated in advance and occurs more completely, thereby promoting the formation of Σ3n twin boundaries.Within the grain boundary engineering (GBE) of alloys, a mixed grain boundary network with random grain boundaries interrupted by twin boundaries, contributes to enhancing the overall grain boundary-related properties. The higher density of twin boundaries is pursued herein. Furthermore, a two-stage deformation method, i.e., prior cold deformation followed by thermal deformation, was proposed for improving the mixed grain boundary network in the thermal deformation of Ni80A superalloy. The influence of prior cold deformation on the mixed grain boundary network was investigated through a series of two-stage deformation experiments. The analysis of the stress–strain curves shows that the critical strain for dynamic recrystallization (DRX) and peak strains decrease significantly under the effect of prior cold deformation. In comparison to the necklace-like microstructures that occur after a single thermal deformation, the microstructures apparent after a two-stage deformation are characterized by finer DRX grains with abundant Σ3 Duendaries” ,7,8. It  network ,10, formsistance , corrosisistance  and creesistance  of the an twin boundaries. Shi et al. [During the thermal deformation process of an alloy, dynamic recrystallization (DRX), grain growth, and twin evolution mechanisms coexist and interact with each other. The evolution of twin density is governed by the comprehensive effects of these mechanisms. In order to improve the mixed grain boundary network, it is a prerequisite to describe the microstructure-evolution mechanisms associated with Σ3i et al. , Aghaie-i et al. , Ebrahimi et al. , Li et ai et al. , etc., ii et al.  investigi et al.  construci et al.  found thi et al.  and Quani et al.  develope−1, the grain boundary migration rate is relatively slow, and there is not enough stored energy to sufficiently activate the DRX process, resulting in a lower nucleation rate of the DRX grains and twins [However, it is noteworthy that the DRX cannot occur fully in all cases where a specimen has deformed under a wide deformation parameter range. At a relatively low temperature and a mediate strain rate of 0.1 snd twins ,24,25,26nd twins ,27,28,29n twin boundaries. Until now, most investigators have analyzed the effects of the TMP processing parameters on twin boundary evolution, such as pre-deformation amount, annealing temperature, annealing time, iterative times, etc. Wang et al. [n twin boundaries increases from this by about five times. Several such works reveal that TMP can improve the density of Σ3n twin boundaries in nickel-based alloys. However, the magnitude of the deformation in the traditional TMP processing route is limited to low or medium levels, and higher twin densities rely on multiple iterations of cold deformations followed by annealing. For the Ni80A superalloy, components fabricated by the thermal deformation processes, such as electric-upsetting and forging, it is impracticable to improve the mixed grain boundary network through the existing TMP processing route.The primary processing route for GBE is a thermo-mechanical process (TMP), i.e., a single step or multiple iterative cold deformations followed by short-time annealing treatments ,30,31. Tg et al.  processeg et al.  investigg et al.  discusseg et al.  reportedCombining the TMP processing route for GBE is a new attempt to improve the mixed grain boundary network through a two-stage deformation method, i.e., prior cold deformation followed by thermal deformation. Further, the influencing mechanisms of prior cold deformation on the mixed grain boundary network in thermal deformation were studied systematically. Firstly, the two-stage deformation experiments for the Ni80A superalloys were conducted, wherein the specimens were subjected to cold deformation under different strains, and this was followed by isothermal compression under the same condition. After this, the flow behaviors were analyzed from the obtained true stress–strain curves, and the microstructures during the two-stage deformation process were characterized. The variations of DRX volume fraction, grain size, and twin density with prior cold strain were quantitatively analyzed. Finally, the influencing mechanisms for improving the mixed grain boundary network via a two-stage deformation method were uncovered. This investigation could provide a theoretical foundation for the design of GBE routes for high-temperature alloys.ϕ 10 mm × 15 mm, were cut from an as-forged billet by wire electrical discharge machining (WEDM). Four of these were prepared for two-stage deformation experiments, in which the specimens were subjected to cold deformation with different true strains, followed by thermal deformation. During the cold deformation stage, the specimens were cold compressed under the true strains of 0.163, 0.223, 0.288, and 0.357 , respectively. Following this, the cold deformed specimens were machined by WEDM to obtain the standard cylindrical specimens, with a size of ϕ 8 mm × 10 mm. During the WEDM process, the central axis of the separated specimens should coincide with that of the cold deformed specimens. Subsequently, the separated specimens were prepared for isothermal compression experiments. During the thermal deformation stage, the separated specimens were heated to 1323 K under a heating rate of 5 K/s and held at this temperature for 180 s to ensure uniform temperature distribution. Then, the heated specimens were isothermally compressed into the fixed true strain of 0.916 (height reduction of 60%) under a strain rate of 0.1 s−1, immediately followed by water quenching to retain the elevated temperature microstructures. These compression experiments were carried out on a Gleeble-3500 thermo-mechanical simulator, and more experimental details are included in [−1, with a true strain of 0.916 to characterize the microstructures in single thermal deformation.In this work, the studied material was Ni80A superalloy, for which the detailed chemical composition (wt.%) is listed in 4:CH3COOH:H2O = 1:1:8 (vol.) with a voltage of 20 V at −15 °C for 22 s. The deformed microstructures were characterized using electron back-scattered diffraction (EBSD) technology. For the specimens subjected to the two-stage deformation process, an area of 480 × 360 pixels was scanned using a step size of 0.5 μm. As for the cold-deformed specimens, an area of 520 × 390 pixels was scanned with a step size of 2 μm. Oxford instruments channel 5 software was employed to construct the EBSD maps: inverse pole figure (IPF), EBSD map highlighting the DRX volume fraction and grain boundary network, band contrast map with Σ3n twin boundaries, and kernel average misorientation (KAM) map. The grain size was detected as a region being completely bounded by high angle grain boundary (HAGBs) without considering Σ3n twin boundaries. The critical misorientation angles of 3° and 15° were used to identify the sub-grain and grain boundaries, respectively. The local intragranular misorientation was calculated up to the fifth nearest neighbor, with a maximum misorientation angle of 5°. The Σ3n  twin boundaries were identified according to the Brandon criterion [n twin boundaries  was introduced to evaluate the proportion of the annealing twins; this can be calculated using the equation (Np × Δ)/A [Np constitutes the number of pixels of Σ3n twin boundaries, Δ represents the step size in μm, and A represents the scanning area in μm2.Afterwards, all of the deformed specimens were symmetrically sectioned into two semi-cylinders, along the compression axis, and the geometric centers of the sectioned surfaces (as shown in p × Δ)/A , where Nn twin boundaries -stage, softening stage, and the steady stage. In the first stage, since dynamic recovery (DRV) is too weak to offset the WH induced by the multiplication and accumulation of the dislocations, flow stress increases sharply with strain increasing. In the second stage, when the accumulated dislocation density exceeds a critical value, i.e., the strain reaches the critical strain for the onset of DRX, DRX takes place and then accelerates the annihilation of the dislocations. Under the competition behavior of WH and DRV as well as DRX, the flow stress continuously increases to a peak; meanwhile, its increasing rate decreases. When the flow stress reaches peak stress, the softening behaviors caused by DRX and DRV are enough to overcome WH. With the continuously increasing strain, DRX takes the predominant role in the softening behaviors, thus, resulting in the decreasing of flow stress. Finally, the flow stress keeps a steady state due to the dynamic equilibrium between WH and softening. In addition, from θ/dσ with θ/dσ reaches the maximum, which corresponds to the inflection point of θ − σ curves, the critical stress for DRX initiation can be identified, and then the critical strain, εp, can be identified from the true stress–strain curves directly. The obtained critical strain, εp, are listed in −1, the critical strain and peak strain are identified as 0.080 and 0.230, while the values of these two points decreases significantly when the specimens are subjected to two-stage deformation. This indicates that DRX is activated in advance under the effect of prior cold deformation, while the effect gets stronger with increasing cold strain. In addition, it can be inferred from From the obtained true stress–strain curves in  plotted b. AccordA kernel average misorientation (KAM) map is usually used to qualitatively reflect the uniformity of plastic deformation. The microstructures with higher KAM values indicate a greater degree of plastic deformation or higher dislocation density . Figure −1 with the fixed true strain of 0.916. The influence of prior cold deformation on the mixed grain boundary network in thermal deformation was discussed. This contributes toward uncovering the mechanisms for improving mixed grain boundary network in the thermal deformation of Ni80A superalloy.In this section, the microstructures after two-stage deformation were characterized; the specimens with different cold strains were subjected to thermal deformation under a temperature of 1323 K and a strain rate of 0.1 s−1. In n twin boundaries, resulting in a lower local intragranular misorientation in the microstructures. It also can be inferred that DRX has occurred more completely under the effect of prior cold deformation.Aiming at the development of sub-grain network in thermal deformation, the KAM maps were characterized, as shown in −1, when the specimen is subjected to a single thermal deformation, the grain boundary migration rate is relatively slow, and there is not enough stored energy to fully activate the DRX process, resulting in the longer incubation period. However, for those specimens subjected to a two-stage deformation, it can be seen that the sub-grain boundaries with high-density dislocation form within the microstructures after cold deformation , Σ3, Σ9, and Σ27 twin boundaries were statistically analyzed, with their variations against cold strain shown in n twin boundaries: Σ3 + Σ3 = Σ9, Σ3 + Σ9 = Σ3, Σ3 + Σ9 = Σ27 [In order to quantitatively analyze the evolution of the Σ3Σ9 = Σ27 ,40,41. An twin boundaries (BLDΣ3n), after two-stage deformation, was estimated. The relationships of BLDΣ3n with the DRX volume fraction and grain size were also demonstrated, as in Σ3n were calculated as 0.0252, 0.1980, 0.2455, 0.2429, and 0.2282 μm−1, respectively. Compared to the BLDΣ3n value, after a single thermal deformation, the BLDΣ3n values after two-stage deformation increase remarkably, improving by about nine times, with the cold strain of 0.223. It can also be noted from Σ3n evolution is synthetically affected by the DRX volume fraction and grain size. In Σ3n exhibits an approximately similar tendency to that of the DRX volume fraction. In Σ3n and grain size; with an increase in cold strain, grain size decreases constantly, while BLDΣ3n increases firstly and then decreases gradually. On the one hand, during grain boundary migration, the probability of forming Σ3 twin boundaries increases with the occurrence of DRX. On the other hand, the formation of Σ3 twin boundaries can accelerate the bulging of the original grain boundary, leading to the newly formed DRX grains separating from the parent grains, contributing to the nucleation and development of the DRX grains [n twin boundaries being formed. This is why BLDΣ3n decreases with decreasing grain size.Additionally, the density of the Σ3X grains ,17,18. HΣ3n and finer grain size in the thermal deformation process can be obtained by adjusting the prior cold deformation.Based on the above experimental results and analysis, it can be concluded that the mixed grain boundary network in the thermal deformation of Ni80A superalloy can be significantly modified through a two-stage deformation method. The microstructures with higher BLD−1, the necklace-like microstructures, with lower twin density, are formed. This is mainly due to the fact that the stored energy for activating the DRX process is insufficient; meanwhile, the probability of forming twins decreases under the relatively slow grain boundary migration rate [During the dynamic thermal deformation process of nickel-based alloys, DRX behaviors and twin evolution mechanisms are strongly associated with stored energy, influenced by the processing parameters, including temperature, strain, and strain rate ,22,32,42ion rate . When thBased on the analysis above, it can be confirmed that, for a fixed thermal deformation condition, the mixed grain boundary network with a finer grain and higher twin density can be achieved by adjusting the prior cold deformation. It is worth mentioning that the thermal deformation temperature and strain rate will also affect the grain boundary migration in the thermal deformation process; the mixed grain boundary network in thermal deformation may be improved by comprehensively coordinating the thermal deformation parameters and the prior cold deformation. Further study and discussion of the effect of the thermal deformation parameters will be carried out in future work. Following this, the quantitative models describing the evolution of grain size and twin density in the two-stage deformation process will be developed to precisely adjust the cold and thermal deformation parameters for achieving the desired microstructures of the alloys. In addition, the cold deformation experiments in the present work were performed at room temperature. It is worth studying whether the lower cold deformation temperature can improve the mixed grain boundary network in the thermal deformation processing of alloys more significantly.n twin boundaries in the thermal deformation process of alloys. The influencing mechanisms of the prior cold deformation on the mixed grain boundary network from the thermal deformation of Ni80A superalloy were investigated. Some conclusions drawn from this study are as follows.In this work, a two-stage deformation method, i.e., prior cold deformation followed by thermal deformation, was proposed to achieve a mixed grain boundary network with a higher density of Σ3(1)The flow behaviors of Ni80A superalloy are significantly influenced by prior cold deformation. The critical strain for the onset of DRX and peak strain decreases remarkably under the effect of prior cold deformation, implying that DRX is activated in advance. These two indicators decrease with increasing cold strain;(2)n twin boundaries and a significantly improved value of BLDΣ3n of about nine times. With increasing cold strain, the grain size decreases continuously, while BLDΣ3n increases at first and then decreases gradually;In comparison to the necklace-like microstructures from the single thermal deformation, the microstructures of the two-stage deformation process are characterized by finer and equiaxed DRX grains accompanied by abundant Σ3(3)Σ3n against prior cold strain exhibit an approximately similar tendency to that of the DRX volume fraction, with it increasing at first and then decreasing gradually with grain refinement. This indicates that the relationship between BLDΣ3n and grain size is non-liner. The mixed grain boundary network, with higher BLDΣ3n and finer grains under thermal deformation, can be obtained by adjusting the prior cold deformation;The variations of BLD(4)n twin boundaries.The mechanisms for improving the mixed grain boundary network by a two-stage deformation method have been uncovered. The sub-grain boundaries formed in prior cold deformation stimulate the nucleation of the DRX grains and twins; meanwhile, the driving force for grain boundary migration is enhanced due to the prior stored energy. In these cases, DRX is activated in advance and occurs more completely, thereby promoting the formation of Σ3


Lacticaseibacillus rhamnosus and Lentilactobacillus buchneri) and the addition of four corn flour proportions  in different treatments, on the composition and function of the bacterial community in whole-plant paper mulberry silage. The different treatments promoted Lactiplantibacillus, Lentilactobacillus, and Lacticaseibacillus growth, but the microbial species responsible for fermentation differed among the treatments. High species diversity and various Gram-negative bacteria, such as Flavitalea sp., Pantoea agglomerans, Acinetobacter pittii, Turicibacter sanguinis, and Ralstonia pickettii, were found in the uninoculated LAB treatments. A beneficial bacterium, Lactobacillus johnsonii, was discovered for the first time in whole-plant paper mulberry silage. LAB inoculation simplifies the microbial community structure, and beneficial Lactobacillus as a key species aggregates in the inoculated treatment group. However, L. rhamnosus inoculation alone may have limited bacteriostatic activity against in whole-plant paper mulberry silage. Compared with silage lacking corn flour, amino sugar and nucleotide sugar metabolism, galactose metabolism, the phosphotransferase system and the pentose phosphate pathway metabolic activity were increased in corn flour-containing silage. Whole-plant paper mulberry can be used as a high-quality silage to provide high-quality feed resources for sustainable ruminant livestock production. Moreover, additive use is necessary for preparing paper mulberry silage.The purpose of this study was to investigate the effects of inoculation with two lactic acid bacteria (LAB) strains ( High elevations, mountainous areas, and severe soil erosion are the primary features of the karst landscape in southwestern China . EcologiBroussonetia papyrifera L.) was originally widely planted in karst mountainous areas due to its strong application prospects in ecological restoration, environmental improvement, water conservation, sand prevention, and sand fixation  strain (BDy3-10), which had the highest acid production rate, and Lentilactobacillus buchneri (LB) strain (TSy1-3), which had the fastest growth rate, were selected as the LAB additives in this study  and analyzed. In the end, a total of 130 bacterial species were isolated, of which 49 were identified as LAB. After all strains were isolated and purified, their acid production and growth rate in 24 h were measured on MRS medium (anaerobic at 37°C) . Lacticais study . As showis study .8 cfu/ml, the dosage is 10 ml/kg) and corn flour as additives were used to establish the following treatments on a fresh basis. Mix the chopped whole-plant paper mulberry with the treated corn flour at the ratio of 0, 3, 6, and 9% to make 4 different types of premixes. Each ratio premix makes 12 servings (contains 3 repeats). The treatments for each premix type were NA , LR (Inoculation with LR), LB (Inoculation with LB) and M (Combined inoculation of LB and LR). For each treatment, 500 g of sample was mixed with additives and placed in a polyethylene silage bag (30 cm × 40 cm), which was vacuum sealed . In total, 48 bags (4 inoculums × 4 proportions × 3 repeats) were stored at a constant temperature (approximately 25°C) in the laboratory away from light for 60 days before unsealing.The raw whole-plant paper mulberry materials in this study were collected from Changshun County, Guizhou Province, China on August 27, 2020 . The test material was the third harvest of whole-plant hybrid paper mulberry (4–5 harvests can be cut throughout the year). Nine points were randomly selected over a natural planting area (no fertilizers and pesticides were used) of 2 hectares (three field replicates were performed after mixing) for mowing. The cutting height was 15–20 cm above the ground (the height of the whole plant was 150–180 cm). After the plants were wilted for 30 min, a straw cutter was used to chop the material into small pieces measuring 1.5–2.5 cm . Corn flour from traditionally grown corn was passed through a 20-mesh sieve before use. All prepared raw materials were immediately taken to the plantation plant for silage production. Sterilize the accurately calculated corn flour with different ratios , and prepare with sterilized distilled water when adding, so that the moisture content of the whole-plant mulberry silage is consistent. LAB suspension (1 × 103-N) content was determined by phenol-hypochlorite colorimetric method . The remaining extracts were subjected to high-performance liquid chromatography  to detect lactic acid (LA), acetic acid (AA), propionic acid (PA), butyric acid (BA), and other organic acids. Ammonia nitrogen  medium agar  cultured at 37°C under anaerobic conditions . Coliform counts were performed on purplish red bile agar (under aerobic conditions at 30°C). Yeasts and molds were counted on malt extract agar .Microbial populations were roughly counted with reference to the plate count method described by Based on the PacBio sequencing platform , single molecule real-time sequencing (SMRT Cell) was used to sequence the marker genes. Raw data were processed using the RS_Readsofinsert.1 protocol in the SMRT Link version 8.0 software.1). Then, the adapter and primer sequences were removed using Cutadapt 1.9.1  magnetic bead method genomic kit, A full-length 16S rRNA gene was amplified using primers  and sequenced on the PacBio sequencing platform with single-molecule real-time sequencing (SMRT Cell). The raw circular consensus sequencing data were demultiplexed using lima  and to predict the functional potential of the microbial community during the phylogenetic process based on the correlation between phylogeny and function  were classified, Sequences at 97% (default) similarity level were clustered using USEARCH (version 10.0), OTUs were filtered with 0.005% of all sequences sequenced as a threshold and downstream analysis. The taxonomy was assigned to features using SILVA (release 132) as reference. Abundance analysis, alpha diversity, and beta diversity were calculated by QIIME2 . Correlafunction .P < 0.05 was regarded as a significant difference, and the results are expressed as the average value. Permutation multivariate analysis of variance (PERMANOVA) was performed to compare the differences in microbial communities between treatments. Differentially enriched taxa between four different types of silage  were selected and displayed using LEfSe discriminant analysis, and significant differences were considered by a LDA ≥ 3.0 and P < 0.05.The effects of the additives on the fermentation characteristics and chemical composition of whole-plant paper mulberry silage were evaluated using one-way or two-way analyses of variance (ANOVA), with Duncan’s multiple range tests. Alpha diversity indices were compared for significance using Student’s T-test. All statistical procedures were conducted using IBM SPSS 26.0 software. 2 cfu/g of FM).The nutritional composition and microbial population of fresh whole-plant paper mulberry and corn flour before ensiling are shown in P < 0.01) impacts on organic acids , DM, CP, and pH. The addition of different corn flour ratios had significant (P < 0.001) effects on the contents of DMloss, NDF, ADF, ASH, and WSC. The addition of LAB had no significant effects on the measured parameters except CP (P < 0.001). The percentage of corn flour (P < 0.001) and its interaction with the LAB treatment (P = 0.002) had significant effects on IVDMD (in vitro DM digestibility). Although there were some differences in the contents of AA and NH3-N, these differences were not significant.The fermentation and chemical characteristics of whole-plant paper mulberry silage are shown in P < 0.05). Compared with NA, LA content increased significantly (P < 0.05) in the 0 and 3% corn flour treatments but decreased significantly (P < 0.05) in the 6 and 9% corn flour treatments; the LA/AA value was higher (P < 0.05) at corn flour ratios of 0 and 3% but lower (P < 0.05) at a corn flour ratio of 9%; the CP content increased significantly (P < 0.05) in the 6% corn flour, 0% LB, and 0% M treatments. In addition, PA content was highest in the LB0, M0, NA3, M3, NA6, LR6, and LB6 treatments. The DM content increased significantly (P < 0.05) as the corn flour ratio increased (P < 0.05). DMloss was positive only when the corn flour ratio was 0%. NDF and ADF contents decreased significantly (P < 0.05) with increasing ratios of corn flour (P < 0.05). Low ASH content was detected in silage with 9% corn flour. The WSC content of the 0% corn flour treatment was significantly lower (P < 0.05) than that of the other treatments. IVDMD was generally higher in the treatments with corn flour ratios of 3% and 9%. BA was not detected in any of the treatments in this study (not listed in the table).With the exception of NA0, the pH was less than 4.2 in all treatments. Except for the M treatment with 6% corn flour, the pH decreased gradually as the ratio of corn flour increased (P < 0.01), and the Simpson and Shannon indices of some ratios of corn flour, such as NA3 and NA9, were significantly higher than those of the other treatments (P < 0.01). There was no general difference in the alpha diversity index with the addition of strains . Except for LB0, LR0, LR3, and LR6, clearly separation of the treatments with LAB inoculants and treatments with corn flour was observed.Amplification and sequencing of the full-length 16S rRNA of silage bacteria generated an average of 11,955 CCS was retained per sample for downstream analysis. The average effective CCS sequences for analysis after removing chimeras was approximately 92.03%. The sequencing coverage of all samples was higher than 0.99, indicating that sequencing adequately captured the majority of bacteria . Except  strains . PrincipLactobacillus taxonomy classification , and the abundance of A. muciniphila decreased as the corn flour ratio increased. The relative abundances of L. buchneri and L. rhamnosus increased significantly with the addition of a single bacterial agent and corn flour ratios of 3 and 9%.The compositions of the bacterial communities in the paper mulberry silages are shown in fication , severalProteobacteria that were unfavorable for fermentation were enriched in the NA treatment. At the genus level, Flavitalea, UTCFX1, Turicibacter, Sphingomonas, and Steroidobacter, all of which belong to the phylum Proteobacteria, were the most different genera in NA-treated silage, while the most different genera in silage inoculated with LB, LR or M were Lacticaseibacillus, Lactobacillus, or Lentilactobacillus, respectively. At the species level, Flavitalea sp., Acinetobacter pittii, Turicibacter sanguinis, uncultured_bacterium_g_UTCFX1, uncultured_bacterium_g_Steroidobacter, and uncultured_ bacterium_g_MND1 were identified as differential species in NA-treated silage, while L. rhamnosus, L. johnsonii, and L. buchneri were identified as differential species in silage inoculated with LB, LR, or M different, respectively.To explain the effects of different corn flour ratios and inoculation on the microbial community of paper mulberry silage, a latent Dirichlet allocation (LDA) effect size (LEfSe) analysis was performed to find markers of differences between the groups . No signL. pentosus, L. buchneri, and L. rhamnosus were identified as key species. L. pentosus was negatively correlated with L. buchneri, and positively correlated with W. cibaria, Levilactobacillus brevis, L. guizhouensis, L. manihotivorans (P < 0.05). L. rhamnosus was negatively correlated with Lacticaseibacillus pantheris and L. guizhouensis (P < 0.05)   ; A. haemolyticus was positively correlated with 12 bacteria including Proteiniclasticum ruminis (P < 0.05)  . LR sila < 0.05) . M silag < 0.05) . NA sila < 0.05) .P < 0.05). The metabolic activity of the two-component system, carbon metabolism, and biosynthesis of secondary metabolites were higher in uninoculated silage than in inoculated LAB silage (P < 0.05). The metabolic activities of glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, galactose metabolism, the phosphotransferase system (PTS), and the pentose phosphate pathway were lower in silage with corn flour than in silage without corn flour (P < 0.001).Different additive treatments changed the composition of the bacterial community in silage and, accordingly, its functional characteristics. To determine the functional characteristics or metabolic pathways of different bacterial communities, PICRUSt2 was used to predict gene function (based on the third level of the KEGG database). We compared differences in the metabolic pathways between treatments inoculated with and without LAB and those with and without corn flour . MetabolIn this study, RDA was used to evaluate the influence of fermentation characteristics on the composition of bacterial communities . ShorterL. rhamnosus was negatively correlated with pH , and Acinetobacter sp. was positively correlated with AA  and negatively correlated with PA. L. buchneri and L. rhamnosus  were positively correlated with PA. LA and LA/AA were positively correlated with uncultured_bacterium_f_Cyclobacteriaceae (P < 0.01), uncultured_bacterium_g_Woeseia (P < 0.01), uncultured_bacterium_o_Actinomarinales (P < 0.01), L. rhamnosus and L. buchneri (P < 0.05) but negatively correlated with P. agglomerans (P < 0.01). Among all treatments containing 3% corn flour . Moreover, LA was positively correlated with L. buchneri  and Lacticaseibacillus pantheris  but negatively correlated with Acinetobacter haemolyticus, Acinetobacter sp., Acinetobacter indicus, A. faeciporci, and R. pickettii (P < 0.05). AA was negatively correlated with A. indicus. Among all treatments with 6% corn flour (L. pentosus and uncultured_bacterium_c_Subgroup_6 (P < 0.01) and negatively correlated with L. buchneri (P < 0.01). pH was negatively correlated with A. faeciporci, L. brevis, and P. formosensis (P < 0.01). Among all treatments with 9% corn flour , A. faeciporci, A. indicus, and Acinetobacter sp. LA was negatively correlated with L. buchneri and L. rhamnosus and positively correlated with uncultured_bacterum_o_Chloroplast (P < 0.01). LA/AA was negatively correlated with L. rhamnosus (P < 0.001) and positively correlated with uncultured_bacterum_f_Microsillaceae (P < 0.01), uncultured_ bacterum_o_Chloroplast, and uncultured_bacterium_c_ Subgroup_6.The correlations between bacterial species and fermentation characteristics were evaluated using a heatmap . Among arn flour , pH was rn flour , LA and rn flour , pH was 3-N. Moreover, Lactobacillus fermentation in whole-plant paper mulberry silage. L. buchneri, which enabled the conversion of LA to AA and 1,2-propanediol. This phenomenon was also indicated by the abundance structure of the main microbial species L. pentosus, L. rhamnosus, and L. buchneri in whole-plant paper mulberry silage, which is reported for the first time in the present study . L. rhamnosus abundance is strongly correlated with LA and CP levels, indicating that inoculation with L. rhamnosus inhibits the metabolism of deaminated and decarboxylated amino acids by Enterobacter and thus reduces the production of ammonia and biogenic amines, which improves the quality of paper mulberry silage   . Our papL. plantarum in whole corn silage identified some Gram-negative bacteria as key populations, certain bacteria from Proteobacteria  were also identified as key species in LR-treated silage. This suggests that L. plantarum and L. rhamnosus, both homofermentative, may have similar functions during silage fermentation. For example, their bacteriostatic mechanism may be limited to rapid acid production, lowering pH, and inhibiting the growth of acid-sensitive microorganisms, instead of directly producing antibiotics  silage had a more complex structure of microbial associations, and many microorganisms that were not conducive to fermentation  were identified as key species. And the key LAB species L. pantheris was positively correlated with 14 unfavorable microorganisms , and there was also a positive correlation between unfavorable microorganisms . These above represent poor fermentation quality and potential microbial pathogenicity of NA.Numerous studies have shown that there is a complex correlation network in the silage fermentation micro-ecosystem , while sibiotics , so the her LABs . In the In general, most metabolic categories increased over time, which indicates that the metabolic intensity of the microbial community increased as microbial diversity and abundance increased . High abStudies of silage microbial communities commonly use RDA to evaluate the influence of fermentation parameters on bacterial communities . In all Lactobacillus. In silage with 0% corn flour  can be found below: CRW: investigation, conceptualization, methodology, data curation, formal analysis, visualization, and writing — original draft. WTS: investigation, data curation, and formal analysis. YH: investigation and data curation. SD: investigation and software. CP: investigation and methodology. YLZ: resources and investigation. CC: resources and validation. JH: resources, supervision, conceptualization, writing — review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.


A multitude of diseases presenting a wide variety of phenotypic appearances are included in neurodegenerative disorders. They are mainly characterized by the loss of specific populations of neural cells. Moreover, several pathophysiological mechanisms can lead to neuronal dysfunction and death, and thus, to neurodegenerative disorders. The accumulation of pathologic amyloid, dopaminergic neuron loss, excitotoxicity, lysosome dysfunction, mitochondrial dysfunction, defective axonal transport, oxidative stress, and defective microglia function are among them .Both genetic and environmental factors confer susceptibility to neurodegenerative diseases . For insWith the increase in the life expectancy of the population, the incidence of neurodegenerative diseases is constantly rising. Current therapeutic options for neurodegenerative disorders are mainly limited to the management of symptomatology . Therefohttps://www.mdpi.com/journal/neurolint/special_issues/Advances_Neurodegenerative_Diseases (accessed on 17 March 2022) aims to cover the latest advancements in the field of neurodegeneration and neurodegenerative diseases. We invite authors to contribute original studies, reviews, meta-analyses, and related case reports regarding neurodegenerative diseases. All article types are welcome.As such, highlighting the results of ongoing research and collecting the current available evidence are now more important than ever. The present Topical Collection (“Advances in Neurodegenerative Diseases”, available online:


In the Republic of Serbia, pig selection in recent decades has been based on genetic improvement of growth and carcass quality traits. Genetic improvement of reproductive traits of pigs was based on the so-called phenotypic selection. The introduction of modern information systems and the availability larger dataset have opened the possibility to perform genetic estimation of reproductive traits within the main breeding programme of the Republic of Serbia. Using the methods of gene flow and connectedness evaluation, our study investigated the possibility of improving the reliability of estimating the breeding value of reproductive traits in highly productive sows. We believe that these methods could lead to a systematic improvement of the genetic value of reproductive traits in sows. Thus far, none of the methods for estimating the degree of connectedness between herds in pigs has been used in the preparation of the National Breeding Programme of the Republic of Serbia.This study investigated the influence of the degree of connectedness on the reliability of the estimated breeding values (EBVs). The focal trait in the study was the number of piglets born alive (NBA) from sows of the highly prolific Large White and Landrace sows. An analysis included total of 58,043 farrowing’s during the 2008–2020 period. BLUP procedure was used to estimate the breeding values for NBA for the three herds separately and after merging all three herds into one herd. The model for EBV estimation included the following fixed factors: parity, genotype, seasons, litter sire, herds, sow age at farrowing, weaning-conception interval, length of previous lactation, and the following random effects: common litter environment, permanent litter environment, and direct additive genetic effect of animal. Heritability values for NBA ranged from 0.048 to 0.097, depending on the data included in the analysis. The connectedness between herds was analysed using the connectedness rating (CR) and the gene flow (GF) methods. CR among the observed herds ranged from 0.245 to 0.994%, depending on the data included. The exchange of genetic material between all three herds was determined using GF method. The high degree of connectedness determined by the CR and GF method had a strong effect on EBV reliability. The average EBV reliability ranged from 0.520 to 0.867, depending on the data included. The increase in average reliability was observed in both cases when the data were added, both in the analysis of average reliability for purebred animals and when crossbreeds were added, where an increase in this value was also observed. The increase in average EBV reliability is a consequence of the greater amount of information included in the joint evaluation. In conclusion, we believe that our research will improve EBV reliability and help in further selection work in the Republic of Serbia. Litter size traits are relatively easy to measure and monitor under production conditions and are, therefore, suitable for inclusion in selection programmes. Low heritability, negative correlation between direct additive genetic and maternal influence, expression only in females, and a relatively late age at which these traits are expressed in animals are the main reasons for slow improvement of litter size traits in the past. On the other hand, the presence of a relatively large direct additive genetic variance for litter size and the availability of data on numerous relatives allow successful selection for litter size. An important step in this direction is the use of the Linear Mixed Model (LMM) to estimate breeding value in domestic animals. The LMM method is now a standard procedure in pig production to evaluate the parameters of phenotypic and genotypic variability using the Restricted Maximum Likelihood (REML) method and to EBV of animals based on Best Linear Unbiased Prediction (BLUP) ,2.Numerous studies have confirmed that genetic progress in the overall reproductive performance of sows can be most effectively achieved by selection for litter size ,9,10,11.The estimation of breeding values using BLUP method in countries with developed and modern pig production is today a routine method for selecting the best individuals. This method makes it possible to estimate the breeding value of animals and their ranking within and between herds. The reliability of the breeding value estimation of animals from different herds depends on the degree of connectedness between them. The higher the connectedness, the more reliable the comparison of individuals from different herds seems to be. Higher genetic connectedness of the herd results in both more reliable EBV and reduced bias. If herds are insufficiently connected the comparison of breeding values between animals from different herds may be biased. Therefore, measuring the across-herd connectedness is important in order to achieve a more reliable genetic evaluation among pig herds (between a large number of farms/herds) ,17,18,19The effect of connectedness in terms of differences between EBVs between and within herds depends on trait heritability . A heritThe aim of measuring the connectedness between herds is to obtain an indication of accuracy and to reduce bias when estimating breeding value in different herds. Several methods have been developed for this purpose. One of the most adequate methods of measuring connectedness is the prediction error variance (PEV) of the difference in EBVs between individuals in different herds . As thisCR is expressed as a correlation between the assessments of herd effects, and it is strongly associated with PEV ,22. In aThe CR method has been routinely used in national genetic evaluation in Canada, Australia, and many other countries. The advantages of the practical application of the CR method are multiple. They are primarily reflected in the simplicity of calculation . The CR According to a previous practice in the Republic of Serbia, the estimation of breeding value of reproductive traits in sows has been conducted individually per herds. By introducing a unique information system, the possibility of simultaneous genetic evaluation of larger number of herds has been opened. The results obtained in this research may indicate certain steps to be taken in order to increase reliability of estimating breeding value in highly prolific sows in the Republic of Serbia. The model applied and genetic parameters estimated could be used during a practical estimation of breeding value in sows.Data on litter size and pedigree data were provided by the Department of Animal Husbandry, Faculty of Agriculture, University of Novi Sad. The dataset included three farms  over a period of 14 years (2007–2020). The reproductive traits of the four most common breeds on farms in the Republic of Serbia  were analysed. The complete dataset (herd-ABC) contained records of 58,043 farrowings . The NBAThe pedigree file required for this analysis was created for at least three generations of ancestors. The complete pedigree file  for all fijklmnopq—a manifestation of the observed trait of the individual animal in sows’ litters, Fi—fixed effect of parity, Sj—fixed effect of the season of successful mating, presented as combination of the year and month, Gk—fixed effect of the female’s genotype Bl—fixed effect of the litter sire, Hm—fixed effect of the herd , b1i nested within the parity, o—random effect of the common litter environment, pijklmnopq—random effect of the permanent environment, aijklmnopq—direct additive genetic effect of animal, i.e., breeding value, and eijklmnopq—residual.In order to determine the significance of the fixed effects on the dependent variable, data processing was performed using the GLM procedure of the software package AS Inst., Inc., Cary, NC, USA . The sysl, Zp, and Za—event matrices that connect phenotype records with corresponding effects.The model used to estimate the components of variance and breeding value for the NBA can be presented in matrix form as follows:The assumptions are as follows:The extent and direction of gene flow between herds was estimated using the gene flow (GF) method . The conConnectedness rating measure (CR) was used to assess the association between herds . The CR The number of farms, individuals, and fertility data used to calculate CR are shown in 2) was calculated as follows:Reliability of EBV  was used for the estimation of variance components, breeding values, and their reliability in order to allow a proper comparison of the breeding advantages between the farms. The components of variance were first assessed for each farm separately and then together for all three farms. VCE 6  softwareVariance components were estimated both for each individual herd and after merging all three herds into one dataset. First, variance components were estimated for the datasets containing the pure breeds Large White and Landrace . All estUpon adding crossbreed data to the purebred dataset, the variance components for each farm separately and upon merging data into one dataset have been estimated. Measuring the degree of across-herd connectedness is very important for efficient estimation of breeding value between herds. The connectedness between herds  for Landrace and Large White is shown in The level of connectedness between the observed herds was quite high for the Large White breed, ranging from 0.864% to 0.983%. The highest CR value for the Large White breed was 0.983% between herds B and C. The correlation between herds A and C was slightly lower at 0.864 %. A high CR between B and C herds was also found for the Landrace breed (0.895%), while the correlation between A and B flocks or A and C herds the connectedness was lower, ranging from 0.245% to 0.274%.The connectedness upon merging into one database for pure Landrace and Large White as well as the pure breeds and their crossbreeds is shown in Gene flow, i.e., the genetic contribution of animals from one herd to another, is shown in After the addition of crossbreeds, the largest GF observed in the period from 2008 to 2020 was between B and C herds. The share of genes from herd B found in herd C was 0.74%. A large percentage of genes derived from farm B was also found on farm A (0.65%). The lowest number of exchanged animals was between herds A and C (0.09 and 0.02%). The percentage of genes originating from other farms on farm B ranged from 0.02% to 0.10%. Herd C had the lowest genetic contribution to the other herds.Increasing the accuracy of EBV for reproductive traits in sows is important because traits from this group have low heritability and manifest relatively late in females. The reliability of breeding value estimation for sow reproductive traits depends on the amount of information used in the estimation, in addition to the indicated level of connectedness between herds. The average reliability of the EBV when using purebred data is shown in Comparison of the reliability of breeding value obtained by BLUP method for datasets containing pure breeds and crossbreeds is shown in The NBA in litter is affected by a number of factors which must be included into models in order to estimate variance genetic component as accurately as possible. Of all the random effects identified, the additive genetic variance had the highest proportion of total phenotypic variance. The values obtained for the additive genetic variances (0.508 to 0.970) indicate that it is possible to perform successful selection in the studied population based on the NBA. A random effect of the litter in which the sows were born/raised and a permanent environmental effect both contributed to a more accurate estimate of the variance component. The proportion of common litter environment variance being 0.181 in total phenotypic variance of NBA is deemed significant since only 10.634 of total variability can be explained by random effects included into the model in case when all the three farms were simultaneously analysed.Heritability values for NBA ranged from 0.048 to 0.095 when purebred data were used, and when crossbreeds were added, values ranged from 0.077 to 0.097. These values are similar to the results found in many research studies ,33. HeriThe presence of some degree of connectedness between herds is a basic requirement for estimating the breeding value with satisfactory accuracy and reliably comparing animals between herds ,18. The In the results presented, there is a high degree of connectedness between all the herds included in the analysis. This high degree of connectedness is explained by the fact that herd B has an AI centre which provides boar semen to the other two herds. There is also trade in breeding animals between the herds. Such a high connectedness between herds indicates that EBVs can be compared with high reliability between herds, which is also shown in the following studies ,14,15,19The first objective of this study was to determine the connectedness between herds of highly prolific sows and to determine its significance. The CR method of determining connectedness between herds was chosen because studies by many authors have shown that indicating connectedness using this method is less computationally demanding ,15,16. LAfter merging data from pure breeds and crossbreeds into one dataset and analysing the data, an increase in CR was observed between all analysed herds, especially after the addition of crossbreeds, which was not the case in a previous study . This shIn order to determine the direction of gene flow between herds the GF method was used ,21,35. TThis method is suitable when the only purpose is to determine whether there was gene flow between herds. When data from three farms were included in the analysis of genetic variability in a dataset, the model determinant weighed zero and the programme found the dependence of the data in the inverse matrix, so that the parameters could not be calculated because there were no genetic links between the herds . In suchThe highest GF in the herds studied was from herd B towards herds A and C in both sets of data, which is consistent with the results obtained using the CR method. Additionally, the results of the CR method showed the greatest connectedness between these herds. This is explained by the fact that herd B has an already-mentioned AI centre and distributes boar semen to the other herds. The only case where there was no exchange of genetic material was from herd C to herd A for a pure breed dataset. In the opposite direction, a small percentage of genes were found in herd A. Among these herds, there was no exchange of genes for the reason that they are commercial-type farms that primarily produce high-yielding gilts of F1 generation and piglets for fattening. When crossbreeds were added to the dataset for analysis, there was a flow of genes from herd C towards herd A.When selecting animals raised in different herds, the accuracy of the estimation of breeding value can be strongly influenced by the connectedness between the herds. If several herds are genetically evaluated together, the estimated EBVs may be overestimated. If the herds are well connected, the reliability of the EBVs can be expected to be higher in a joint evaluation, as information from one herd contributes to the other ,19,36. TIncreasing the reliability of EBV is very important for the litter traits, because they manifest only in female individuals and have low heritability. Publications ,15,20 stThe results of this study show that the estimation of breeding values between herds that are genetically connected can strongly influence the genetic progress of the population under selection. If genetic association is not considered when estimating breeding values of animals from different herds, it is assumed that the average genetic values of the analysed traits of all the herds are the same. This assumption may have a negative impact on the estimation of breeding values .In addition to across-herd connectedness, an important factor influencing the reliability of the EBVs was the increased number of individuals in the pedigree and phenotype records. Upon merging the herds into a single dataset, there occurred an increase in the average EBV reliability. In both cases, when the average reliability for the pure breed was analysed and after the addition of crossbreeds, this value increased. The increase in the average reliability of EBVs is probably a consequence of the increased volume of information included in the joint evaluation, as reported in the research of many authors ,36,37,38Since the increased amount of data in the breeding value estimation has a positive effect on the reliability of EBV, further research of this type should be conducted on a larger number of farms. The inclusion of a larger number of farms would increase the population on which the selection is carried out. In this way, the response of the selection would be directly influenced, i.e., the NBA would increase. The inclusion of more farms would also allow smaller farms to compare their animals with larger farms ,41. The The heritability values (0.048 to 0.097) obtained for the NBA indicate that successful selection is possible in the investigated pig population of the Republic of Serbia. The sufficient connectedness level and the direct genetic links allow the estimation of the breeding value with high reliability. High values of average reliability from 0.520 to 0.867 in this study confirm this. Reliability increased with the addition of pedigree information and phenotype data. This shows that an increase in EBV reliability is possible by including additional data. High values for EBV reliability are associated with the large amount of data and good connectedness between farms. High reliability has an important role, especially in the selection for traits with low heritability. The high values of CR between the analysed herds in this study suggest that the use of artificial insemination in the selected population may indirectly increase EBV reliability. In addition to the increased reliability of EBVs, high CR values indirectly affect the positive selection response. This is important because the effects of selection are directly related to the accuracy of breeding value estimation. It should be noted out that besides the lack of systematic selection an average NBA in the analysed population is not low. For this reason, we should be cautious when making recommendations regarding future pig breeding decisions. We believe that our study can contribute to a better understanding of breeding value estimates in highly productive sows in the Republic of Serbia. Furthermore, the results obtained could have an impact on the future direction of pig breeding.


Amorphophallus konjac (AK) flour in different concentrations. This experimental study is divided into three steps: preparation of the gluten-free bread formulations, sensory analysis, and statistical analysis. The addition of Konjac flour in a gluten-free bread formulation was tested in different proportions, 12.5%, 25%, 37.5%, and 50% of the flour content. The checking all-that-apply (CATA) was conducted with 110 panelists; among these, 43 were consumers of gluten-free bread. Sensory analysis was conducted using a 9-point hedonic scale for color, aroma, texture, flavor, appearance, and overall acceptability. The AK flour influenced the sensory characteristics of gluten-free bread. Bread with characteristics closer to those found in bread with gluten was the one with 12.5% of konjac flour for both the acceptability analysis as the attributes raised through a detailed CATA map. The control sample is located next to features like dry appearance, dry texture and grainy, dark color, and salty. Therefore, 12.5% AK gluten-free bread is closer to the characteristics of the control sample, such as light crust color, light crumb color, soft and moist texture, cohesion, and brightness. The bread with the highest percentage of overall consumer acceptance was 12.5% konjac with 93% and 96% acceptance among consumers and non-consumers of gluten-free bread, respectively.This study aimed to evaluate the sensory profile of gluten-free bread with  Bread is a worldwide staple food, present in many civilizations since most antique eras, and is still largely consumed today. Its consumption revolves around 24 kg per capita/year worldwide, with evident growth trends for consumption and market value . A similGluten-free bread tends to present reduced volume and alveoli structure, brittle texture, light crust color, and usually, loaves present heavy, tenacious, and elastic mouthfeel ,7. IngreAmorphophallus konjac (AK) is a mountain-based plant native to southern Asia and Africa, with numerous corms from which a water-soluble, non-cellulosic polysaccharide of high molecular weight is extracted [Among the studied alternatives, xtracted . Glucomaxtracted ,11. AK’sxtracted . Becausextracted ,14,15.Amorphophallus konjac flour in different concentrations.Laignier et al.  tested AThis exploratory and quantitative study was performed in two steps: (I) Sample preparation, and (II) Sensory Analysis. The Check-all-that-apply test (CATA) was used since it is a rapid descriptive method that serves as a simple and effective alternative to screen the sensory attributes and drivers of liking various products . In addiThe samples were developed at the University of Brasilia, Federal District, Brazil, according to the formulations developed by Laignier et al. . The con®, Parsippany, NJ, USA), and the yeast was pre-activated with sucrose and warm water (38 °C) for 10 min. Rice flour, potato starch, konjac flour (in modified samples), and salt were mixed in another recipient. Afterwards, eggs, water, and oil were mixed with the dry ingredients. The activated yeast was added, and the dough was kneaded and rested for 50 min (27 °C). Afterwards, the dough went through a second kneading and modeling (40 g spheres). Finally, the samples were baked for 40 min. in a preheated (180 °C) gas oven Brastemp®-Sau Paulo, Brazil [The water content was adjusted due to the AK flour’s capacity to absorb more water than the gluten-free flour used in control bread. Therefore, the water was used in 131%, 228%, 297%, and 406%, based on the weight of the flour basis. The formulations can be accessed in , Brazil .After baking, the samples were removed from the trays and cooled down in polyethylene containers. Bread samples were codified with 3-digit random numbers . SamplesFirst, the sensory descriptors for CATA were elaborated by applying the Kelly’s Repertory Grid Method  with fivBread samples were also submitted to an evaluation using a 9-point hedonic scale  to measure the acceptance for appearance, flavor, texture, aroma, and global acceptance ,21. BothBoth CATA descriptors and samples’ order were presented in a randomized and balanced order in the sensory forms. Samples were presented monadically. The panelists answered the forms according to their perception of the suggested descriptors’ presence and the attributes’ intensity.110 untrained consumers participated for both simultaneous tests, and 43 (39%) were regular gluten-free bread consumers. Of the 110 participants, 68.18% were female, and 31.82% were male, from 18 and 59 years. Participants were recruited through invitations posted on social media. The individuals who arrived at the laboratory through the invitation to perform the sensory test and met the inclusion criteria were included in the sample without gender balance.n = 110) and another with only regular consumers of gluten-free bread . The acceptance percentage was obtained from the scores given by the panelists in the acceptance test. A product was considered accepted when it obtained at least 70% approval  [Two groups were formed to overview the differences between the perception of all consumers—one with all the consumers  .p < 0.05) was performed to compare the attributes evaluated in the hedonic scale acceptance test. SPSS  was used in this step.A two-way ANOVA followed by Tukey’s test  to assess whether there were significant differences in consumers’ perception of any given attribute among the different samples. Then, a multiple pairwise comparison test was performed using Bonferroni (McNemar) procedure with 5% significance. A test of independence between rows and columns was carried out with 5% of significance. Additionally, correspondence analysis (CA) based on chi-squared distances was performed to summarize a sensory map of samples.For the CATA analysis, descriptors were initially compared with non-parametric Cochran’s Q test (n = 110) with a hedonic scale. Gluten-free bread that used 12.5% AK flour replacing part of the gluten-free flour presented the highest mean for appearance, texture, and global acceptance. Regarding flavor and aroma, this sample (12.5%) and the control also presented the highest means.n = 20) were perceived as different among all samples by the consumers of Group I   of the descriptors were perceived as different by members of Group II   presented the highest frequency of the term “Dry Crust Appearance”, while in Group II, the same was also true, while all the remaining samples did not present differences between them.“Light crust color” was more frequent in sample 25, in group I, and equal in frequency in samples 25 and 37.5 in group II; however, differences between all samples were present in both groups. The term “Dark Crumb Color” was most frequent in sample 50; however, in both groups, sample 25 was not significantly different for the same term, although presenting a lower absolute frequency proportionally. The control sample presented a higher frequency for the term “Pretty” in all groups, though, in group I, sample 12.5 did not show significant differences for the same term.Regarding the aroma of bread, in both groups, “Pleasant scent” presented the highest frequency among all samples, given that sample 12.5 presented the highest value for this descriptor. However, no differences were seen between samples 12.5 and control in group I, while in group II, sample 12.5 was different from all the remaining others. As for the descriptor “Seafood scent,” sample 12.5 was the only significantly different from the others in both groups.“Pleasant flavor” and “Unpleasant flavor” presented similar distributions in both groups. Samples 12.5 and control did not statistically differ, while samples 25, 37.5, and 50 were in the same group, with a “Seafood flavor” also highlighted as a frequent descriptor. Samples 12.5 and control presented no differences while the other samples were in the same group. In group I, “Yeast flavor” was a frequent descriptor, with significant differences between samples. In group II, no differences for the same descriptor were found.The control sample presented the highest frequency for “Sweet flavor” in groups I and II. In group I, no difference was found between control and 12.5%. In group II, differences were present only between control and 25%. The highest frequencies for “Savory flavor” were in the control sample in both groups. Differences were found only between control and 50%.Regarding texture-related descriptors, control presented the highest frequencies for “Dry”, “Grainy”, “Stiff”, “Compact”, and “Buttery” in both groups I and II. The term “Moist” was most frequent in samples 12.5% and 37.5% in group I and 37.5% and 50% in group II. The control sample presented the lowest frequency for this descriptor in both groups.In group I, “Stiff” was most frequent in the control sample, presenting differences compared to all remaining samples. In group II, sample 25% was statistically equal, although showing a much lower frequency. “Rubbery” was present in samples 25%, 37.5%, and 50%, with no significant differences between these samples in both groups.“Soft” was frequent in sample 12.5%, presenting differences in all remaining samples in groups I and II. The remaining samples did not present differences between them for the same descriptor. In groups I and II, the descriptor “Rubbery” did not present differences regarding control and 12.5%, while samples 25%, 37.5%, and 50% were different from these other two and statistically the same.A heterogeneous distribution was evidenced for “Grainy”. The control sample presented the highest frequency for this term in both groups. However, in group I, control and 50% did not present differences, while in group II, control differed only from the sample 12.5%. The descriptor “Dry” was most frequent in control, differing from all remaining samples in all groups.Correspondence analysis was carried out to generate a sensory map of samples and their relation with attributes. Groups 1 and 2 Principal Coordinate Analysis (PCA) and Symmetric Plot are in Amorphophallus konjac flour in higher proportions (12.5–50%) of gluten-free flour, which makes it difficult to compare the results with other studies. Amorphophallus konjac flour in proportions of 12.5%, 25%, 37.5%, and 50% of the gluten-free flour content was previously tested in gluten-free bread, but only the nutritional and physicochemical properties were evaluated [Hydrocolloids like AK flour in gluten-free bread are used to mimic viscoelastic and crumb texture performed by gluten to improve sensory characteristics. Our study first evaluated the sensory profile of gluten-free bread using valuated .A study evaluated the acceptance of gluten-free bread with AK glucomannan but used in lower amounts . The autRegarding flavor, bread with percentages above 25% presented lower acceptance means than control and 12.5%. Therefore, the addition of konjac flour negatively influenced the flavor of bread above 12.5%. This could be explained by the water absorption capacity of AK flour. The final volume of the dough increases because of the need for water addition. It decreases the concentration of ingredients that provide greater palatability to food, such as sugar, fat, and salt . A potenThere was no difference in aroma acceptance between the control bread and the 12.5% in this study. However, there is a difference between the control bread and the bread with AK flour percentages above 37.5%, indicating that the higher the percentage of AK flour less accepted the aroma of the gluten-free bread. This was probably due to the characteristic odor of the AK flour or the lower concentration of sugars and proteins, which prevented chemical reactions that produce aroma, such as the Maillard reaction and caramelization, from adequately occurring. Regarding the aroma of konjac bread, some comments could be extracted from the evaluation forms filled in by the evaluators, “Despite the smell of fish, it has a normal bread texture”; “although there is a smell of seafood, the taste is good”; “Taste good, but bad aroma”. This was confirmed by the CATA test, in which “Seafood Scent” was most frequent among samples with 25% to 50% of AK flour addition. Considering CATA, the control bread had the lowest score for seafood aroma, and in the AK flour bread, more AK flour led to more consumers perceiving this odor. The 50% bread was most punctuated for seafood aroma, and, in the sensory analysis, 50% AK flour bread had the lowest average for aroma. This can be explained by the characteristic odor of konjac flour that resembles a fish [The bread samples were considered statistically equal in texture, except for the 12.5% bread which presented the highest acceptance rate. This bread sample was the one that presented the highest instrumental hardness in a previous study ; howeverThe sample with 12.5% of AK flour obtained a percentage of global acceptance among evaluators who habitually consume gluten-free bread of 93.03% and among non-consumers of 95.52%. This result was the opposite of that obtained by another study . Gluten-Considering the CATA test, the data show that the dark color crust obtained a significantly higher frequency for the control bread, reflecting the same result obtained in the instrumental analysis in a previous study, indicating that this was the darkest crust color bread . The posThe term dry appearance was considerably more frequent for the control bread, which was statistically different from the others. The least pointed to this term was 12.5% bread, which was not expected since our previous study showed that, among AK flour samples, the 12.5% presented the lowest moisture content . HoweverFor light crust color, 25% AK flour bread was more frequent. The highest frequency was found in 12.5% AK flour bread for light crumb color, and dark crumb color was more observed in 50% bread. In our previous instrumental evaluation , the 50%n = 110) (n = 110) . ConsideA previous study  on AK flThis research presents, as a limitation, the absence of the descriptive analysis (DA), a more robust and reliable evaluation tool when precise definitions and quantification of the sensory attributes of products are required ,30. HoweAmorphophallus konjac flour interfered with the sensory characteristics of gluten-free bread. Adding up to 12.5% of AK flour  was feasible on the gluten-free bread formulation used in this study. The most desirable attributes in the studied bread were aroma, pleasant taste, beautiful appearance, and clear crumb, found in gluten-free bread with 12.5% AK flour. In this way, the study demonstrates that it is feasible to use AK flour in higher concentrations than being used as an additive, up to 12.5%, helping to improve sensory characteristics of gluten-free products. Further studies should be conducted to evaluate the glycemic index of these gluten-free bread, determine the shelf life, and determine the purchase intention of the products so that they can go to the consumer market.