My approach tremendously simplifies the calculation of the affectional status distribution compared to the conventional method, which requires the parental mating information, and could be applied to disease with both dichotomous trait and quantitative trait locus (QTL). This distribution is shown to be independent of relative relationship and be employed to develop the marker IBD distributions for relative relationship. In addition, three linkage tests: the proportion, the mean test, and the LOD score test are proposed for different relative pairs based on their marker IBD distributions. Among all three tests, the mean test for sib pair requires the least sample size, thus, has the highest power. Finally, I evaluate the significance of different relative relationships by a Monte-Carlo simulation approach.I introduce a novel approach to derive the distribution of disease affectional status given alleles  Penrose first considered the covariance of the quantitative sib pair trait phenotype and genetic marker in the linkage analysis  affectional status and (marker) alleles  linkage . The proz et al. , 9. The bilities , 11. Thebilities . Anotheries  distributions at marker locus given disease affectional status for siblings, uncle–nephew, grandparent–grandchild, half sibs, and first cousin pairs. By taking advantage of the ITO matrices, I first demonstrate that the probabilities of dichotomous disease status given trait IBD score are independent of relative relationships. Then, I fully derive the marker IBD distributions given dichotomous disease affectional status for various relative relationships by utilizing the relative pairs’ joint probabilities of IBD scores at both trait and marker loci. I also calculate the marker IBD distributions given extreme discordant relative pairs at a quantitative trait locus (QTL) for different relative relationships by my novel ITO method. Next, I examine the power to detect the presence of a significant disease susceptibility locus through linkage analysis by perturbing the conditional marker IBD distribution. Specifically, three tests, the proportion test, the mean test, and the logarithm of odds (LOD) score test, were applied to obtain the sample size required to achieve significance level p with different power. Finally, the Monte-Carlo simulation studies have been conducted in order to evaluate the performance of my methods. I assume Hardy–Weinberg equilibrium, random mating and the marker locus to be completely polymorphic such that all matings are informative.In this research, I adopt a novel 2T/t) at the trait locus are linked to alleles (M/m) at a marker locus through recombination fraction θ and assume that the marker locus is completely polymorphic. Additionally, the diallelic frequencies are p and q for alleles T and t, where p + q = 1. I denote penetrance frequencies, i.e., the probability of the affected relative given genotypes TT, Tt, or tt by f1, f2, or f3, respectively. The prevalence of the trait in the population is defined as PK = p2f1 + 2pq f2 + q2f3, in addition to the additive variance (AV = 2pq[p(f2 − f1) + q(f3 − f2)]2) and dominance variance (DV = p2q2(f1 − 2f2 + f3)2). I assume no major gene by residual interaction and no epistasis, i.e., the non-allelic interaction of different genes.Let us consider the situation where alleles  given IBD score at trait locus for generalized relative pairs, I reckon the genotype information of relative pairs derived from the ITO matrices, as shown in Table Let k k = 0, , 2 givenTIBD  probabilities has been partitioned on all possible genotypes of relative pairs, i, iGT = 1, 2, …, 9: TT − TT, TT − Tt, TT − tt, Tt − TT, Tt − Tt, Tt − tt, tt − TT, tt − Tt, and tt − tt, as shown in equation  as in Table 1 of Suarez was reproduced here in Table ITO method  , for sib pair, grandparent–grandchild, uncle–nephew, half sib, and first cousin are given in Table Pr(X) = ΣtPr(X |TIBD = t)⋅Pr(TIBD = t), I give the marginal affected status probabilities for different relative relationships from a randomly mating population in Table The probabilities of IBD scores at the marker and trait loci be denoted by MIBD and TIBD, respectively. The joint probabilities for a relative pair to have IBD scores at the marker locus M and the number of affected relative pair is calculated as equation (r (relationship) refers s (sib), g (grandparent–grandchild), u (uncle–nephew), h , and f (first cousin). One notices that conditional probabilities Pr(X = x|TIBD = t) are independent of the relative relationships. Hence, the differences among relative relationships of the joint probabilities Pr are due to the contribution of Pr. Combining Pr [see Table θ is the recombination fraction between the trait and marker loci, parameter ψ defines θ2 + (1 − θ)2.Let the equation .3)Pr(IPr(IIBD s2.2QTL)  with gene frequencies p and q, respectively  for the population distribution of the trait is a mixture of three normal distributions:ϕ(s) is the standard normal density function. Next, the probability of one relative’s phenotype falls in the top decile and the other relative’s in the bottom decile given their trait genotypes, Pr(T1B1|iGT) , is given asϕ is the bivariate normal density function, G1, G2 take 1, 0, or −1 as their genotypes are TT, Tt, or tt, respectively. Thus, the probabilities of the general extreme discordant relative pair given allele IBD sharing at trait locus is obtained through ITO method:Pr(T1B1|iGT) is integrated according to equation (Pr(iGT|TIBD = t) are the ITO matrices given in Table QTL given TIBD are partitioned over their genotypes through the ITO approach. Similar to the discrete case, Pr(T1B1|TIBD = t) is also independent to the relative relationships. If one regards the extreme discordant relative pair with QTL as the continuous case for X = 1, then the probabilities of Pr(T1B1) and Pr(MIBD|T1B1) could be derived in a similar fashion as in the discrete case.Risch and Zhang have shown that sib pairs from opposite tails of the phenotypic distribution have substantial power to detect linkage for a quantitative trait locus (QTL) , 22. Assectively . Let a bgiven asPrT1B1|GB1|GQTL (3T/t) and marker (M/m) loci decreases. Here, I refer the perturbation as the absolute deviation of the conditional probabilities in Table i.e., PK value as 10% and focus on doubly affected relative pairs. In order to compare the test power between the full sib pair and other relative relationships, I let DV = 0.01 such that the perturbation of sib pairs is increasing as AV increases  as the counts of doubly affected relative pairs with the dichotomous trait or extreme discordant relative pairs with QTL, which share j marker allele(s) IBD among total N relative pairs sampled. The Wald test statistic isjN is approximately normally distributed withIBD probabilities of relative relationship found in Table I define IBD perturbations are monotonic based on the parameters chosen, the proportion tests are one-sided: rW > αZ for doubly affected relative pairs and rW < −αZ for relative pairs with QTL. The required sample size rN for this test to have the power of 1 − β is Nr=±3.2jN  is the following:Following previous notation, the kernel of the likelihood of θ any more, but jN, the count of relative pairs sharing j allele(s) IBD. With T for the likelihood ratio test based on equation , and assuming level-α test with 1 −β power, I obtain {j, NN} for each relative relationship as the critical size of relative pairs sharing allele IBD and total required sample size, respectively. One can check easily that T is an increasing function of jN when Ns are fixed. In other words, for an each N, I reject the null hypothesis if the counts of allele IBD are greater than jN. Usually, the LOD score test use more strict criterion than the proportion test does. Here, the total required sample size N of the 90% power, level α = 0.001 LOD score test power is plotted as a function of the recombination fraction θ for both doubly affected relative pairs with the dichotomous trait and extreme discordant relative pairs with QTL  IBD, I weight N1 with Since Ws−m is approximately normally distributed withIBD probabilities for sib pair sharing two or one allele(s) IBD, respectively. For sib pair, one expects the increased allele sharing under the alternative hypothesis, the level-α one-sided mean test is: s–mW > Zα for doubly affected sib pairs with the dichotomous trait and s–mW < −Zα for extreme discordant sib pairs with QTL. Following similar procedure as the proportion test, I obtain the required sib pair sample size s–mN for level-α mean test with power 1−β Ns−N of doubly affected sib pair with the dichotomous trait for all three test at α level of 0.05 with 90% power: the proportion test (solid line), the mean test (dotted line), and the LOD score test (medium dash). The mean test for sib pair requires the least sample size than other two. For example, the required sample sizes are {157, 128, 176} for the proportion, mean and LOD score tests, respectively, when θ = 0.1. Here, the mean test demonstrates the largest test power among all three tests  perturbation is closely related to the power to linkage tests. When AV = 0, the non-zero perturbation of full sib pair is due to the DV term. However, there are no DV term in the perturbations of other relative relationships, i.e., the perturbations are always zero, whenever AV hits zero for relative relationship, grandparent–grandchild, uncle–nephew, half sibs, and first cousin. Among all the relative relationships, only the perturbation of grandparent–grandchild shows linear dependence upon the recombination fraction, θ, while the remaining perturbations are higher order polynomial functions of θ. One also notices that the condition of θ = 0 and N = 211 at θ = 0  process to generate the counts of crossovers and their locations along a chromosome  IBD. The empirical type I error rates generated by both programs are consistent with the nominal α levels (results not shown). However, neither SLINK nor SIMULATE could track allele segregation unambiguously under the alternative hypothesis. Therefore, I constructed Monte-Carlo simulation directly from Table There exist programs that could evaluate the type I error rate of the three statistical tests under the null hypothesis of no linkage. The marker genotypes of each relative pair are independently generated by either enotypes , 24, whiromosome . Once thIBD in affected relative pairs to detect linkage, informativeness of the marker IBD determines the accuracy of linkage analysis. A marker is highly informative for linkage studies, if any individual chosen at random is likely to be heterozygous for that marker. Nonetheless, in almost all applications, the biallelic IBD value can not be determined unambiguously, but has to be estimated. Previous work has been shown that increased information of allele shared IBD of sib pair can be achieved by analyzing two or more linked marker loci simultaneously  and IBD missing at random (MAR) into the test statistic which has equal power as those in Kong and Cox  linkage analysis and advantageous over traditional model based methods. Thus, this method does not require specification of the disease model and could be readily applied to either early- or late-onset disease. In practice, samples collected for affected relative pair will likely contain three or more affected relatives, such as siblings, grandparent–grandchild, uncle–nephew, half sib, or first cousin. However, most commonly used methods, restrict the linkage analysis to sib pair only. Thus, a large amount of information contained in the data is discarded. The simple way to achieve larger power is to include all available affected individuals from each relative type. Since the possible selected pairs are no longer independent, several weighting schemes were applied to sib pair , 31. TheQZ contributed to the research topic, derived model formulation, carried out the numerical simulation, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


B-cell adaptor for phosphoinositide 3-kinase (BCAP). BCAP has a sequence previously proposed as a “cryptic” TIR domain. Here, we present the structure of the N-terminal region of human BCAP and show that it possesses a canonical TIR fold. Dimeric BCAP associates with the TIR domains of TLR2/4 and MAL/TIRAP, suggesting that it is recruited to the TLR signalosome by multitypic TIR-TIR interactions. BCAP also interacts with the p85 subunit of PI3K and phospholipase Cγ, enzymes that deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), and these interactions provide a molecular explanation for BCAP-mediated down-regulation of inflammatory signaling.Ligand binding to Toll-like receptors (TLRs) results in dimerization of their cytosolic Toll/interleukin-1 receptor (TIR) domains and recruitment of post-receptor signal transducers into a complex signalosome. TLR activation leads to the production of transcription factors and pro-inflammatory molecules and the activation of phosphoinositide 3-kinases (PI3K) in a process that requires the multimodular  Phosphoinositide 3-kinases (PI3Ks) are crucial mediators of various cellular processes including growth, survival, proliferation, migration, metabolism, and membrane transport. Dysfunction in the PI3K signaling network is a major driver of oncogenesis –4. The w–Drosophila protein Dof, where it is required for FGF-dependent signaling ,ignaling , 8, and ignaling .L, and the second largest splice variant is designated BCAPS.The expression and cellular distribution of BCAP suggest that it is involved in immunological processes in addition to regulation of BCR , 11. In L is recruited into activated complexes of Toll-like receptors (TLRs) and associated signaling adaptor proteins. TLRs are pattern recognition receptors that respond to microbial products such as lipopolysaccharide from Gram-negative bacteria. The TLRs and the signaling adaptors MyD88 and MAL/TIRAP contain TIR domains, and these interact via homo- or heterotypic interactions to form complexes that constitute the post-receptor signalosome. A recent study predicted the existence of a TIR domain in the N-terminal region of BCAPL that is required for negative regulation of inflammatory signaling by TLRs . The TIR domain of BCAP is composed of five parallel β-strands (βA–βE) surrounded by five α-helices(αA–αE). The third α-helix has a kink and forms two helices, αC and αC′, and the fourth is distorted. The loop regions, with the exception of the biologically important BB-loop, which connects the βB-strand to the αB-helix, were well defined in the electron density, and unlike the structure of the MAL-TIR domain , although the sequence identity between the TLR2-TIR domain and BCAP-TIR is only 12%. Other close homologues include the TIR domain of the interleukin-1 receptor, IL-1RAPL , Leu-32 (αA), Pro-49 (βB), Leu-101, and Leu-102 (βD) . The electrostatic potential of the solvent-accessible surface of BCAP (E and F) reveals distinct regions of negative and positive potential in addition to hydrophobic patches, suggesting that the TIR domain of BCAP may interact with diverse TIR domain-containing proteins. For example, the TIR domain of MAL/TIRAP is predominantly hydrophobic but has acidic and small basic patches  suggest that Pro-49 is not required for the interaction. Both the D75A and L401A mutations exhibited reduced binding, whereas the D117A mutation abolishes the interaction between BCAP and MAL/TIRAP. Asp-117 is located in a region of acidic surface potential sandwiched between the αD-helix and βE-sheet, and it is likely that this region interacts with a basic patch on MAL/TIRAP. The reduced association observed with the L401A mutant suggests that the determinants of the BCAP-MAL/TIRAP interaction may not solely reside within the TIR domain, and it is likely that there are other regions involved in the BCAP-MAL/TIRAP hetero-association.Given the necessity of TIR-TIR interactions to signal propagation, several mutations were made to explore possible regions on BCAP that may be involved in its association with MAL/TIRAP. Sequence alignments of BCAP and proteins known to interact with MAL/TIRAP  revealed three conserved residues . Two of these (Leu-70 and Asp-117) lie within the TIR domain, and all were mutated to alanine. Additionally, the highly conserved βB loop proline residue (Pro-49), which directs adaptor specificity in TLRs , was mutL and an N-terminal region spanning residues 7–142, which contains the TIR domain. The sedimentation velocity data, the residuals after fitting, and the c(S) distribution are shown in −13s and 1.82 × 10−13s for BCAPL and the N-terminal domain, respectively. The qualities of the fits were very good with root mean square deviation values of 0.007 (BCAPL) and 0.005 . Additionally, the frictional coefficient was well determined, and this allows estimation of molecular masses from the sedimentation velocity data. Using c(M) analyses, the molecular masses are 165 and 18 kDa for BCAPL and the TIR domain, respectively. These values are within ∼10% of those expected for dimeric BCAPL and monomeric N-terminal domain on the basis of their amino acid sequence. Weight-averaged molecular masses obtained from an analysis of the SEC-MALS  data are consistent with those obtained by AUC.Analytical ultracentrifugation (AUC) experiments were performed on purified samples of full-length BCAPL self-association lie outside this region. To clearly define the regions responsible for BCAPL oligomerization, the splice variant (BCAPS) and a truncated molecule comprising the first 330 residues of BCAPL (BCAP330), which contains the TIR and DBB domains, were expressed, and their hydrodynamic behavior was studied by sedimentation velocity. L, are dimeric. SEC-MALS data for BCAP330  also confirm that it is dimeric. Because BCAP330 contains the DBB domain, this confirms that it is, at least in part, responsible for the self-association of BCAPL. The ankyrin repeats are also known to facilitate oligomerization  but interacted with FLAG-SARM (B) and also with Myc-MyD88 and the Myc-MyD88-FXXM mutant (C).TIR domains associate via homotypic interactions, and the presence of an N-terminal TIR domain in BCAP could potentially enable binding with the cytosolic adaptor proteins involved in the TLR pathway. To determine whether BCAPMyc-TRAM A but intLAG-SARM B and alsM mutant C.XXM motifs present in the full-length protein, and the TIR, DBB, and ANK domains. BCAP-TDA was tested against full-length constructs of MAL/TIRAP and its mutants D96N and S180L, the TIR domains of TLR2 and TLR4, and also the regulatory p85 subunit of PI3K. In these experiments, all proteins tested interacted with BCAP-TDA . Interestingly, the disease-associated MAL/TIRAP variants S180L . This construct includes the dimerization site, two of the four Yts S180L  and D96Nts S180L  also bints S180L .L is involved in the BCR-mediated activation of PLCγ2 and PI3K  were carried out in the presence or absence of either human YFP-tagged Syk or GFP-tagged Lyn. The results presented in Fig. 5A show that FLAG-BCAPL associates with PLCγ2-V5 in the presence and absence of either YFP-Syk or GFP-Lyn. Moreover, the addition of Syk or Lyn did not increase the apparent binding of BCAPL to PLCγ2, suggesting that the interaction between BCAP and PLCγ2 may be promoted by endogenous tyrosine kinases in these experiments. However, it is apparent from the presence of a higher molecular mass band of BCAP that it is phosphorylated by the transfected Lyn kinase. BCAP was also able to pull down both Lyn and Syk in the absence and presence of PLCγ2 . Consequently, BCAP is phosphorylated by Lyn and forms a complex containing either Syk or Lyn kinases and PLCγ2 irrespective of its activation by kinase.To address these questions, pulldown assays using BCAPB).We also investigated the ability of PLCγ2 to interact with the TIR domain-containing proteins SARM, MAL/TIRAP, and MyD88. Both MyD88 and MAL/TIRAP were able to interact with PLCγ2. In contrast, SARM appears not to associate directly with PLCγ2 B.Information flow through the TLR pathway is initiated by ligand binding to leucine-rich repeats on the receptor ectodomain followed by receptor dimerization. Subsequently, homotypic associations of cytoplasmic receptor TIR domains and those of various adaptor molecules including MyD88, TRIF, MAL/TIRAP, TRAM, and SARM ensue, setting in motion a cascade of molecular interactions that result in many cellular responses including the production of pro-inflammatory molecules . Until rL paralogue, BANK1, which regulates CpG-induced IL-6 production  and negative regulation of MAL/TIRAP-dependent TLR signaling.Together our findings provide evidence that BCAP is part of a membrane-bound network that down-regulates inflammatory signaling by the TLRs. Thus, activation of PI3K and PLCγ2 results in the conversion of PIP2 to PIP3 and inositol 1,4,5-triphosphate (IP3), respectively, leading to depletion of MAL/TIRAP from the membrane B and negThe association between BCAP and PLCγ2 appears to be more stable than the transient interaction observed between BANK1 and PLCγ2  and may 39–et al. . After nickel-nitrilotriacetic acid purification in 20 mm Tris, 20 mm NaCl, pH 7.5, samples were treated with tobacco etch virus (TEV) protease to remove the hexahistidine tag, and further purification was performed using S200  size exclusion chromatography in the same buffer supplemented with 5 mm DTT. BCAPL (1–805), BCAPS, and BCAP330 were cloned into the same vector with a Strep-tag at the C terminus. Proteins were purified using a 5-ml StrepTrap column , and proteins were eluted using 2.5 mm desthiobiotin (Sigma) dissolved in the same buffer with final purification on S200 size exclusion as described above.The N-terminal domain of human BCAP comprising residues 7–142 was cloned into pMCSG7 (DNASU) and expressed as an N-terminal hexahistidine-tagged fusion protein in + and Optilab T-rEX instruments, respectively (Wyatt Technology), and data analysis was performed using the ASTRA (v6.1) software package from the manufacturer. Protein concentrations were 4, 2, and 0.8 mg/ml for the TIR, BCAP330, and BCAPL, respectively.Sedimentation velocity measurements were performed using a Beckman XL-A ultracentrifuge equipped with absorbance and interference optical systems. Sample concentrations ranged from 0.3 to 1 mg/ml. Data were acquired every 300 s at rotor speeds of 30,000–40,000 rpm using an An60Ti rotor (Beckman Coulter). Buffer density and viscosity were estimated using SEDNTERP, and the experimental data were analyzed using SEDFIT . SEC-MALm HEPES, 100 mm NaCl, 2 mm DTT, pH 7.5. Following lysine dimethylation 2SO4, and 1 mm 5-amino-2,4,6-triiodoisophthalic acid monohydrate (I3C)  into 50 mhylation , sampleste (I3C) .Diffraction data were collected on the IO3 beam-line . Data reduction and scaling were performed using XDS  and AimlThe antibodies used were mouse α-Myc (Santa Cruz Biotechnology), mouse monoclonal α-FLAG M2 (Sigma-Aldrich), rabbit α-FLAG (Sigma-Aldrich), and α-V5-HRP conjugate (Invitrogen).m 3-amino-1,2,4-triazole (3-AT) plates for assessing the interaction. Negative controls were the empty vectors of pGBKT7 and pGADT7 treated similarly.The bait plasmid for yeast two-hybrid assay was generated by inserting the first 434 amino acids of human BCAP (Source Bioscience) in-frame into the pGBKT7 vector (Clontech) to be expressed as a GAL4-DBD fusion protein. The prey plasmids were generated by inserting either the full-length proteins or different protein mutations and truncations in-frame into the pGADT7 vector (Clontech) to be expressed as GAL4-AD fusion proteins. The TIR domain of TLR2 contained residues 636–784, whereas the TIR domain of TLR4 comprised residues 671–820. Protein-protein interactions were assayed by transforming the bait plasmid into the yeast strain AH109 (Clontech) and the prey plasmids into the yeast strain Y187 (Clontech) using standard lithium acetate transformation  with them Tris, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, pH 8.0, which was supplemented with 1% Triton X-100, 0.5% n-octylglucoside, 1 mm PMSF, and 1× protease inhibitor cocktail . After incubation on ice for 10 min, the mixture was transferred to an Eppendorf tube, and lysis was continued for an additional 30 min at 4 °C with agitation, after which the mixture was centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was collected for analysis by Western blotting or for immunoprecipitation with anti-FLAG M2 beads . 250 μl of the extracted protein sample was incubated with anti-FLAG beads for 2 h at 4 °C with constant rotation, after which non-specifically bound proteins were removed by repeated centrifugation at 1000 × g and resuspension of the beads with 500 μl of lysis buffer. After the final wash, excess solution was aspirated, and 30 μl of 1× SDS loading buffer was added to the beads and heated at 100 °C for 10 min. Finally, the protein samples from whole cell lysate and immunoprecipitation were analyzed by Western blotting using the indicated antibodies.Human BCAP and splice variant was cloned into either p3XFLAG-CMV-10 or pCMV/Myc vectors. Protein constructs were transfected into HEK 293T cells  (70–80% confluence) using JetPEI (Polyplus-transfection SA) according to the manufacturer's recommendations. 3 μg of plasmid DNA was transfected per well in a 6-well plate. When required, pcDNA3.1 was included to ensure that the total DNA transfected was 3 μg. The transfection mixture was incubated for 24 h, after which cells were washed once in 1× PBS and lysed using 300 μl of a solution containing 20 msupplemental Fig. 3. M. C. M. wrote most of the paper and performed AUC analysis and X-ray structure determination and SEC-MALS. N. J. G. initially conceived the project and helped with writing the paper.S. H. produced proteins, conducted Y2H and pulldown experiments and assisted with the AUC and crystallographic analysis and writing. E. S. and B. V. supplied the data for


The effect of supplementation on the mineral contents of biscuits was also significant (p < 0.05). Potassium, magnesium, calcium, iron and zinc contents increased from 105.30, 14.65, 43.91, 3.74 and 0.94 to 145.00, 26.64, 79.60, 7.93 and 1.60 mg/100 g, respectively. Sensory evaluation revealed that the quality score of biscuits in terms of color, taste, texture and overall acceptability decreased with increased supplementation. The present research work confirmed that a maximum of 10% alfalfa seed flour supplementation in refined wheat flour could produce acceptable biscuits with an appropriate nutritional profile.The effect of alfalfa seed flour supplementation on the quality characteristics of refined wheat flour-based biscuits was studied. The proximate composition of refined wheat flour and alfalfa seed flour was determined. Refined wheat flour contained 12.43% moisture, 11.52% crude protein, 1.61% crude fat, 0.71% crude fiber, 1.43% ash and 70.83% nitrogen free extract, while alfalfa seed flour contained 5.79%, 29.49%, 12.71%, 5.53%, 4.80% and 41.73% moisture, crude protein, crude fat, crude fiber, ash and nitrogen free extract correspondingly. Alfalfa seed flour at 5%, 10%, 15% and 20% supplementation levels was incorporated in refined wheat flour to produce composite flour. The biscuits prepared were subjected to quality evaluation. Physical analysis of biscuits disclosed that supplementation of alfalfa seed flour decreased the width from 47.25 to 42 mm and the spread factor from 62.7 to 53.12, while it increased the thickness from 7.53 to 8.10 mm. Supplementation of refined wheat flour–based biscuits with alfalfa seed flour at different inclusion levels significantly ( The word biscuit is derived from the Latin “bis coctus”, which means twice cooked. They are first positioned in a hot oven to bake and then transferred to dry out in a cool oven . BiscuitA number of causes for such broad fame of biscuits are low price among other baked foods and straightforward accessibility . BiscuitThough the consumption of biscuits is very popular, the inferior quality protein of wheat flour has been an important concern in its utilization . Its proMedicago sativa L.) is an ancient crop. It belongs to the pea family Fabaceae [Legumes such as beans, peas and alfalfa are essential sources of dietetic protein for a large area of the world’s inhabitants ,17. TheyFabaceae . The chaFabaceae .Alfalfa seeds are a rich source of protein and other nutrients on average; they contain 7.40% moisture, 30.10% crude protein, 3.60% crude fat, 6.90% crude fiber, ash 3.90% crude fiber and 48.10% nitrogen free extract . AlfalfaRefined wheat flour, alfalfa seeds and other ingredients used in biscuits preparation including sugar, shortening (vegetable ghee), skimmed milk powder, glucose, raising agents (sodium bicarbonate and ammonium bicarbonate), and emulsifying agents (lecithin and glycerol-mono-stearate) were obtained from local market.Alfalfa seeds were cleaned and freed of broken seed, dust and other foreign materials manually and were washed with tap water. Clean alfalfa seeds were milled in laboratory mill with the size of 0.07 mm (Tecator Cyclotec sample mill Model No. 1093) and the flour was packed in thick polyethylene bags.Composite flour was prepared from refined wheat flour supplemented with alfalfa seeds flour at different levels and biscuits were prepared from supplemented flour with a little modification of the method described in  is indicA known weight of vegetable ghee and powdered sugar was mixed together at medium speed to get light and soft cream. Then, the flour, skimmed milk powder, glucose, raising agents (sodium bicarbonate and ammonium bicarbonate) and emulsifying agents (lecithin and glycerol-mono-stearate) were added to the cream paste and mixed for obtaining uniform smooth dough. The dough was rolled out using a rolling pin. Biscuits were cut out into round shape of about 4.5 cm in diameter with a biscuit cutter. Baking was done at 175–185 °C for 10–12 min in baking oven. Biscuits were permitted to cool at ambient temperature for 8–10 min and were sealed in polyethylene bags for quality evaluation.SF) was calculated with the following formula. Where, D is the diameter, T denoted thickness and CF is a correction factor at constant atmospheric pressure and its value is 1.Physical characteristics of biscuits, such as diameter (width), thickness and spread factor were determined using the standard method described in . Spread Chemical analysis of biscuits was determined according to the standard method of . DiffereMineral contents were determined according to standard wet digestion method described in . The mixA panel of 10 well-trained judges was selected to assess acceptability of biscuits. The biscuits were tested for different sensory quality characteristics such as, color, taste, texture and overall acceptability with the use of nine-point hedonic scales of .The experiment was carried out using a completely randomized design (CRD) with three replications with the using of software MSTAT-C for analysis of variance (ANOVA) and tested at the 5% significance level by .The proximate composition of the refined wheat flour and alfalfa seed flour was determined as shown in S0 whereas the minimum mean value (42.89 mm) was noted in MS4 (p < 0.05) decreased with the gradual increase in alfalfa seed flour supplementation. The decrease in the diameter may be attributed to an increased protein content in the flour blend as reported by [S4 whereas the minimum mean value (7.53 mm) was found in MS0  increased compared to that of control, except MS1 which contained 5% alfalfa seed flour supplementation. The results achieved are in line with the findings of [S0 whereas the minimum mean value (53.12) was noted in MS4 (p < 0.05) decreased with the progressive increase in alfalfa seed flour supplementation. The decrease in the spread factor may be attributed to the effect of the composite flour which forms aggregates with an increased number of hydrophilic sites struggling for the limited free water in the biscuit dough [Statistical analysis disclosed that the thickness of supplemented biscuits had significantly  was noted in MS4 (p < 0.05) decreased compared to that of the control biscuits, except MS1 which contained 5% alfalfa seed flour supplementation. The moisture content of the flour blend decreased with the addition of alfalfa seed flour, because the moisture content of the alfalfa flour is lower than that of the refined wheat flour. The present results are in agreement with the findings of [S4 while the minimum mean value (10.19%) was observed in MS0 (p < 0.05) increased with the gradual increase in the alfalfa seed flour supplementation. The crude protein content increased in the flour blend due to the addition of the alfalfa seed flour, because of the higher crude protein content in the alfalfa seed flour as compared to the refined wheat flour. The present results are in complete agreement with the findings of [The average results of the moisture content of control and supplemented biscuits are shown in d in MS4 . Statistdings of . Banurekdings of  found thd in MS0 . Statistdings of . Onoja edings of  reportedS4 whereas the minimum mean value (0.73%) was noted in MS0 (p < 0.05) increased with the progressive increase in alfalfa seed flour supplementation. The crude fiber content in the flour blend increased due to the addition of alfalfa seed flour, because the alfalfa seed flour has a higher crude fiber content than refined wheat flour. Comparable results were achieved by [S4 while the minimum mean value (17.46%) was found in MS0 (p < 0.05) increased with the gradual increase in the alfalfa seed flour supplementation. The crude fat content of the flour blend is increased with the inclusion of alfalfa seed flour, because the crude fat content of alfalfa seed flour is high compared to refined wheat flour. These results are in accordance with the findings of [For the crude fiber content, the average results of the control and supplemented biscuits are shown in d in MS0 . Statistieved by ,40 foundd in MS0 . Statistdings of ,42 foundS4 while the minimum mean value (1.37%) is observed in MS0. Statistical analysis disclosed that the ash content of biscuits had significantly (p < 0.05) increased with the progressive increase in alfalfa seed flour supplementation. The increase in ash content could be attributed to the increased inclusion of alfalfa seed in the flour blend. This may be due to the fact that the ash content of alfalfa seed flour is high compared to refined wheat flour. The results obtained are in complete agreement with the findings of [S0 whereas the minimum mean value (56.32%) was noted in MS4 (p < 0.05) increased with the progressive increase in alfalfa seed flour supplementation. The increase in the nitrogen free extract could be due to increased incorporation of alfalfa seed in the flour blend. This may be due to the fact that the nitrogen free extract of alfalfa seed flour is low compared to refined wheat flour. These results were in agreement with the findings of [dings of ,44 indicd in MS4 . Statist value 1.% is obsedings of  found thS4 while the minimum mean value (105.30 mg/100 g) is observed in MS0. Statistical analysis disclosed that the potassium content of biscuits had significantly (p < 0.05) increased with the increase in alfalfa seed flour supplementation. The increase in potassium content could be due to the increased inclusion of alfalfa seed in the flour blend. This may be due to the fact that the potassium content of alfalfa seed flour is high compared to refined wheat flour. The present results are in complete agreement with the findings of [The mineral content of wheat flour and alfalfa seed flour were checked and are shown in dings of ,40,41,42S4 while the minimum mean value (14.65 mg/100 g) was found in MS0 (p < 0.05) increased with the gradual increase in alfalfa seed flour supplementation. The increase in the magnesium content could be due to the increased inclusion of alfalfa seed in the flour blend. This may be due to the fact that the magnesium content of alfalfa seed flour is high compared to refined wheat flour. These results are in accordance with the findings of [Similarly, the average result of the magnesium content of the control and supplemented biscuits is shown in d in MS0 . Statistdings of ,40,41,42S4, while the minimum mean value (43.91 mg/100 g) was noted in MS0 (p < 0.05) increased with the progressive increase in alfalfa seed flour supplementation. The increase in the calcium content could be due to the increased incorporation of alfalfa seed in the flour blend. This may be due to the fact that the calcium content of alfalfa seed flour is high compared to refined wheat flour. Comparable results were achieved by [The maximum mean value of the calcium content (79.60 mg/100 g) was observed in Md in MS0 . Statistieved by ,46 foundS4 while the minimum mean value (3.74 mg/100 g) was observed in MS0 (p < 0.05) increased with the gradual increase in alfalfa seed flour supplementation. The increase in iron content could be due to the increased inclusion of alfalfa seed in the flour blend. This may be due to the fact that the iron content of alfalfa seed flour is high compared to refined wheat flour. Similarly, the maximum mean value of the zinc content (1.60 mg/100 g) was found in MS4 while the minimum mean value (0.94 mg/100 g) was noted in MS0 (p < 0.05) increased with the progressive increase in alfalfa seed flour supplementation. The increase in the zinc content could be due to the increased inclusion of alfalfa seed in the flour blend. This may be due to the fact that the zinc content of alfalfa seed flour is high compared to refined wheat flour. These results are in agreement with the findings of [However, for iron content, the maximum mean value (7.93 mg/100 g) was recorded in Md in MS0 . Statistd in MS0 . Statistdings of ,44,45,46S0 while the minimum mean value (5.60) was recorded in MS4. Statistical analysis showed that the supplementation had significantly (p < 0.05) influenced color of the biscuits. The dark brown color dominated with the gradual increase in alfalfa seed flour. This may be due to the reaction between reducing the sugar and amino acids (Maillard reaction) and caramelization as reported by [S0 whereas the minimum mean value (5.00) was found in MS4 (p < 0.05) between the treatments. Results exhibited a decrease in the quality of alfalfa seed flour–supplemented biscuits in terms of taste. This decrease may be attributed to the beany flavor of alfalfa seed flour. Beany flavor is usually linked with legumes [orted by . These rorted by ,45 repord in MS4 . Statist legumes . In legu legumes ,50. The  legumes ,48,49,50S0 whereas the minimum mean value (5.38) was noted in MS4 (p < 0.05) decreased with the progressive increase in alfalfa seed flour supplementation. This decrease may be due to the increased crude fiber content of alfalfa seed flour in the flour blend. The results obtained are in accordance with the findings of [The average results for the texture of the control and supplemented biscuits are shown in d in MS4 . Statistdings of ,32 foundS0 while the minimum mean value (5.32) was observed in MS4 (p < 0.05) between the treatments. Results revealed a decreased quality of alfalfa seed flour–supplemented biscuits in terms of overall acceptability. Results obtained are in line with the findings of [Therefore, the maximum mean value of the overall acceptability (8.10) was found in Md in MS4 . Statistdings of ,32 also The present research work confirmed that the recommended supplementation of refined wheat flour should be up to a maximum of 10%, which could produce acceptable biscuits with an appropriate nutritional profile. The physical characteristics of alfalfa seed flour–supplemented biscuits portrayed variable results. The thickness of the biscuits increased while the width and spread factor decreased. Supplementation of alfalfa seed flour significantly influenced the proximate composition of refined wheat flour–based biscuits. Crude protein, crude fiber, crude fat and ash contents increased whereas moisture content and nitrogen free extract decreased. Mineral contents  of biscuits increased with the increase in alfalfa seed flour supplementation. Sensory evaluation of the biscuits showed that the quality score in terms of color, taste, texture and overall acceptability decreased with the increased supplementation of alfalfa seed flour.


A widely used method for hot forming steels and alloys, especially heavy forging, is the process of multistage forging with pauses between stages. The well-known effect which accompanies multistage hot forging is metal plasticity recovery in comparison with monotonic deformation. A method which takes into consideration the recovery of plasticity in pauses between hot deformations of a billet under isothermal conditions is proposed. This method allows the prediction of billet forming limits as a function of deformation during the forging stage and the duration of the pause between the stages. This method takes into account the duration of pauses between deformations and the magnitude of subdivided deformations. A hot isothermal upsetting process with pauses was calculated by the proposed method. Results of the calculations have been confirmed with experimental data. A widely used method for hot forming steels and alloys, especially heavy forging, is the process of multistage forging with pauses between stages (stress relaxation technique). During these pauses in the forging process, the stress relaxation in metal allows decreased forging force. This technique is used extensively in the upsetting process in heavy forging as it is one of the most energy-intensive operations. There has been considerable research concerning this technique. The works of Sokolov and Efimov (Ukraine) Sokolov  have madAnother important and well known effect which accompanies multistage hot forging is increased metal plasticity in comparison with monotonic deformation. First of all this effect was manifested and used in forging special steels and alloys. Russian researcher Dzugutov  marked tDzugutov established experimentally that during forging of hydraulic press billets from pearlitic structural alloyed steel, a value of 30–60% of relative strain on one stage of multistage forging can be achieved. During forging of high alloyed steels with low plasticity, an acceptable value of relative of strain on one stage of forging would be 6–10%.ZhS6KP only multistage forging with a one-stage relative strain of 2–4% and frequent heating allowed the achievement of positive results and acceptable values of limiting strain. It was proposed to determine the value of relative strain on one stage of forging experimentally.Dzugutov  marked tEI319 and EI437 resulted in significantly increasing plasticity. By Legrand’s data the multistage hot forging of materials can increase plasticity by 2–2.5 times.Legrand  shows thDzugutov  refers tEfimov established that during pauses in multistage forging the dislocation density in the forged billet decreases Fig. . This reOne of the types of multistage forging is vibration forging (upsetting)  with low used plasticity ψi on i-th stage of deformation is equal ψi and with a high value of used plasticity (close to 1) the recovery plasticity approaches 0. In this case the coefficient K of Eq. , characterizes the material, and can be found experimentally. To determine the function f it is necessary to conduct a set of experiments. Specimens should be deformed under tensile conditions with certain prescribed temperature and velocity. Experiments should include deformation of the specimen with a value of ψi in the range of 0.1–0.9. This allows the determination of parameter Δψ and parameter m that simply define f for Eq.  the value of recovery plasticity and used plasticity after two stages of upsetting can be found as follows: During the pause after the second stage of upsetting, plasticity will be recovered which was used during the second stage of upsetting ɛi at point T on the relative height deformation ɛh a simulation of the upsetting process was made. A finite element method (FEM) (Zhbankov and Perig 2K). Materials simulation parameters are described in Table For determination of the dependence of logarithmic strain Results of the simulation are fields of equivalent plastic strain in the longitudinal section of the billet and the graphical dependence of equivalent plastic strain on relative height deformation of the billet Fig. .Fig. 5DiAfter processing the obtained data, the analytical dependence of equivalent plastic strain on relative height deformation can be calculated, which is expressed for the given temperature and speed conditions in the form:−1 was considered. By the data which was obtained experimentally by a monotonic isothermal tensile test of cylindrical billets with temperature 1100 °C, and strain rate 0.00066 s−1, the logarithmic strain in the fracture ɛp was equal to 0.65. We now assume that the stress state at the peripheral surface of the upset workpiece barrel is the same as the stress state in the tensile test. So the workpiece fracture in monotonic upsetting would occur at the instant, when the equivalent plastic strain at point T reaches the critical value of 0.65. This critical value of equivalent plastic strain at point T corresponds to a relative height deformation of the billet ɛh equal to 0.63, which could be found on the basis of Eq. (Δɛh equal to 0.25 and the pause duration between stages 60 s on the basis of Eqs. (For the calculation of the maximum height deformation of the billet during multistage hot upsetting, the option of forging the billet from steel with 0.70% C in isothermal conditions with temperature 1100 °C, and strain rate 0.00066 ss of Eq. . We now  of Eqs. .m is equal to 1 − ψi in Eq. (Coefficient i in Eq.  for a gi for Eq.  is equalentclass1pt{minimaΔɛh. Obtained results are confirmed by experimental data, which are described in Figs. Using the above relations and assumptions it was determined that multistage upsetting with one stage height deformation equal to 0.25 it is possible to increase the maximum height deformation from 0.63 to 0.75 for two stages with pauses for 60 s. Total height deformation in the upsetting can be increased by reducing deformation on one stage A method for calculation of recovery plasticity in hot multistage forging has been proposed. This method allows the prediction of billet forming limits as a function of deformation during the forging stage and the duration of the pause between the stages.A calculation of the isothermal multistage upsetting process of a cylindrical billet was made. This calculation shows that in monotonic upsetting the maximum relative height deformation is equal to 0.63. In multistage upsetting with relative height deformation in one stage equal to 0.25 and pause duration 60 s it is possible to increase the maximum height deformation to 0.75 without fracture.


In this study, AZ91 magnesium alloy rods were used to investigate the effects of torsion deformation on microstructure and subsequent aging behavior. Extruded AZ91 rod has a uniform microstructure and typical fiber texture. Torsion deformation can generate a gradient microstructure on the cross-section of the rod. After torsion, from the center to the edge in the cross-section of the rod, both stored dislocations and area fraction of {10-12} twins gradually increase, and the basal pole of the texture tends to rotate in the ED direction. Direct aging usually generates coarse discontinuous precipitates and fine continuous precipitates simultaneously. Both twin structures and dislocations via torsion deformation can be effective microstructures for the nucleation of continuous precipitates during subsequent aging. Thus, aging after torsion can promote continuous precipitation and generate gradient precipitation characteristics. Both aging treatment and torsion deformation can reduce yield asymmetry, and torsion deformation enhances the aging hardening effect by promoting continuous precipitation. Therefore, combined use of torsion deformation and aging treatment can effectively enhance the yield strength and almost eliminate the yield asymmetry of the present extruded AZ91 rod. Finally, the relevant mechanisms are discussed. As the lightest metallic structural materials, Mg alloys are expected to replace some steel and aluminum alloys in vehicles for the purpose of weight reduction. However, the improvement of strength and anisotropy is still needed to extend the applications of Mg alloys. Precipitation hardening is an important hardening method and has exhibited high competitiveness in the development of high strength Mg alloys ,2. MoreoCold deformation is an effective way to tailor the precipitation behavior of Mg alloys ,11,12,13In previous studies, pre-cold deformation is usually carried out by pre-tension, pre-compression and pre-rolling ,19,20,21−1. The temperature of the ingots was kept at 400 °C during the whole extrusion process. Extruded AZ91 rods were solution treated at 420 °C for 3 h, then quenched into water at room temperature. The solution-treated AZ91 rods were subjected to free-end torsion deformation. Dog-bone-shaped specimens with a gauge length as Φ 4 mm × 28 mm were prepared for the torsion deformation. The longitudinal axis of the specimen is parallel to the extrusion direction (ED). The torsion test was carried out at a constant rate of 2 rpm, which resulted in a maximum equivalent strain rate of 0.9 × 10−2 s−1 at the sample surface. The starting AZ91 material was an extrusion billet. Cylinders with a dimension of 85 mm × 180 mm were cut from the as-cast AZ91 alloy and homogenized at 400 °C for 24 h and then hot-extruded into rods with a diameter of 16 mm. The extrusion ratio was about 28:1 and extrusion rate was about 20 mm·s−3 s−1. After torsion, some of the dog-bone-shaped specimens were used for the tension test, and the cylindrical specimens with nominal dimensions of Φ 4 mm × 8 mm were cut from the dog-bone-shaped specimens for the compression test. The yield strength was measured as 0.2% proof stress and the extent of yield asymmetry was described as the ratio of compressive yield stress to tensile yield stress (CYS/TYS).Tensile and compressive tests along ED of the twisted samples were performed at room temperature at a strain rate of 1 × 10Microstructures were characterized using scanning electron microscopy  and electron backscatter diffraction (EBSD). EBSD measurements were carried out on cross-sections of the samples and characterized regions were near the surface of rods (edge position) and near the cores of rods (center position). The characterized region for edge position is about 1.8 mm from the rod axis and suffers a shear strain of approximately 20%. Surfaces of samples for EBSD measurement were prepared by mechanical grinding using silicon carbide papers, followed by electro-polishing in the AC2 electrolyte for 60 s at 20 V. EBSD analyses were carried out by using the HKL Channel-5 software .It has been reported that torsion deformation can introduce a gradient strain along the radius direction . Thus, tc-axis and ED, the inverse pole figures of various samples are shown in c-axes of most grains are perpendicular to the ED, as shown in Clearly, torsion deformation can generate gradient microstructure on the cross-section of as-extruded Mg rod. From center to edge on the cross-section of rod, both stored dislocations and {10-12} twins gradually increase and the basal pole of texture tends to rotate towards ED direction.17Al12 phases exhibit a non-uniform distribution in SA sample. The coarse discontinuous precipitates (DP) and fine continuous precipitates (CP) usually simultaneously precipitate within the same grain, as shown in 2, the area fractions of DP (f-DP) and CP (f-CP) were evaluated and listed in f-DP is 48% for the SA sample. Torsion deformation can generate a gradient microstructure on the cross-section of AZ91 rod, as shown in f-DP is reduced to 34% in center position of the STA sample. For the edge position of the STA sample, both un-twinned and twinned regions can be the favorable nucleation sites for the continuous precipitation (f-DP = 11%). Moreover, it is noticed that the continuous precipitates in un-twinned and twinned regions of each grain exhibit different morphologies. This can be attributed to the different observation crystallographic plane between un-twinned and twinned regions due to that {10-12} twining causes a grain rotation of ~86.3° [pitation c, resultf ~86.3° . ClearlyN is the number of rotation, r is the radial position in the sample, l is the sample length and ε is the equivalent strain. This shows that torsion deformation can generate a gradient distribution of strain and the equivalent strain linearly increases from core to surface of rod. As discussed in c-axis of texture tends to rotate towards ED direction. Previous reports have revealed that dislocation slips are dominant deformation mechanism of torsion deformation for extruded Mg alloys [c-axis of texture rotate toward ED during torsion. The ideal orientation for Mg alloys under torsional shear deformation is known as B fiber texture with c-axis// ED [c-axes of most grains are perpendicular to ED. On this occasion, torsion deformation will induce the c-axes to rotate towards ED [In this study, torsion deformation can introduce a gradient distribution of microstructure. It could be attributed to the nature of torsion deformation, i.e., inhomogeneous deformation. Torsion can generate a gradient strain within the samples. The equivalent strain in torsion can be calculated as :γ = 2πg alloys ,30,35. Txis// ED . For extwards ED .17Al12 phases are favorably formed within extension twins introduced by pre-strain [17Al12 phases can also be favorably formed within un-twinned regions as well as within extension twins and the coarse discontinuous precipitates can be almost completely inhibited.For the SS sample, the coarse discontinuous precipitates and fine continuous precipitates can simultaneously precipitate, as shown in e-strain ,11,13,17e-strain ,11,13,17Generally, lattice defects via pre-deformation can promote the nucleation and suppress coarsening of precipitates ,11,13,170 is a frictional contribution, σHP is the contribution from refinement hardening, σd is the contribution from dislocation hardening, σppt is the contribution from precipitation hardening and σori is the contribution from orientation hardening.Torsion deformation can introduce dislocations and twin lamellae and cause textural change. Dislocations and twin boundaries can generate dislocation hardening and refinement hardening effects, respectively . Thus, ic-axis⊥ED), the basal slip with the lowest critical resolved shear stress (CRSS) is hard to activate owing to the low SF for deformation along ED. Under this occasion, it is generally considered that yielding in tension and compression along ED are dominated by prismatic slip and extension twinning, respectively. The large yield asymmetry of as-extruded magnesium alloy is attributed to the fact that the CRSS for extension twinning is far less than that for prismatic slip [Textural change will influence the Schmid factor (SF) of dominated deformation modes. For extruded magnesium alloy rods with fiber texture (i.e., tic slip . Torsiontic slip ,42,43. T17Al12 phase with basal plates in Mg-Al alloys. It has been reported that the Mg17Al12 phase can generate a higher Orowan hardening effect on extension twinning than on prismatic slip, leading to a reduction in yield asymmetry [17Al12 precipitates, but also retains the torsion-deformed texture and twin lamellae. It could be the reason why the yield asymmetry of STA is almost eliminated.Both torsion deformation and aging treatment can reduce the yield asymmetry. Firstly, textural change via torsion deformation can reduce the yield asymmetry . Moreovesymmetry . The subIn this study, it has proved that torsion deformation is an effective method to improve the mechanical properties of extruded AZ91 rods. It shows that both twins and dislocations via torsion deformation can be favorable nucleation sites for the continuous precipitations. It can contribute to the enhancement of aging-hardening response and the reduction of yield asymmetry of extruded AZ91 rod. Recently, some works have focused on the enhancement of age-hardening response of Mg alloys by pre-inducing dislocations or twins ,11,12,13The SS sample has a uniform microstructure and typical fiber texture. Torsion deformation can generate gradient microstructure evolution on the cross-section of as-extruded AZ91 rod. After torsion, from the center to the edge on the cross-section of rod, both stored dislocations and area fraction of {10-12} twins gradually increase, and the basal pole of the texture tends to rotate towards ED direction.Direct aging usually generates coarse discontinuous precipitates and fine continuous precipitates simultaneously. Torsion deformation can promote continuous precipitation. Both twin structures and dislocations via torsion deformation can be effective microstructures for the nucleation of continuous precipitates.Twin structures are favorable nucleation sites only for the continuous precipitation, while the precipitation behavior of un-twinned regions largely depends on the strain amount. High strain can generate a mass of dislocations to promote continuous precipitation in un-twinned regions. Thus, ST sample exhibits gradient precipitation characteristics due to the gradient distribution of twins and dislocations.SS sample exhibits a large tension-compression yield asymmetry. Aging treatment can enhance the tensile and compressive yield strength simultaneously and reduce yield asymmetry. Torsion deformation can also reduce the yield asymmetry owing to the generation of twin lamellae and textural change. Moreover, torsion deformation can enhance the aging hardening effect. Finally, combined use of torsion deformation and aging treatment can effectively enhance the yield strength and almost eliminate the yield asymmetry of the present extruded AZ91 rod.In this study, an AZ91 magnesium alloy rod was used to investigate the effects of torsion deformation on microstructure and subsequent aging behavior. Moreover, tension and compression properties of various samples were also discussed. Following conclusions can be drawn:


Vibrio cholerae. CdgH has two N-terminal tandem periplasmic substrate-binding (PBPb) domains for its signal recognition; however, the role of the tandem PBPb domains remains unclear. Here, we reported the crystal structure of the periplasmic portion of CdgH, which indicated that both tandem PBPb domains consist of typical interlobe ligand-binding architecture. Unexpectedly, the PBPb-I domain binds an L-arginine which apparently has been co-purified from the E. coli expression system, whereas the PBPb-II domain is in an unliganded open state. Structural comparison with other amino acid-binding proteins indicated that despite similar ligand-binding pockets, the PBPb-I domain possesses two ligand-binding residues (E122 and Y148) not conserved in homologs and involved in hydrophilic and hydrophobic interactions with L-arginine. Isothermal titration calorimetry indicated that the PBPb-I is primarily an L-arginine/L-lysine/L-ornithine-binding domain, whereas the PBPb-II domain exhibits a preference for L-glutamine and L-histidine. Remarkably, we found that the periplasmic portion of CdgH forms a stable dimer in solution and L-arginine binding would cause conformational changes of the dimer.Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger involved in bacterial signal transduction and produced by diguanylate cyclases (DGCs) generally containing highly variable periplasmic signal-recognition domains. CdgH is a DGC enzyme that regulates rugosity associated phenotypes in  Vibrio cholerae and is responsible for the infection of 3 to 5 million individuals and 100,000 to 120,000 deaths annually1. In the aquatic phase of its life cycle, V. cholerae is a planktonic, free-swimming organism, but prefers to form a sessile biofilm on the chitin surfaces of aquatic organisms4. The biofilm matrix aids the survival of V. cholerae by enabling it to overcome nutrient limitations, as well as providing protection from other environmental stressors5. After entering a human host along with contaminated water or food, V. cholerae responds to changes in environmental conditions by transiting from an aquatic bacterium to a human pathogen and producing a toxin-co-regulated pilus and cholera toxin that cause host illness7. The responses of V. cholerae to environmental signals involve surface attachment, biofilm formation, orbiting and roaming motility, virulence-gene expression, and life cycle progression5.Cholera is a severe diarrheal disease caused by the Gram-negative bacterium V. cholerae12. Increases in c-di-GMP levels induce increased expression of various factors necessary for the establishment and maintenance of biofilm communities, whereas decreased levels usually lead to enhanced expression of virulence and motility factors related to biofilm degradation14. C-di-GMP is synthesized from guanosine triphosphate by diguanylate cyclase (DGC) enzymes and hydrolyzed to 5′-phosphoguanylyl-(3′ → 5′)-guanosine by c-di-GMP-specific phosphodiesterases (PDEs)16. Proteins with a conserved C-terminal GGDEF domain act as DGCs18, whereas proteins containing EAL or HD-GYP domains act as PDEs19. The V. cholerae genome typically encodes 31 proteins with a GGDEF domain10. Interestingly, most DGCs are not only the producers of c-di-GMP but also sensors of environmental signals. In addition to the conserved C-terminal GGDEF domain, these DGCs also contain highly variable N-terminal conserved signal-recognition domains20. The significant number of different DGCs implies that each DGC can sense and respond to specific environmental signals and thereby produce c-di-GMP as a second messenger5. However, little is known about the molecular mechanisms associated with this possible activity, and the functions of the variable N-terminal signal-recognition domains in most DGC enzymes remain unidentified.3′,5′-cyclic diguanylate monophosphate (c-di-GMP) is a ubiquitous secondary messenger that plays a key role in response regulation and lifestyle conventions of pathogenic bacteria, including V. cholerae CdgH is a DGC enzyme containing the conserved C-terminal cytoplasmic GGDEF domain that allows it to engage in DGC activity in vivo10. CdgH differs from other V. Cholerae DGCs by having two N-terminal tandem periplasmic substrate-binding (PBPb) domains as its signal recognition domain21; however, the role of these tandem PBPb domains remains unclear. As previously reported, CdgH positively regulates rugosity associated phenotypes in V. cholerae, and V. cholerae CdgH mutants form less corrugated and flatter colonies as compared with those formed by wild-type variants10. CdgH also plays a major role in c-di-GMP synthesis by responding to the presence of bile acids21. CdgH deletion reduces bile-mediated induction of c-di-GMP and biofilm formation21. Although CdgH regulates significant V. cholera-related phenotypes, the environmental signals sensed by CdgH remain unknown, and the mechanisms associated with how substrate binding triggers the DGC activity of CdgH also remain elusive.V. Cholerae CdgH. Our structure indicated that both tandem PBPb domains of CdgH contain a typical interlobe ligand-binding structural architecture. Intriguingly, PBPb-I domain is in complex with L-arginine. Our results indicated that the PBPb-I and -II domains have different amino acid binding specificity. Moreover, the periplasmic portion of CdgH was found to form a dimer in solution which could undergo conformational changes upon L-arginine binding.Here, we reported the crystal structure of the periplasmic portion of 322 space group, with one molecule in the asymmetric unit. Most residues  were well traced in the final model  of CdgH containing a C-terminal His-tag Fig. , and sol23  domains. Structural superposition revealed that although the PBPb domains of CdgH and the VFT domains of BvgS are both typically bi-lobed domains, the orientations and arrangements of the tandem periplasmic domains of the two proteins are obviously different : 3.1], and the PBPb-II domain was the histidine kinase sensor domain of HK29S26 , indicating that both CdgH PBPb domains contained a typical interlobe ligand-binding architecture, despite sharing relatively low sequence identities.Meanwhile, DALI searches also revealed that the highest scoring result for the PBPb-I domain was the ancestral arginine-binding protein AncQRd = 1.21 × 10−9 M; Fig. d = 14 × 10−9 M)27. This unexpected bound L-arginine may be co-purified from the E. coli expression system. The interlobe ligand-binding pocket agrees with many other reported bacterial ligand-binding structures. However, we observed no similar density in the PBPb-II domain, indicating that the PBPb-I domain of our structure is in a ligand-binding conformation, and the PBPb-II domain is in a ligand-free conformation  and hydrogen bond interactions with the main-chain nitrogen atoms of T104 and T147. Additionally, the L-arginine side chain (NH1 And NH2) forms two salt bridges with E56 and E122 Fig. . In addi25 (PDB: 4ZV1), a synthetical bacterial Glutamine/Arginine-binding protein that is involved in amino acid transport, LAOBP28 (PDB: 1LST), a bacterial periplasmic binding proteins involved in substrate transport and chemotaxis in Salmonella typhimurium and GlnBP29 (PDB: 1WDN), a component of the ligand-specific periplasmic glutamine permease system in Escherichia coli29  based on structural details of the ligand-binding site. These wild-type PBPb-I domain and mutants are all soluble by itself in solution. Then we tested their affinities for L-arginine by ITC , PBPb-IR109A (Kd = 8.77 × 10−6 M), and PBPb-IE122A (Kd = 2.92 × 10−5 M) mutants resulted in 51.5-, 242.9-, and 808.9-fold reductions in binding affinity, respectively, relative to the wild-type PBPb-I domain (Kd = 3.61 × 10−8 M). Moreover, the PBPb-IE56A/R109A/E122A triple mutant led to no measurable binding ability, implying the essential functions of these three residues for recognizing L-arginine.To confirm the importance of the key residues involved in ligand recognition, we expressed and purified the PBPb-I domain 46–250) alone in 50 alone d = 6.67 × 10−7 M), which is about 18-fold weaker than that with L-arginine. Moreover, the PBPb-I domain binds L-ornithine (Kd = 4.17 × 10−6 M), L-histidine (Kd = 2.32 × 10−5 M) and L-glutamine (Kd = 6.76 × 10−5 M) with lower binding affinities, and L-aspartate with no measurable binding affinity. These findings suggested that the CdgH PBPb-I domain exhibits the highest affinity for binding L-arginine, L-lysine, and L-ornithine, but also possesses the ability to bind L-glutamine and L-histidine.To determine the ability of the CdgH PBPb-I domain to bind a broad range of ligands in addition to L-arginine, we employed ITC to test the binding affinities of the wild-type PBPb-I domain with various amino acids  Fig. . The resE56A, PBPb-IR109A, PBPb-IE122A) with different ligands  by ITC  to test the ligand-binding properties of the PBPb-II domain by ITC experiments. The results showed that PBPb-IE56A/R109A/E122A/PBPb-II binds L-glutamine (Kd = 7.94 × 10−5 M) and L-histidine (Kd = 9.25 × 10−5 M) with the highest affinities , L-lysine (Kd = 2.26 × 10−3 M), and L-ornithine (Kd = 3.18 × 10−3 M) with much lower affinities  Fig. , suggest2 (type I dimer) and 1039.2 Å2 (type II dimer), respectively, and values of CSS  are 1 and 0, respectively, suggesting the interface of the type II dimer seems to be a result of crystal packing only. In addition, we analyzed the interfaces of both dimer types. In the type I dimer, the lobe-II′ of the PBPb-II domain of each protomer interacts with the hinge region of the PBPb-I domain of the opposing protomer , is larger than that of the refolded protein without L-arginine (77.2 kD) Fig. , revealiInterestingly, BvgS, the only other reported structure containing tandem periplasmic domains found by DALI server, also forms a dimer with a ligand-free state, which is consistent with our analytical ultracentrifugation result that the tandem PBPb domains of CdgH without L-arginine also form a dimer in solution. Despite the differences in the arrangements of the tandem periplasmic domains of CdgH and BvgS, their dimeric architectures both require the entire tandem periplasmic domains, as the PBPb-II/VFT2 domain of each protomer is to interact with the PBPb-I/VFT1 domain of the opposing protomer to form the dimer Fig. . Moreove34. However, little is known about the detailed mechanisms associated with how these DGC domains activate the C-terminal GGDEF domains in response to environmental signals to initiate subsequent signal transduction pathways. There are 31 DGC proteins found in V. cholera; however, to our knowledge, no structure of V. cholerae DGCs has been reported. Sequence analysis indicated that the periplasmic portion of the CdgH DGC employs two tandem PBPb domains21, but the function of the periplasmic portion of CgdH remains elusive.C-di-GMP is a second messenger in bacterial signal transduction. Most DGCs contain conserved C-terminal GGDEF domains enabling synthesis of c-di-GMP, as well as highly variable N-terminal signal-recognition domains for responding to specific environmental signals, such as PAS (Per-Arnt-Sim), GAF , REC (receiver) domains and so onV. cholera DGC. Unexpectedly, our structure shows that the PBPb-I domain is in complex with L-arginine which may be co-purified from the E. coli expression system, whereas the PBPb-II domain shows no ligand bound. Furthermore, our results indicated that the PBPb-I domain binds various amino acids, with a higher affinity for L-arginine, L-lysine, and L-ornithine, whereas the PBPb-II domain exhibited higher affinities for L-glutamine and L-histidine.Here, we solved the crystal structure of the periplasmic portion of CdgH containing tandem PBPb-I and -II domains. To our knowledge, this is the first structure of the periplasmic tandem PBPb domains of a Structural analysis revealed that the binding of L-arginine resulted in a conformational change in the PBPb-I domain from open to closed. We also observed dimerization of the periplasmic portion of CdgH both in solution and in the crystal-packing alignment. Moreover, both calculated molecular masses of the unrefolded wild type protein (87.3 kD) and refolded protein with excess L-arginine (86.8 kD) are larger than that of the refolded protein without L-arginine (77.2 kD), revealing that the liganded dimer is more compact than the unliganded dimer upon L-arginine binding.P. aeruginosa by controlling the intracellular levels of c-di-GMP35. In addition, S. Typhimurium was shown to specifically respond to L-arginine with an increase in c-di-GMP, and the responses required the regulating periplasmic domain of the diguanylate cyclase STM198736. Together with these previous reports, our structural and functional studies suggest that L-arginine might also function as a small molecule modulator for the DGC activity of CdgH. Under low concentrations of L-arginine in environmental conditions, the periplasmic portion of CdgH exhibits as a loose dimer with both open apo conformations of the PBPb-I and -II domains. Upon L-arginine binding, the dimer undergoes conformational changes from the loose state to the compact state, subsequently causes rotation and translocation of the transmembrane helices, thereby enables rearrangement of the C-terminal GGDEF domains and affects subsequent enzyme activation.Meanwhile, it has been reported that amino acids, including L-arginine, can modulate biofilm formation and swarming motility of V. cholerae and may facilitate the development and optimization of anti-biofilm drugs for the treatment of infections.The novel ligand-binding mode of the periplasmic portion of CdgH illustrated here provided new insights into the selectivity for various amino acids among different ligand-binding proteins. Moreover, this work revealed the first structure of a tandem PBPb-containing domain related to rugosity and biofilm formation in V. cholerae CdgH were cloned into the pET22b vector  via the NdeI and XhoI restriction sites, with a constructed C-terminal His6, respectively. Site-directed mutagenesis was performed using a QuikChange kit  according to manufacturer instructions.The periplasmic portion (residues 46–491), the PBPb-I domain (residues 46–250) and the PBPb-II domain (residues 251–491) of E. coli BL21 (DE3)-CodonPlus-RIPL strain in LB medium. Protein expression was induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside at an OD600 of ~0.8, followed by incubation of the cell cultures overnight at 16 °C. The cells were subsequently harvested by centrifugation and stored at −80 °C.The proteins were overexpressed by the Cell suspensions were thawed, resuspended in binding buffer , and homogenized using a high-pressure homogenizer . After centrifugation by 16,000 rpm for 30 min at 4 °C, the supernatant was loaded onto a 5-ml Ni-affinity column  equilibrated with binding buffer.E56A/R109A/E122A/PBPb-II mutant that is purified without refolding, other proteins were first purified by Ni-affinity chromatography with on-column refolding which ensured complete removal of endogenously bound ligands. The Ni-affinity column pre-loaded with the proteins was washed with 50 ml binding buffer followed by 50 ml unfolding buffer . Proteins were refolded on-column by application of a gradient from unfolding buffer to binding buffer over 100 min at a flow rate of 2 ml/min using an FPLC , then eluted with elution buffer . Then the refolded proteins were exchanged into buffer A , loaded onto an anion-exchange column  equilibrated with buffer A and eluted by a gradient from buffer A to buffer B . Finally, the proteins were purified by a size-exclusion column  in Tris buffer .For ITC experiments, except for the PBPb-IE56A/R109A/E122A/PBPb-II mutant for ITC assays were purified by Ni-affinity chromatography without refolding, followed by an anion-exchange column  and a size-exclusion column .The periplasmic portion of CdgH for crystallization and the PBPb-IAll of the purified fractions were collected and concentrated to ~40 mg/mL in Tris buffer , followed by freezing in liquid nitrogen and storage at −80 °C.The purified periplasmic portion of CdgH without refolding was used for crystallization. Crystal screening was performed with commercial screening kits  using the sitting-drop vapor diffusion method, and positive hits were optimized using the hanging drop vapor diffusion method at 293 K. SeMet crystals of the periplasmic portion of CdgH protein were obtained and optimized in buffer containing 0.1 M Tris-HCl (pH 8.0) and 10% w/v polyethylene glycol 8000 using the concentration of 10 mg/ml. After soaking for 3 min in cryoprotection solution (well solution complemented with 15% sucrose), the crystals were cooled by plunging them into liquid nitrogen. A single diffraction dataset (360 images with an oscillating range of 1°) of the best crystal (diffracting at 2.60 Å) was collected at a wavelength of 0.97848 Å and a temperature of 100 K on beamline BL18U at the Shanghai Synchrotron Radiation Facility (SSRF).37. The crystal space group is P6322, with one molecule in the asymmetric unit. The phase was determined using single-wavelength anomalous diffraction (SAD) method based on the selenomethionine-substituted protein crystal. Electron-density maps were calculated using PHENIX38, and model building was performed using COOT39 and refined using PHENIX. refine40. The final structures were analyzed with PROCHECK41. The structure was refined to final Rwork of 22.92% and Rfree of 28.85%. A Ramachandran analysis indicated that 91% of residues are in most favored region, 7% in additionally allowed region and 1.9% in disallowed region. Data collection and refinement statistics are presented in Table http://www.pymol.org). Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.pdb.org) under accession codes 5 GZS.Diffraction images were indexed and scaled using the HKL2000 software programE56A/R109A/E122A/PBPb-II whose binding ability of the PBPb-I domain is eliminated, other proteins were all refolded and purified as described above. ITC experiments were performed in a buffer composed of 20 mM Tris (pH 8.0) and 150 mM NaCl at 25 °C using VP-ITC or iTC200 calorimeters . For VP-ITC calorimetry experiments, L-Arg (0.08 mM) was titrated into the tandem PBPb domains (0.02 mM), and the titration sequence included a single 2 µl injection, followed by 55 injections of 5 µl each, with a 210 s interval between injections and a stirring rate of 307 rpm. In addition, various amino acids (0.5 mM) were titrated into the PBPb-I domain and its mutants (0.05 mM), respectively, and the titration sequence included a single 2 µl injection, followed by 27 injections of 10 µl each, with a 210 s interval between injections and a stirring rate of 307 rpm. For iTC200 calorimetry experiments, various amino acids (8 mM) were titrated into the PBPb-IE56A/R109A/E122A/PBPb-II protein (0.2 mM), and the titration sequence included a single 0.5 µL injection, followed by 19 injections of 2 µL each, with a 2 min interval between injection and a stirring rate of 1000 rpm. Each ITC experiment was performed twice. Calorimetric data were analyzed using OriginLab software . For titrations with c > 1, where c is the product of the association constant (Ka) for the interaction and the protein concentration in the cell, the stoichiometry (n), enthalpy (ΔH), and Ka for the interaction were determined. For titrations with c < 1, n was fixed at 1 while ΔH and Ka were determined. The ITC data are presented in Table For ITC experiments, except for PBPb-I42.Sedimentation velocity measurements (SV-AUC) were performed on a Beckman ProteomeLab XL-I  at 25 °C. All protein samples were diluted to a concentration of 0.7 mg/ml in 20 mM Tris (pH 8.0) and 150 mM NaCl. The data of PBPb-I domain were collected at 60,000 rpm, while the tandem PBPb domains unrefolded and refolded with or without excess L-arginine at 40,000 rpm every 3 min at a wavelength of 280 nm. Interference sedimentation coefficient distributions were calculated from the sedimentation velocity data using SEDFITsupplementary data


Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM) targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities. Escherichia, Pseudomonas, and Yersinia, are also structurally very diverse, and a major subset of these molecules adopt a modular organization characteristic of polymorphic toxins. They consist of a receptor-binding domain (RBD) allowing docking onto the surface of a target cell, a moiety enabling transfer across the membrane, and a variable carboxy-terminal toxin domain mediating killing by different mechanisms  that attach to cognate toxin modules with strict specificity and high affinity , 10. UpoE. coli is one of the smallest modular bacteriocins studied to date. Its carboxy-terminal catalytic domain (ColM) displays phosphatase activity and cleaves the peptidoglycan building block lipid II, accessible from the periplasm. This leads to accumulation of undecaprenol (C55-OH) and 1-pyrophospho-MurNAc (N-acetylmuramic acid)-GlcNAc (N-acetylglucosamine), with MurNAc carrying the stem pentapeptide, which cannot be reused for murein biosynthesis . Notably, whereas colicin M genes are plasmid encoded in E. coli and some other Enterobacteriaceae (such as Citrobacter and Shigella), usually joined upstream by a colicin B pore-former–immunity gene tandem  is highlighted in red. The partially conserved proline residue likely isomerized by FpkA is colored blue. Abbreviations: Acat, Acidovorax cattleyae; Bamb, Burkholderia ambifaria; Bcep, Burkholderia cepacia; Bglu, Burkholderia glumae; Bokl, Burkholderia oklahomensis; Brgoo, Brenneria goodwinii; Bsp, Burkholderia sp.; Bubo, Burkholderia ubonensis; Cfre, Citrobacter freundii; Dchr, Dickeya chrysanthemi; Ecol, Escherichia coli; Enaer, Enterobacter aerogenes; Enkob, Enterobacter kobei; Ermal, Erwinia mallotivora; Ersp, Erwinia sp.; Ilim, Inquilinus limosus; Kpne, Klebsiella pneumoniae; Kvar, Klebsiella variicola; Lyeo, Luteibacter yeojuensis; Paana, Pantoea ananatis; Pacon, Pantoea conspicua; Paer, Pseudomonas aeruginosa; Pbre, Pseudomonas brenneri; Pced, Pseudomonas cedrina; Pecar bra/car, Pectobacterium carotovorum subsp. brasiliense/carotovorum; Pflu, Pseudomonas fluorescens; Pput, Pseudomonas putida; Psp, Pseudomonas sp.; Psyr (ace/tom/mor), Pseudomonas syringae (pv. aceris/tomato/morsprunorum); Sent (ent), Salmonella enterica (subsp. enterica); Semar, Serratia marcescens; Shboy, Shigella boydii; Stmal, Stenotrophomonas maltophilia. Download FIG S1, TIF file, 7.1 MB.Multiple sequence alignment of ColM domains. One representative bacteriocin per genus was selected in the case of highly homologous sequences (>80% pairwise amino acid identity for full-length proteins). Gray shading reflects the degree of sequence conservation. The ColM signature motif  (A), PaeM (PDB 4G75) (B), and pectocin M2 (PDB 4N58) (C). ColM domains are shown in wheat color; the FhuA- and FiuA-targeting sequences from colicin M and PaeM, respectively, are colored in blue; amino-terminal parts comprising the TonB box are shown in lemon color; the FusA-targeting ferredoxin module in pectocin M2 is dark green; and the helix connecting the ferredoxin module and ColM domain in pectocin M2 is dark gray. ColM domains require the presence of a coordinating divalent cation for catalytic functionality . The ferredoxin module binds 2Fe-2S (yellow). Side chains of residues constituting the ColM motif are shown as black sticks. The proline residue (P176) likely targeted by FkpA in colicin M is indicated. Download FIG S2, TIF file, 0.8 MB.Cartoon representation of colicin M  is fused at the C terminus with a ColN-related pore-forming domain —involved in membrane translocation of numerous nuclease bacteriocins (5LK) in this second hybrid (tentatively named PmdH) suggest that in this protein the phosphatase function is abrogated  extracted from characterized bacteriocins and homologues reveals that, with few exceptions, they are affiliated with one of two major clades, here termed ColMα and ColMβ ; Fig. 1.ectocins  and pseuectocins , but theectocins . Two notg domain , 35, 36.g domain . Conceiveriocins —and a toeriocins . The lateriocins , 10, 40.brogated .Burkholderia and Pseudomonas, with some Pseudomonas strains carrying bacteriocin genes of both types, i.e., a ColMα- and a ColMβ-type gene, at unlinked locations   . No crysM302280) , not parcolM in Escherichia coli, nor is this the case for chromosome-carried colM genes  exposed at the cellular surface. Colicin M anchors to (certain loops of) the ferrichrome transporter FhuA and utilizes this OMP for translocation into the periplasmic space  17, 46), 4617, 447–led FiuA . Evolutiled FiuA . Based oled FiuA . Such anled FiuA .Pectobacterium and related genera (Brenneria and Dickeya), the RBD has been replaced by a ferredoxin module (Pfam PF00111) , a different receptor is expected since its RBD is significantly longer and lacks sequence similarity with respective domains from FiuA-binding ColM-type pseudocins  , and forPF00111) . As oppoPF00111) , 52. Theeudocins  25). Th. ThPectoentative ) and disEnterobacteriaceae and other proteobacteria, and a similar strategy is exploited by numerous bacteriocins and phages. At this point, a straightforward prediction of the receptors likely targeted by other ColM-type bacteriocins is complicated by the general lack of known domains (except for the above-mentioned ferredoxin-containing pectocins). Diversifying recombination events have led to a tremendous heterogeneity among nuclease bacteriocins cis-trans-isomerase FkpA . Fk. Fkcis-t(E. coli . In supp killing . It cannE. coli tRNase effector delivered to target cells via CDI requires the cysteine biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A) for modulation of growth inhibition . These bacteriocin genes are associated  with a gene encoding an immunity protein with a similar topology as Cmi but lacking the YebF signature domain.Interestingly, pseudomonads do not carry  genomes . Instead genomes  24), an, ancmi-l genomes , 8, 76.  genomes , PmiA liBurkholderia and Pseudomonas, respectively , encoded downstream in the opposite direction, adopts yet another organization with six TMHs , this may suggest that immunity by this type of protein was obtained later in evolution. (iii) The observations that the key residues for Cmi immunity are located at the carboxy terminus of the protein and that PmiAs sharing very poor sequence similarity with the cognate immunity protein may provide noncognate immunity  As lipid II tends to appear in regions of increased fluidity (RIFs), hereby forming “atoll”-like regions in the inner membrane, different immunity protein topologies may be the result of preferential (genus-dependent) association or integration in these areas to protect lipid II from enzymatic digestion (A number of options can be considered. (i) Part of the answer will likely be provided by the interaction between the ColM domain and its chaperone(s) and the structural consequences thereof. At this point, it cannot be excluded that ColMs (in different genera/clusters) require different permissive factors. This is a reasonable assumption considering the absence of a universally conserved proline in ColM domains (see above). The interacting chaperone(s) may ultimately define which type of immunity partner is required. (ii) Different immunity protein topologies may point to immunity at different cellular locations, i.e., fundamentally different mechanisms would exist to impede lipid II-degrading activity, targeting different patches of the ColM domain for interaction, directly or indirectly impeding the enzymatic function. Considering that integral membrane proteins in the ColMα group arise only in pseudomonads and a small subset of immunity , 72 couligestion .It will also be of future interest to explore whether kin selection is involved in ColM-immunity divergence. This model states that genealogical relatives have more alleles in common than nonrelated strains; therefore, the probability of maintaining a gene in subsequent generations will increase if it is associated with an enhanced fitness for the genealogical relatives . Toxin-i82–E. coli ColM system along with exploration of related systems from other proteobacterial genera has revealed ColM’s catalytic site and noncatalytic activity, allowed delineation of structural domains that may be the target of diversifying recombination and selection events, dissected the contribution of a first permissive factor, and revealed a whole array of topologically distinct membrane-associated immunity proteins. The features emerging from this broadened view are indicative of a genuine polymorphic toxin family and a patchwork of immunity partners, giving rise to several new scientific questions and hypotheses. The essentiality of lipid II for cell viability makes ColM bacteriocins an attractive candidate drug worth exploring further.Continued scrutiny of the


Stipules are leaf structures common in many groups of plants that can take a variety of forms. In Gentianales, interpetiolar stipules are considered a synapomorphy of Rubiaceae but in this study we detected two lateral expansions at the base of the leaf primordia (stipules) that give rise to colleters in many genera of Apocynaceae, similarly to Rubiaceae species. Through ontogenetic study it was possible to prove for the first time that Apocynaceae is a stipulate family whose stipules are modified into colleters, and their absence might be a secondary loss, changing the interpretation of stipule evolution in Gentianales. Stipules are leaf structures common in many groups of plants that can take a variety of forms. In Gentianales, interpetiolar stipules are considered a synapomorphy of Rubiaceae; however, some reports in the literature refer to their presence in other families. The goal of this study was to analyze the development of leaf primordia to investigate the possible presence of reduced or modified stipules in Apocynaceae. Shoot apices of 12 genera were analyzed under light and scanning electron microscopy comparatively with one species of Rubiaceae. Early in their development, leaf primordia form two lateral expansions at the base of the petiole (stipules) that give rise to colleters in 11 of the 12 genera of Apocynaceae studied, similarly to the Rubiaceae species. The basal genera have pairs of stipules modified into colleters positioned laterally to the petiole, while other species belonging to the derived subfamilies have interpetiolar stipules that each project towards the opposite stipule and merge, forming a sheathing stipule and from this arc the interpetiolar colleters originate. The ontogenetic study proved for the first time that Apocynaceae is a stipulate family whose stipules are modified into colleters and their absence might be a secondary loss, changing the interpretation of stipule evolution in Gentianales. Stipules are usually small, inconspicuous projections of the petiole base of leaves with protective function mainly in the bud, and there might be other functions as well. Often they appear as a pair of leafy appendages located on each side of the leaf base, but in some plants they may be modified into thorns or glands .Stipules occur in many families of Eudicots and are especially important in some orders, such as Gentianales, which is composed of the families Apocynaceae, Gelsemiaceae, Gentianaceae, Loganiaceae and Rubiaceae . Among tThose reports raise doubts about the occurrence and development of stipules in this group, especially in relation to Apocynaceae, generally characterized as not having stipules . ApocynaIn spite of the centuries of knowledge of the group and several structural studies performed in Apocynaceae, few studies have suggested the presence of morphologically distinguishable stipules in Apocynacean species , with EnColleters are secretory structures present in vegetative and/or reproductive organs that produce a sticky fluid, composed only of mucilage and/or lipophilic compounds, which protects meristems . In ApocOn the leaves, colleters were recorded on the apex or base of the petiole and along the margin and/or midrib of the lamina in many genera of Apocynaceae belonging to all five subfamilies . HoweverThe homology among the traits of different taxa may be identified by different survey techniques, but when the morphology is profoundly altered, the only studies on the developmental mechanisms are able to perceive their common origin . TherefoThe goal of this study was to verify the presence of reduced or modified stipules in the leaves of 12 genera of Apocynaceae belonging to different subfamilies, focusing on the beginning of leaf development and the ontogeny of interpetiolar colleters occurring on the petiole base to evaluate their possible stipular nature. One species of Rubiaceae was selected to compare the colleter/stipule origin.Mussaenda erythrophylla, was sampled at Universidade de São Paulo, São Paulo, SP, Brazil for comparison.Twelve species belonging to 12 genera were selected for this study  to repreShoot apices were collected and promptly fixed in FAA  for 24h  or BNF  and subsThe anatomical analyses were performed under a Leica DMBL light microscope  using the Scan System Images (IM50).Morphology: The leaves of all species have opposite phyllotaxis, being alternate only in Aspidosperma. The morphological analysis of leaves was not able to detect any stipule in the species of Apocynaceae studied, but colleters are easily observed on leaves of all species  the stipules grow as a pair laterally to the petiole and become glandular with the base fused to the stem  near the petiole, and the tip of the stipules is also modified in colleter  and Universidade de São Paulo.D.D. designed the research, B.A.R. and D.D. performed experiments and analyzed the data, and N.V.C. and D.D. interpreted the results and wrote the paper.None declared.