Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, we produced an allelic series of single codon substitutions in the Drosophila insulin receptor, InR. We generated InR substitutions using homologous recombination and related each to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. We propose the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.Mutations of the  Early work extended lifespan through mutations of the insulin-like receptors osophila , which cC elegans and Drosophila while these invertebrates produce many insulin-like ligands  when heterozygous over a P-element insertion allele   . We thenDrosophila InR mediates aging in independent ways. One affects reproduction and thereby extends lifespan through reduced survival costs, the other affects mechanisms to assure longevity independent of reproduction. (2) The biochemical impact of substitutions within InR may be understood in a context of asymmetric transphosphorylation (We propose three conclusions. (1) rylation  that robInR is on the third chromosome. Lines with EMS-induced mutations in InR, balanced by the third chromosome inversion InR+TM3, were provided by M. Frasch (The Mount Sinai School of Medicine)   background using the balancer stock Sam 1; Sam 2; InR+TM3/TM6B .The Drosophila insulin/IGF-receptor Drosophila melanogaster caught at the Raleigh Farmer’s Market  . In addition, the InRNC442 line was used to normalize survival data across test blocks.Iso-female lines were established from wild-type InRE19 (using adult InRE19/InR+TM3) and the nine viable Frasch InR EMS-alleles (using InREMS/InR+TM3), and between the 18 InRNC wild-type alleles (using InRNC/InR+TM3). Offspring from reciprocal crosses were pooled to measure phenotypes. Phenotypes of every InREMS/InRE19 and InRNC/InRE19 offspring were compared to a contemporaneous cohort of the allele when complemented by wild-type InR (InREMS/InR+TM3 or InRNC/InR+TM3).Quantitative complementation testing measures the phenotypic difference of one allele complemented to a standard hypomorph relative to when it is complemented to a standard wild-type allele . We madeSupplementary Methods). Cohorts were initiated with three replicate demography cages of ∼150 newly eclosed adults at ∼1:1 sex ratio per genotype. In total, we assessed the lifespan of 16,057 males and 17,223 females. Each 2 days, food vials were changed and dead flies were removed and counted. Life tables collected in five test blocks (j = 1–5) were made with data from replicate cages combined. Within each test block, we included the wild-derived line InRNC442 to normalized genotypes across blocks. One life table was generated for each InREMS allele and InRNC allele expect for duplicates generated in independent blocks for InR211(EMS), InR74(EMS), and InR353(EMS).Life tables (number of deaths per 2-day census period) were generated for adult offspring of each genotype (details in ij)β within each block j for each InR allele i (EMS or NC) when complemented to InRE19 relative to when complemented to +TM3InR. In each block, we estimated adjusted mortality coefficient for each InR allele as iInR/InRE19 relative to iInR/(+TM3InR); values near zero indicate there is no effect on mortality and values greater than zero indicate there is increased mortality. The transformation InR allele complemented to InRE19 relative to when it is complemented to +TM3InR. Life expectancy was also standardized among blocks: for each block j we calculated the difference in life expectancy between InRNC442/InRE19 and InRNC442/(+TM3InR) denoted InR allele i is We used proportional hazard analysis  to conduInREMS mutant and InRNC wild-type allele when complemented to InRE19 and to +TM3InR. Adults were reared simultaneously on standard diet from vials seeded with 100 eggs, and collected across three replicate vials. The relative body size of each tested iInR allele was measured as (mean of iInR/InRE19)/(mean of iInR/+TM3InR).Head capsule width was measured from 20 females from each InRE19, InR211, InR74, and InR353 based on their ability to reduce mortality, and InR246 as a contrast where QCT showed effects body size but not mortality. Ron Kohanski  provided cDNA sequences for the InRE19, InR74, and InR246 alleles that respectively contained codon substitutions as Val810Asp, Ile1543Phe, and Val1384Met (InR353 and InR211 were reported by wDah background. We made five single replacement lines (designated InRE19(HR), InR74(HR), InR211(HR), InR246(HR), InR353(HR)). We produced independent, replicate accessions for InRE19(HR) (denoted InRE19(HR)-2, InRE19(HR)-14, InRE19(HR)-22) and for 353InR(HR) (denoted InR353(HR)-8, InR353(HR)-15, InR353(HR)-20). We replaced the native wDah sequence with itself to produce the wild-type control InR+(HR) (accession denoted “29B”). We generated a double substitution allele ), and a null InR allele (InRnull(HR)).To validate and extend the results of QCT, we focused on the EMS alleles l1384Met . Codon sSupplementary Materials. Targeting arms for InR cloned from wDah . Targeting arms contained single nucleotide substitutions to produce the desired amino acid replacements. Flies with the +white eye color marker mapped to the first or second chromosomes were used for homologous recombination. These lines were crossed to flies expressing flipase and the restriction enzyme I-Sce1 to execute excision and linearization of the transgenic construct. Candidate insertion lines were selected based on +white mapped to chromosome 3. Chromosomes with replaced alleles were crossed to flies expressing cre recombinase to remove the +white marker. The +white marker was retained in an accession of the wild-type replacement allele to produce InRnull(HR). All replacement alleles were validated by sequencing.Detailed methods for the homologous recombination are provided in the InR(HR) alleles. Four females and males were housed for mating and to lay eggs over 24 h in replicate vials with yeast-supplemented standard media. We recorded stage of lethality  and the number of emerging adults each subsequent 24 h. Adult size (thorax length) was measured from 20 off-spring females of every viable cross.Reciprocal crosses were made among all Newly eclosed males and females were collected, transferred to bottles with fresh medium and kept at 25°C for 2 days (details in Supplementary Methods). Mated females were transferred to demography cages maintained at 25°C at an initial density of 125 females per cage. Three to five replicate cages were initiated for each genotype. Food was changed every two days, at which time dead flies were removed and counted. Life tables were constructed with genotype replicate cages combined for survival analysis. In total, we assessed the lifespan of 17,184 homologous recombination females.wDah males into three replicate demography cages, modified for egg collection (details in Supplementary Methods). For the following 15 days, egg plates were supplemented with yeast paste and changed twice a day at which time eggs were counted. Dead females were removed and counted daily. Daily fecundity (mean eggs per day) is the number of eggs summed across replicate cages per surviving female. Average fecundity is the mean of daily fecundity across the sample period. Ovariole number was measured from fifteen 10-day old females of each genotype. Ovaries were dissected in PBS, fixed with 4% paraformaldehyde, and scored for total number of ovarioles across both ovaries.Fifteen newly eclosed females of each genotype were placed with 15 4-Akt Cat#4054, pan-Akt Cat#4691). Blots were imaged and quantified with ImageLab (Bio-Rad).We evaluated the ability of IRs to induce phosphorylation of Akt from larval fat bodies stimulated by human insulin, as in Supplementary Figure S1 provide amino acid sequence annotation of Drosophila InR, and human IR and IGF1R. We number the human IR as the mature long isoform of Drosophila InR beginning at the first translation initiation site in wDah wildtype (deposited in GenBank as accession MT_563159), which is +45 amino acids relative to the TIS reported by wDah sequence also differs from that of Fernandez by an insertion/deletion (wDah lacks H144) and the substitution H146Q. This H144/H146 polymorphism segregates along latitudinal clines of Drosophila populations , eclosion time distribution , and the verified sequence alignments of the homologous recombination alleles . Supplementary Figure S3 provides conceptual diagrams of dimer complementation and a model for receptor domain function in aging. Tables in the Supplemental Materials provide survival statistics of the QCT (Supplementary Table S1), survival data for wild-type alleles in QCT (Supplementary Table S2), survival data of hemizygotes (Supplementary Table S3), and survival data of all HR alleles and accessions (Supplementary Table S4). https://doi.org/10.25386/genetics.13377023.Supplementary material is available at figshare DOI: InR EMS alleles  affect aging. InRE19 relative to when complemented to wild-type InR (InR+TM3) (Supplementary Table S1). Mortality rates in all cohorts increased exponentially once the death rate exceeded the lower limit of demographic power (InRNC442 measured in each to control for block effects (Supplementary Table S2). Relative to InRNC442 within each block, the EMS mutations variously reduced mortality (block adjusted iβadj = -1.80), had little effect (iβadj  iβadj = 1.64) (Supplementary Table S1). Because InRNC442 is just one instance of a wild-type allele, we applied QCT to a collection of 18 wild-type InR alleles extracted from the North Carolina Market population (NC series) and evaluated how each EMS mutant performed relative to the wild-type distribution (Supplementary Table S1). Six EMS InR mutants did not significantly reduce mortality relative to wild-type alleles , while InR327 strongly increased mortally. InR74 and InR211 significantly reduced mortality in males and females. The InR353 allele reduced female mortality but not significantly so relative to the wild-type distribution. The transformation InR74 and InR211 alleles reduced male and female mortality 2.6- to 6-fold, while InR353 reduced female mortality about 2-fold  used for complementation; each tested InR may contain unidentified substitutions; allelic effects are relative rather than absolute; and the measured effect of InR is confounded by a deleterious effect of the TM3 balancer chromosome. Our newly generated series of homologous recombination InR alleles resolves these issues.Quantitative complementation testing has limitations: it only measures recessive allelic effects; it confounds epistatic interactions with potential co-segregating mutations; the tested alleles were not derived from the wild-type Supplementary Figure S2). Every mutation had normal size and development time when heterozygous with +InR(HR). All mutations were homozygous lethal except for rare escapes in the case of InRE19(HR)/InRE19(HR). As expected from our QCT, all kinase domain mutations (InR74(HR), InR211(HR), InR246(HR), and InR353(HR)) produced small adults with delayed development when heterozygous with InRE19(HR) (an ectodomain mutation). Several kinase domain mutations were viable as trans-heterozygotes: InR74(HR)/InR353(HR) and InR74(HR)/InR211(HR), and these adults were small and developed slowly.We evaluated viability, eclosion time, and adult size for all allele combinations . Because homozygotes of mutant alleles are inviable, we infer homodimeric mutant receptors are nonfunctional. Viable mutant trans-heterozygotes must produce functional heterodimers, but they will also make fewer functional dimers because half of all potential receptors are mutant homodimers. To determine if simply having fewer functional receptors extends longevity, we evaluated the survival of hemizygote +InR(HR)/InRnull(HR) adults, which produce about 25% less InR mRNA (quantitative PCR in Supplementary Table S3). These adults increased life expectancy between 2 and 4 days when tested in replicate trials (Supplementary Table S3). This difference provides an approximate benchmark: we may infer a genotype affects receptor function when its gain in longevity is more than that caused by numerical loss of receptors modeled in InR hemizygotes.Insulin receptors are preformed dimers assembled with protomers translated from either allele (InRE19(HR)/InR+(HR) does not extend lifespan (InRE19(HR) accessions was 43.3 days, compared to 44 days for wildtype (Supplementary Table S4). Likewise, the intracellular kinase domain alleles InR74(HR), InR211(HR), and InR246(HR) when heterozygous with +InR(HR) did not extend lifespan more than expected from InR hemizygotes (Supplementary Table S4). In contrast, in replicated trials with independent accessions, InR+(HR)/InR353(HR) increased life expectancy on average 12.5 days by decreasing mortality about fourfold (Supplementary Table S4).The heterozygote lifespan : median InRE19 relative to when complemented to InR+. From the homologous recombination alleles, we now estimate the absolute allelic effect of each mutation. Across replicate trials, the genotypes InR74(HR)/InRE19(HR) and InR211(HR)/InRE19(HR) extended lifespan 6–14 days (InR74(HR)/InR211(HR) extended lifespan (InR hemizygote (Supplementary Table S4).In QCT, kinase domain alleles reduced mortality when complemented to –14 days . We alsolifespan . In everInRE19(HR)/InR74(HR) slowed aging, we asked if the mutations of these individual alleles extend lifespan when the substitutions were on the same protomer. They did not: InRE19,74(HR) had little effect on survival when heterozygous over a wild-type allele  when heterozygous with InR211(HR). These adults extended longevity to the same extent as trans-heterozygote InR74(HR)/InR211(HR)  . LongeviInR246 allele did not slow aging although it reduced body size. These observations are confirmed with InR246(HR)/InRE19(HR): adults were small (InR246(HR)/InRE19(HR) intersects that of wildtype, suggesting the mutant genotype may have high age-independent mortality which obscures a beneficial effect of reduced demographic aging.In QCT, the re small  but not re small . HoweverInR353(HR) extended lifespan when heterozygous with wildtype (10–16 days), InR353(HR) also slowed aging when heterozygous with InRE19(HR) (14–15 days). When heterozygous with InR74(HR), InR353(HR) extended lifespan 21–22 days, an effect caused by decreasing age-dependent mortality sixfold (Supplementary Table S4).While InR74(HR)/InR211(HR);  InR74(HR)/InR353(HR); InRE19(HR)/InR353(HR)) and document a dominant longevity benefit conferred by InR353(HR) (InR+(HR)/InR353(HR)).The homologous recombination alleles validate inferences from QCT but now measure absolute effects and are tested in an independent genetic background. We describe new trans-heterozygous genotypes that extend lifespan /InR246(HR) females. Fecundity was reduced in all long-lived trans-heterozygotes. In contrast, long-lived +InR(HR)/InR353(HR) females produced more eggs than wildtype. The elevated fecundity of +InR(HR)/InR353(HR) could arise because these females have more ovarioles (subunits of the ovary) or produce more eggs per ovariole. We therefore measured ovariole number for all genotypes and regressed this trait against daily egg production. Fecundity positively associates with ovariole number with a slope less than one (InR353(HR) allele as a covariate , but the regression intercept for genotypes with one InR353(HR) allele is ∼12 days greater than genotypes without this allele (P = 0.013). The InR353(HR) allele confers longevity that is additive to and independent of how reproduction associates with survival.Drosophila insulin signaling modulates fecundity , we deteoduction . Fecundithan one : differeovariate . A strikInR353(HR) substitution (R1466C) lies in the kinase insert domain, a region of receptor tyrosine kinases thought to modulate substrate interaction rather than receptor kinase activity (+InR(HR)/InRnull(HR) was moderately insulin resistant. Fat body from trans-heterozygotes involving InRE19(HR), InR74(HR), InR246(HR), and InR211(HR) induced little pAkt; they were insulin resistant. In contrast, fat body from +InR(HR)/InR353(HR) induced wild-type levels of pAkt. The InR353(HR) allele dominantly extends lifespan without loss of insulin-stimulated kinase activity.The activity . We theractivity . Fat bodDrosophila InR alleles modulate aging, growth, and reproduction.Mutations of insulin/IGF receptors are thought to slow aging and reduce growth because they diminish the total amount of insulin signaling. Alternatively, mutations of the IR may alter protein-receptor interactions in specific domains that control particular phenotypes, as proposed for receptor tyrosine kinases in general by InR mutations that slow aging. Three alleles reduced mortality more than expected from a sample of natural wild-type alleles. We also quantify how alleles affect growth. While all alleles significantly reduced adult size, there was no association between relative size and relative mortality among genotypes.We used QCT to identify EMS InR mutations do not share a common genetic background. Epistatic interactions with unknown second site mutations on the third chromosome could affect traits attributed to InR. QCT only estimates recessive effects of the InR mutations, and we could not evaluate complementation besides with InRE19. Unidentified polymorphisms within each InR locus might influence the complementation. Finally, QCT measures relative rather than absolute allelic effects, and these relative effects were confounded by how the balancer haplotype itself reduced lifespan.As noted, QCT has caveats . The EMSInR substitutions of selected EMS alleles. These new alleles provide single, defined substitutions derived from a common wild-type progenitor in a controlled genetic background. We produced five homologous recombination (HR) alleles (InR74(HR), InR211(HR), InR246(HR), InR353(HR), InRE19(HR)), an HR-derived wild-type allele, a null allele, and a cis-allele with the E19 and 74 substitutions upon the same protomer ).To address these issues, we used ends-out homologous recombination to reconstitute the putatively causal Supplementary Figure S3). Thus, the lifespan of trans-heterozygote adults may be slowed simply because they have fewer functional receptors. To test this idea, we studied the hemizygote InR+(HR)/InRnull(HR). This genotype reduced InR mRNA about 25% and presumably this reduced the total number of receptors. Hemizygotes moderately impaired phosphorylation of Akt and only modestly affected longevity. The number of receptors in these hemizygotes is expected to be similar to that of recessive lethal alleles when heterozygous with wildtype (InR+(HR)/InRE19(HR), InR+(HR)/InR74(HR), InR+(HR)/InR211(HR)) due to their loss of mutant-mutant dimers . Yet, those adults have normal longevity. Together these data suggest that genotypes with robustly extended lifespan either further reduce the quantity of receptors, or alter the quality or quantity of signaling from their functional receptors.InR is a dimeric transmembrane receptor. Flies with two wild-type alleles generate normal protomers so that every dimer is functional. On the other hand, trans-heterozygotes produce some functional dimers with protomers of complementing alleles, as well as nonfunctional homodimers from protomers of each single mutant allele (InRE19(HR) substitution Val810Asp occurs in a linker sequence between the L2 and FnIII-1 ectodomains, corresponding to Phe475 of human IR and to Phe495 of human IGF-1R . Insulin induces a hinge motion in this linker to swing the fibronectin domain inward, bringing together the intracellular kinase domains of each protomer (810Asp substitution of Drosophila InR will destabilize the fibronectin domain movement (+InR(HR)E19/InR(HR) genotype has wild-type traits because the Val810Asp protomer retains its ability to transphosphorylate the complementing wild-type kinase domain, which is sufficient for full receptor kinase function.The Drosophila insulin/IGF-like receptor is homologous to that of human IR and IGF1R , a seven amino acid sequence containing three tyrosine residues that participate in transphosphorylation  and 13 final residues (InR74(HR) is an Ile1543Phe substitution within the MTRDI motif. This change may reduce the ability for Tyr1544 to be phosphorylated and thus diminish the domain catalytic activity or destabilize the loop binding platform (The activation loop (A-loop) of the InR+(HR)/InR74(HR) has normal phenotypes, suggesting that the Ile1543Phe protomer can transactivate its complementing wild-type protomer to provide full receptor function. In contrast, only the Ile1543Phe protomer can be transactivated in InRE19(HR)/InR74(HR) adults, and the lone hypomorphic function of Ile1543Phe increases life span. This conclusion is consistent with how InRE19(HR)/InR74(HR) reduces insulin-stimulated kinase signaling (pAkt). To further test this explanation, we produced a double mutant where Val810Asp and Ile1543Phe were on the same protomer ). This allele was homozygous lethal but the genotype InR+/InRE19,74(HR) had normal growth and lifespan. The data show that aging is slowed when receptors only activate a protomer with a Ile1543Phe kinase domain.InR246(HR) allele is a Val1384Met substitution at the invariant ATP binding loop of the N-terminal lobe  (InRE19(HR)/InR246(HR) fat body. The normal traits of the heterozygote +InR(HR)/InR246(HR) suggests the InR246(HR) protomer fully transactivates wild-type protomers within heterodimers. In contrast, InRE19(HR)/InR246(HR) adults are small, as expected from their loss of kinase activity. It is unclear why this genotype does not also extend longevity although their cross-over survival pattern suggests these adults suffer high age-independent mortality that could obscure slow demographic aging.The gure S1) . This su1598Arg substitution of InR211(HR) is in the kinase domain C-terminal lobe, a site conserved in human IR (Gly1212 Ebina) and IGF1R (Gly1215). These sites are three conserved residues from the substitution found in daf-2(e1370), the canonical IR longevity allele of C. elegans . The homologous amino acid segment in human fibroblast growth factor receptor FGFR3 stabilizes the dimer interface during transphosphorylation  allele is exceptional. The heterozygote InR+(HR)/InR353(HR) extends lifespan yet adults have normal size and elevated fecundity. InR353(HR) combined with InRE19(HR) and InR74(HR) likewise produce long-lived adults, although these are small and have reduced fecundity.The InR353(HR) contains the substitution Arg1466Cys. This arginine resides at a site found in human IRs (Arg1092)  where Arg1092 stabilizes kinase domain dimerization   (SupplemInR+(HR)/353InR(HR) has wild-type insulin sensitivity, demonstrating these receptors retain kinase catalytic activity unlike other longevity assurance mutations of InR. How then are InR+(HR)/353InR(HR) adults long-lived? The KID of Drosophila InR is 12 amino acids longer than the KID of human IR  (Drosophila Chico (homolog of IRS1-4) where it recruits Grb2/Drk (Grb2/Downstream-of-kinase)   as do nuDrosophila InR kinase insert domain regulates lifespan through its interaction with adaptor proteins. Drosophila InR has been previously recognized to contain adaptor protein binding sites in the C-terminal tail and in the juxtamembrane domain /353InR(HR) presents a paradox: adults are highly fecund and yet long-lived. This positive association, which has been seen in other genetic manipulations of C. elegans and Drosophila .Drosophila limits the rate of egg production within ovarioles (E19InR(HR), 74InR(HR), 211InR(HR) may be long-lived, in part, because their reduced egg production mitigates survival costs-of-reproduction. Genotypes with these alleles are also insulin resistant and small. We propose these growth and life history phenotypes are caused by how each amino acid substitution reduces insulin-stimulated kinase catalytic activity.The first mechanism involves reproductive costs . Reducedvarioles  and incr1466Cys substitution destabilizes this interaction to induce systems that promote somatic survival while maintaining kinase catalytic activity. Thus, +InR(HR)/InR353(HR) gains about 12 days of longevity. The genotypes InRE19(HR)/InR353(HR) and InR74(HR)/InR353(HR) also gain this reproduction-independent longevity, but now in addition to benefits conferred by reduced costs-of-reproduction.The second mechanism confers longevity assurance independent of reproductive costs. We propose the kinase insert domain recruits substrate proteins through its SH2 binding motif. The ArgDrosophila chico to regulate aging  extends fly lifespan without reducing growth or fertility and maintains its ability for insulin to stimulate Akt phosphorylation. Chico mutated at the p85/p60 sites, on the other hand, extends life span but represses growth and reproduction. This suite of traits is seen in genotypes with the alleles E19InR(HR), 74InR(HR), and 211InR(HR), which also do not phosphorylate Akt in response to insulin.Our explanation is consistent with how we currently understand Drosophila IR appears to regulate aging through distinct systems modulated through structure-defined domains. This view reflects an emerging concept whereby protein tyrosine receptors control varied phenotypes through the action of specific protein interactions regulated by structural domains (The  domains . Future


The CIDE domain was initially identified in apoptotic nucleases and now forms a highly conserved family with diverse functions ranging from cell death to lipid metabolism. Based on structural determination of the DREP3 domain, it is suggested that the head-to-tail helical filament structure might be a unified mechanism of CIDE-domain assembly and represents a critical higher-order scaffolding structure that is important for the function of CIDE-domain-containing proteins in DNA fragmentation and lipid-droplet fusion. The cell-death-inducing DFF45-like effector (CIDE) domain is a protein-interaction module comprising ∼80 amino acids and was initially identified in several apoptotic nucleases and their regulators. CIDE-domain-containing proteins were subsequently identified among proteins involved in lipid metabolism. Given the involvement of CIDE-domain-containing proteins in cell death and lipid homeostasis, their structure and function have been intensively studied. Here, the head-to-tail helical filament structure of the CIDE domain of DNA fragmentation factor-related protein 3 (DREP3) is presented. The helical filament structure was formed by opposing positively and negatively charged interfaces of the domain and was assembled depending on protein and salt concentrations. Although conserved filament structures are observed in CIDE family members, the structure elucidated in this study and its comparison with previous structures indicated that the size and the number of molecules used in one turn vary. These findings suggest that this charged-surface-based head-to-tail helical filament structure represents a unified mechanism of CIDE-domain assembly and provides insight into the function of various forms of the filament structure of the CIDE domain in higher-order assembly for apoptotic DNA fragmentation and control of lipid-droplet size. The expression vector was delivered into Escherichia coli BL21 (DE3) cells using heat shock at 42°C, and the transformed bacteria were spread on a lysogeny broth (LB) agar plate containing ampicillin and incubated at 37°C for 16 h. A single recombinant colony was selected and cultured overnight at 37°C in 5 ml LB containing 50 µg ml−1 ampicillin, after which the cells were transferred to a large-scale culture in 1 l medium. Upon reaching an optical density at 600 nm of ∼0.6, 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the culture to induce gene expression. IPTG-treated cells were chilled on ice and further cultured at 20°C for 18 h in a shaking incubator. Subsequently, the bacterial cells were harvested and the pellet was resuspended in 20 ml lysis buffer . After adding phenylmethanesulfonyl fluoride , the cells were disrupted by sonication on ice with six bursts of 30 s each with a 60 s interval between each burst. The cell lysate was centrifuged at 10 000g and 4°C for 30 min to remove cell debris, and the supernatant was collected and mixed overnight with nickel–nitrilotriacetic acid (Ni–NTA) resin  by gentle agitation at 4°C. The resulting mixture was loaded onto a gravity-flow column. The Ni–NTA resin in the column was washed with 50 ml wash buffer  to remove unbound proteins. For elution, 0.5 ml elution buffer  was added to the column five times. The resulting eluate was concentrated to 30 mg ml−1 and subjected to size-exclusion chromatography (SEC) using an ÄKTAexplorer system  equipped with a Superdex 200 Increase 10/300 GL 24 ml column  pre-equilibrated with SEC buffer . The SEC peak fractions were pooled, concentrated to 9 mg ml−1, flash-frozen in liquid nitrogen and stored at −80°C until further use. Protein purity was assessed using SDS–PAGE.The expression plasmid for the DREP3 CIDE domain (corresponding to residues Phe112–Asp195) was constructed by inserting the amplicon, digested with NdeI and XhoI, into a pET-21a vector. The gene sequence information was derived from UniProt  according to the manufacturer’s instructions. Mutagenesis was confirmed by sequencing, and the mutant proteins were expressed and purified using the methods described above.2.3.−1 protein sample in buffer  was mixed with an equal volume of reservoir solution  and the droplet was allowed to equilibrate against 400 µl mother liquor. The crystallization conditions were further optimized and finally adjusted to obtain a buffer composition comprising 0.1 M citric acid pH 3.4, 0.6 M ammonium sulfate. Diffraction-quality crystals appeared within three days and grew to a maximum size of 0.3 × 0.1 × 0.1 mm. For data collection, crystals were soaked in mother liquor supplemented with 40%(v/v) glycerol as a cryoprotectant, mounted and flash-cooled in a nitrogen stream at −178°C. Diffraction data were collected on beamline 5C at the Pohang Accelerator Laboratory (PAL), Pohang, Republic of Korea at a wavelength of 1.0000 Å. The diffraction data were indexed, integrated and scaled using HKL-2000  was interconnected with the ÄKTAexplorer system . The molecular mass of bovine serum albumin was used as the reference value. The absolute molecular mass was assessed using ASTRA (Wyatt Technology). Protein samples at a concentration of 10 mg ml−1 were used in the MALS experiments.The absolute molar mass was determined using SEC-MALS. The purified target protein was passed through a 0.2 µm syringe filter and loaded onto a Superdex 200 10/300 gel-filtration column  pre-equilibrated with SEC buffer. The mobile-phase buffer flowed at a rate of 0.4 ml min2.7.−1 and a 5 s response time. The protein samples were diluted to 0.1 mg ml−1 in buffer consisting of 20 mM Tris–HCl pH 8.0, 500 mM NaCl before use. Four scans were accumulated and averaged.The secondary structures were measured by CD spectroscopy using a J-715 spectropolarimeter at the Korea Basic Science Institute, Daejeon, Republic of Korea. Far-ultraviolet (UV) CD spectra were recorded from 200 to 260 nm at 25°C in a quartz cuvette with a 0.1 cm path length using a bandwidth of 1.0 nm, a speed of 50 mm min2.8.7v6e.Atomic coordinates and structural factors have been deposited in the Protein Data Bank as PDB entry 3.3.1.a). CIDE-domain-mediated DFF40 and DFF45 interactions are important for regulating the nuclease activity of DFF40 during apoptotic DNA fragmentation. The CIDE domain forms a head-to-tail helical filament in solution, the implications of which remain undetermined, although a process related to increased local concentration through filament formation is suspected . Despite this difference, the DREP3 CIDE domain contains two conserved differently charged regions that are critical for hetero-oligomerization and homo-oligomerization .The CIDE domain is a conserved protein-interaction module found in several proteins involved in cell death and lipid metabolism Fig. 1a. CIDE-on Fig. 1b.VM) of 2.76 Å3 Da−1, corresponding to a solvent content of 55.43%, and solved the structure at a resolution of 3.0 Å. Diffraction data and refinement statistics are summarized in Table 1Phaser . The final model contained residues 117–193 for all nine molecules. The five amino-acid residues at the N-terminus (residues 112–116) and two residues at the C-terminus (residues 194 and 195) were not included in the final model due to untraceable electron density in these regions.To further understand this dynamic protein-interaction module, we obtained a crystal of the DREP3 CIDE domain which had a calculated Matthews coefficient . The two helices (α1 and α2) included residues Ile139–Lys149 and Glu169–Thr174, respectively. The four strands comprising residues Lys119–Lys124, Arg131–Ala136, Arg156–Leu159 and Glu180–Val184 are indicated as β1, β2, β3 and β4, respectively . The structure of all nine molecules was nearly identical, exhibiting a root-mean-square deviation (r.m.s.d.) of around 0.3–0.4 Å when they were superimposed . As a common feature, the surface of the DREP3 CIDE domain was also divided into two distinctly differently charged sides .The structure of the DREP3 CIDE domain exhibited the canonical CIDE-domain fold, which comprises an α/β-roll fold with two α-helices and four β-strands Fig. 1d. The tly Fig. 1d. The sed Fig. 1e. As a a). However, the structures of the α1–β3 loop and the C-terminal loop differed .The newly solved structure of the DREP3 CIDE domain was compared with those of structural homologues, including DREP2 and DREP4. Molecule 1 among the nine molecules, which has the lowest temperature factor, was picked and compared by structural superposition; this indicated that the overall structures were nearly identical, as indicated by superimpositions with r.m.s.d.s of 0.7 Å (DREP2 CIDE domain) and 0.9 Å (DREP4 CIDE domain) Fig. 2a. Howevd Figs. 2a–2 ▸c.3.2.M NaCl), corresponding to a molecular weight of 30–40 kDa and indicating that this domain also forms a homo-oligomer in solution. Compared with the CIDE domains of DREP2 and DREP4, which form higher oligomeric complexes and elute at 12 and 13 ml by SEC (in 500 mM NaCl), the DREP3 CIDE domain formed a smaller homo-oligomeric complex . To analyse the dynamic assembly/disassembly of the DREP3 CIDE domain, we determined its concentration-dependent self-assembly using SEC. Three independent SEC experiments performed with protein samples at three different concentrations  showed that larger amounts of the DREP3 CIDE domain produced larger-sized particles that eluted earlier on SEC, indicating that homo-oligomerization of the DREP3 CIDE domain is dependent on the protein concentration . The void peak contained an aggregated sample of the DREP3 CIDE domain and the peak after 24 ml contained various protein aggregates that were produced during the concentration of DREP3 . Additionally, we assessed the salt-dependence of CIDE-domain self-assembly using SEC. As the NaCl concentration was increased the DREP3 CIDE domain eluted later in SEC, indicating that a higher oligomeric complex was dissociated in high-salt conditions. This experiment indicated that self-assembly of the DREP3 CIDE domain was also highly dependent on salt concentration . The accurate molecular mass of various forms of the DREP3 CIDE domain in different concentrations of NaCl was analysed by SEC-MALS measurements, which can calculate the absolute molecular mass of a particle in solution. This experiment revealed that the molecular masses of the DREP3 CIDE domain in 50 mM, 500 mM and 2 M NaCl were 62.14 kDa (1.7% fitting error), 35.46 kDa (2.2% fitting error) and 13.47 kDa (1.3% fitting error), respectively, indicating that the DREP3 CIDE domain forms a higher oligomeric complex in low-salt conditions and this oligomeric complex is dissociated on increasing the salt concentration .The dynamic assembly/disassembly property of the CIDE domain in solution is its primary feature . We observed that all nine helix-related molecules were arranged into a helical structure in the crystal, with nine molecules per turn, a rise of 53.8 Å and a diameter of ∼90 Å . In the crystal lattice, the helical structure was continuous and stacked along the a axis of the unit cell .As found in other CIDE domains, the surfaces of the DREP3 CIDE domain are divided into two oppositely charged surfaces, which are critical for their homo-oligomerization. Additionally, their dynamic assembly/disassembly properties  in solution were similar to those of the previously studied CIDE domains of DREP4 and DFF40 . The protein sample eluted at the void volume in SEC was aggregated as judged by EM.To confirm the helical assembly of the DREP3 CIDE domain in solution and corroborate the crystal structure, we performed EM experiments. The protein sample eluted at 16–17 ml from a Superdex 200 gel-filtration column under salt conditions of 500 mre Fig. 4b. The p3.4.a and 5b). The highly positively charged interface comprising Lys122, Lys132, Lys145 and Lys149 forms a large number of salt bridges and hydrogen bonds with the highly negatively charged interface comprising Asp162, Asp165, Asp167, Asp168 and Glu170 of the neighbouring molecule . This interface buries ∼520 Å2 of accessible surface area per molecule. To validate our findings, we performed a mutagenesis analysis. To disrupt the interfaces, the representative interface residues Lys132 for the positively charged interface and Asp167 for the negatively charged interface were mutated to aspartic acid and arginine, respectively, producing K132D and D167R mutants. Based on SEC, the D167R mutant clearly failed to form a helical oligomeric structure, whereas the wild-type DREP3 CIDE domain predominantly formed oligomers in solution . Further analysis of the D167R mutant by SEC-MALS revealed that the molecular weight of the D167R mutant was 10 804 Da (2.4% fitting error), indicating that the D167R mutant existed as a monomer in solution and failed to form a filament-like structure . This means that disruption of the tentative interface detected in the helical filament structure affects the filament assembly of the DREP3 CIDE domain. For the K132D mutant, the expression levels reduced dramatically. Although the effect of the mutation was less clear than that produced by the D167R mutant, the K132D mutation clearly affected the helical assembly of the DREP3 CIDE domain . To ensure that oligomer disruption was not due to structural alterations resulting from mutagenesis, we obtained far-UV CD spectra data. The wild-type and mutant variants showed similar CD spectral patterns typical of mixed α-helix- and β-sheet-containing proteins, with two pronounced minima at 208 and 222 nm, respectively, indicating that the structures were unchanged by mutagenesis .The helical assembly of the DREP3 CIDE domain is mediated by repetitive head-to-tail polymerization between oppositely charged interfaces . Additionally, nine molecules were involved in one turn of the helical filament in the DREP3 CIDE domain, whereas eight and ten molecules were involved in one turn of the helical filaments of the DREP2 and DREP4 CIDE domains, respectively . At ∼105 Å, DREP4 had the largest diameter of the CIDE-domain helical filaments, compared with 90 Å for the DREP2 and DREP3 CIDE domains. Furthermore, the rise of one helical turn was 50.2 Å for DREP2, 53.8 Å for DREP3 and 56.5 Å for DREP4 .Next, we compared the helical filament structure of the DREP3 CIDE domain with the recently revealed helical structures of the DREP2 and DREP4 CIDE domains by superimposition (Choi c). This helical feature was also observed in the filament structures of the DREP2 and DREP4 CIDE domains . Additionally, the diameters of the holes produced by the spiral assemblies of CIDE domains differed, with the largest being produced by the DREP4 domain (∼57.3 Å) and the smallest by the DREP2 CIDE domain (∼39.9 Å) . These analyses indicated that multiple CIDE domains form helical filament structures in solution, and that these structures vary in length, diameter and helical properties, although all of the domains appear to share a unified mechanism for helical assembly.The electrostatic surface view of the helical filament structure of the DREP3 CIDE domain showed that the two helical ends carried opposite charges, which extended the filament structure Fig. 6c. This 4.et al., 1998et al., 2013et al., 2014et al., 2000et al., 2017et al., 2017et al., 2021et al., 2020abet al., 2007et al., 2021The functions of CIDE-domain-containing proteins have expanded from involvement in apoptotic DNA fragmentation as a hallmark of apoptosis to lipid homeostasis and brain synapse functions (Liu et al., 2021et al., 2017et al., 2017et al., 2017The structure of the DREP3 CIDE domain is another example of a helical filament structure, with an assembly mediated by repetitive head-to-tail polymerization between oppositely charged interfaces. Unlike the more stable and cooperatively formed filament-like structure presented by the DD superfamily (Xia et al., 2017Structural comparison of the DREP3 CIDE domain with recently identified helical filament structures of other CIDE domains revealed the diversity of these structures in length, diameter and helical properties, although the CIDE domains share a helical assembly mechanism. Although the function of the CIDE-domain-mediated filament structure in apoptotic DNA fragmentation has been established in a recent structural study (Choi DREP3, 7v6ePDB reference:


Bacteriophages have evolved a range of anti-CRISPR proteins (Acrs) to escape the adaptive immune system of prokaryotes, therefore Acrs can be used as switches to regulate gene editing. Herein, we report the crystal structure of a quaternary complex of AcrIIA14 bound SauCas9–sgRNA–dsDNA at 2.22 Å resolution, revealing the molecular basis for AcrIIA14 recognition and inhibition. Our structural and biochemical data analysis suggest that AcrIIA14 binds to a non-conserved region of SauCas9 HNH domain that is distinctly different from AcrIIC1 and AcrIIC3, with no significant effect on sgRNA or dsDNA binding. Further, our structural data shows that the allostery of the HNH domain close to the substrate DNA is sterically prevented by AcrIIA14 binding. In addition, the binding of AcrIIA14 triggers the conformational allostery of the HNH domain and the L1 linker within the SauCas9, driving them to make new interactions with the target-guide heteroduplex, enhancing the inhibitory ability of AcrIIA14. Our research both expands the current understanding of anti-CRISPRs and provides additional culues for the rational use of the CRISPR-Cas system in genome editing and gene regulation. Streptococcus pyogenes Cas9 (SpyCas9) (Staphylococcus aureus Cas9 (SauCas9), which is ∼300 amino acids smaller than SpyCas9  ,9. HowevSpyCas9) . Therefo SpyCas9 ,14. It wIn the long process of evolution, phages are challenged by bacterial and archaeal CRISPR-Cas systems, and in order to survive they have evolved a counter-measures ,16. Cas Staphylococcus, inhibits SauCas9 activity  (Novagen) cells and were induced with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at OD600 = 0.6 for 16 h at 18°C. Cells were harvested and resuspended in lysis buffer , disrupted by sonication, and purified on glutathione sepharose 4B (GS4B) beads . Prescission protease was incubated in buffer  overnight at 4°C. The cleaved protein was eluted from GS4B resin. For Cas9 proteins, further fractionated by heparin sepharose column and cation exchange chromatography . For eluted AcrIIA14ct protein, further fractionated by cation exchange chromatography.The cDNA of full-length SauCas9, SpyCas9, NmeCas9 and AcrIIA14 were synthesized and sub-cloned into a expression vector pGEX-6P-1 . Mutants were constructed using a site-directed mutagenesis kit. All proteins were overexpressed in A280 absorbance to 18, as measured by Nanodrop One, before crystallization.To assemble the SauCas9–sgRNA–AcrIIA14ct–dsDNA complex, SauCas9 (N580A/C946A) protein was incubated with sgRNA (73 nt), AcrIIA14ct and dsDNA at the molar ratio of 1:1.2:5:2. Components were added in the order listed and incubated at room temperature for 5 min and 4°C for 40 min before adding the next component. The complex was applied onto size-exclusion chromatography  with buffer  to remove excess sgRNA, dsDNA and AcrIIA14ct. Finally, complexes were concentrated to an In vitro transcription with T7 RNA polymerase and template (dsDNA) for sgRNA was generated by PCR. All relevant sequences are listed in 2, 30 mM DTT, 2 mM spermidine, 2 mM each NTP , 100 μg/ml T7 polymerase and 500 nM transcription template. The sgRNA was purified by gel electrophoresis on a 8% denatured (8 M urea) polyacrylamide gel and followed by running Elutrap system. Finally, the sgRNA was resuspended in DEPC (diethylpyrocarbonate) H2O, stored at –80°C.The hanging-drop vapor-diffusion method was used for crystal growth. Crystallization conditions were screened on a large scale with mosquito robot and then optimized according to the results of the primary screening. All crystals used for diffraction were obtained by mixing 1 μl SauCas9 complex with 1 μl reservoir solution and incubated at 20°C for 7–14 days. Crystals of the SauCas9–sgRNA–AcrIIA14ct–dsDNA complex were grown from 1.0 M sodium citrate dihydrate, 0.3 M imidazol–HCl, pH 10.0, 0.02 M sodium malonate, pH 7.0.Before data collection, the crystals were transferred into cryo-protectant buffer  glycerol) and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at beamline BL-17U1 at Shanghai Synchrotron Radiation Facility (SSRF). All images were collected at the wavelength of 0.9791 Å with 0.5° rotation. The diffraction data for SauCas9–sgRNA–AcrIIA14ct–dsDNA was processed with HKL2000. The structure was determined by molecular replacement (MR) with the program PHASER. Each domain of the SauCas9 (PDB 5CZZ) was used as an individual search model for MR. The initial model was improved by several rounds of structural refinement in PHENIX. The final model was validated through MOLPROBITY and PROCHECK. Data collection and structural refinement statistics were listed in supplementary information, in vitro DNA cleavage assays were performed in 20 μl system containing 500 nM Cas9 RNP complex, 25 nM pUC19 plasmid in the buffer  glycerol). The pUC19 target DNA plasmid (target DNA with PAM sequence cloned into the pUC19 vector in Ssp1 restriction site) was linearized by EcoR1 digestion before the cleavage reaction. To test AcrIIA14 mediated inhibition of Cas9 to cleave target DNA, pre-incubated of AcrIIA14 with Cas9-sgRNA complex for 5 min at 37°C and molar ratios of Cas9 to AcrIIA14 ranging from 1:0 to 1:4. The cleavage assays were performed at 37°C for 30 min. Reactions were quenched by adding 6× TBE–urea gel loading and ran on 1% agarose gels stained with ethidium bromide for production detection.The 2, 1 mM DTT and 5% (w/v) glycerol. Streptavidin sensors were pre-equilibrated in the buffer for at least 10 min before used in experiments. Biotinylated SauCas9 protein (or SauCas9-sgRNA complex) were loaded onto streptavidin biosensors for 60 s. Binding kinetics were determined from binding data obtained with four or five concentrations of sample. The interference from the biotinylated protein with buffer were analysised as a control. Finally, we calculated the binding constant using Octet data analysis software.All ffinities were measured by biolayer interferometry (BLI) using the ctet RED96 system (FortéBio). All experiments were performed at 20°C with buffer containing 20 mM HEPES, pH 7.55, 100 mM KCl, 10 mM MgClIt was previously reported that three inhibitors of SauCas9, AcrIIA13-AcrIIA15, that share a conserved N-terminal HTH domain which can interact directly with the promoter-proximal sequences of the anti-CRISPR protein locus and plays a role in regulating protein expression, but is not necessary for inhibiting the cleavage activity of SauCas9 Figure S1A). The C-terminal domain in contrast, here referred to as AcrIIA14ct, is required and spans residues 60–159  Figure . A DALI KD = 58.0 nM versus 5.58 pM for mutant and wild type, respectively)  , implyinn Figure  and G. Oin vitro DNA cleavage assay showed that SauCas9 mutants R617G, K485G and K489G reduced sensitivity to AcrIIA14ct inhibition . This revealed that AcrIIA14 binding only induces significant conformational changes in the HNH domain and the L1 linker Figure . In detan Figure  and I. In Figure . As expen Figure  and I. TIn the crystal structure of SauCas9–sgRNA–AcrIIA14ct–dsDNA, the distance between the catalytic center of the HNH domain  and the scissile phosphodiester linkage in the target DNA strand (between the third and fourth nucleotides upstream of the PAM sequence) is ∼33.3 Å Figure . This suAccording to the previously reported Cas9 activation model, sequential binding of sgRNA and target DNA, the interaction between non-target strand and RuvC domain destroy the fragile stability of the original conformation between the RuvC, L2, L1 and HNH domain, which drives the movement of HNH domain to the cleavage site ,31. In tAs described above, during Cas9 activation the HNH domain undergoes a large-scale rotation and translocation. Based on the previously solved structure of Cas9 in the activated state ,30, we sFrom the above structural analysis, AcrIIA14ct binding leads to the formation of two new interactions between the HNH domain and L1 linker and the target-guide heteroduplex in the SauCas9 complex. Mutating the residues that mediate these interactions reduced the effectiveness of AcrIIA14ct Figure  and I. TLike AcrIIA14, AcrIIC1 and AcrIIC3 prevent Cas9 protein from cleaving target DNA via direct binding to the HNH domain, whereas their action does not interfere with sgRNA and target DNA binding ,26. To cIn contrast to AcrIIC3, AcrIIA14 binds on a surface of the HNH domain far from the nucleic acid heteroduplex, interacting with the protrusion formed by the two α helices of the HNH domain Figure . This stin vitro cleavage assays  is significantly higher than to sgRNA (KD = 473 pM) or to target DNA (535 pM)  . This alThe atomic coordinates of the crystal structure of SauCas9-sgRNA-AcrIIA14ct-dsDNA have been deposited in the Protein Data Bank (PDB ID 7EL1).gkab487_Supplemental_FilesClick here for additional data file.


Food waste is a current global problem. The aim of this work was to investigate the possibility of reintroducing bread discarded by retailers in the preparation of sugar-snap cookies. Bread flours were obtained from stale breads (white and whole wheat) milled with 200, 500 and 1000 μm sieves. Cookies were elaborated using 100% bread flours and combinations of 50% of bread flour and wheat flour. The rheology of the doughs, the dimensions of the cookies, their texture and colour were evaluated. Bread flour doughs presented higher G’ (elastic modulus), G” (viscous modulus) values than the control, especially with increased particle size. Bread flour cookies had a smaller diameter and a harder texture than the control, but in the case of whole bread flours of larger particle sizes, those differences were reduced. Cookies made with bread flour had a darker colour and higher a* values. The 50% mixtures did not present significant differences with respect to the control in terms of dough rheology, hardness, or lightness. Although the spreading factor was reduced, it was more similar to the control than to 100% bread flour cookies. Wasted bread flour can thus be used to replace wheat flour in cookie formulations. An estimated one third of the food produced in the world is wasted. This waste generates a high expenditure of resources, soil, energy, chemicals and materials needed along the value chain. It also produces a high environmental impact that might be reduced by minimizing food waste or through better food products management practices . In receBread is a product formulated mainly with flour and water. Due to the fermentative processes and the high temperature bread is submitted to, flour undergoes important modifications during the baking process. The high temperature of baking favours the denaturation of proteins and the gelatinisation of starch . TherefoFortunately, breads with lean formulas usually harden before there is any appreciable development of microorganisms, so they are discarded without a high microbial load and can be milled to be reused in the form of flour or semolina.In the production of sugar-snap cookies the gluten network is not developed, nor does the starch gelatinise, due to the absence of sufficient water in the formulation. In fact, these cookies can be elaborated with flours that have undergone heat treatment . Flours The aim of this work was to evaluate the use of flours obtained from stale breads in sugar-snap cookies. For this purpose, two different bread types  were ground with different sieves  to obtain flours of three different particle sizes. Flour colour, dough rheology and dimensions, as well as cookie texture and colour were analysed. A focus group discussion has also been included to evaluate the sensory quality of the obtained cookies.The following ingredients have been used in the preparation of the different cookies: wheat flour  provided by Harinera Castellana S.L. , traditional white bread  and whole wheat bread . purchased at Carrefour , sucrose , Argenta “cream” margarine , sodium bicarbonate  and local potable water.Breads were bought and allowed to dry at room temperature for 24 h prior to milling. Bread flours were obtained using 100% of traditional white bread (TBF) or 100% of whole wheat bread (WBF). Breads were ground with a ZM 200 ultra-centrifugal mill  equipped with three different sieves  to obtain flours of different particle sizes. These particle sizes were evaluated using a Mastersizer 3000  and values of D [Nine different sugar-snap cookies were elaborated. Control was prepared with wheat flour and six samples with 100% of each bread flour. Two other samples were elaborated with 50% of the flours (wheat flour/TBF1000 and wheat flour/WBF1000). These cookies have been called TBF1000 50% and WBF1000 50%, respectively. The cookie control formulation (CF) and the eight different cookies with bread flour were prepared according to the recipe presented in To prepare the cookie, margarine was heated in a microwave for 1 min (1000 watts) and creamed with sugar at speed 4 for 180 s in a Kitchen Aid 5KPM50 mixer  with a flat beater, scraping down every 60 s. Then, water was added, and the batter was mixed at speed 4 for 120 s. The finals ingredients added were flour and sodium bicarbonate. Mixing was performed at speed 2 for 120 s, scraping down very 30 s. After mixing, the dough was allowed to stand for 30 min before lamination in a Salva L-500-J sheeter . Samples were cut  and taken to the oven  at 185 °C for 14 min. The cookies were cooled for 60 min before stored in properly closed plastic bags at a 20 °C food chamber. All cookies were prepared in duplicate.The rheological behaviour of the dough was measured with a rheometer Thermo Scientific HaakeRheoStress 1  and a Phoenix II P1-C25P water bath  that controlled the analysis temperature (set at 25 °C). The rheometer was equipped with a titanium parallel plate geometry sensor PP60 Ti (60 mm diameter and 3 mm space). For the rheology test, doughs of 3 mm thick and 60 mm diameter were used. Panreac vaseline oil was applied  on exposed surfaces to prevent samples from drying out during the test. Samples were left at initial position for 300 s prior to the measurements. The tests were then started by a strain sweep test (0.1–100 Pa) at a constant frequency (1 Hz) to identify the linear viscoelastic region of the sample. The strain value was then employed to carry out a frequency sweep test (10–0.1 Hz), which led to the results of elastic modulus (G′ (Pa)), viscous modulus (G″ (Pa)), complex modulus (G* (Pa)) and loss factor (tan δ). Two repetitions were performed and each of them was analysed in duplicate.L* a* b* colour space [Cookie characteristics were determined 24 h after their elaboration. Width and thicknesses were measured with a calliper. The diameter of each cookie was measured twice perpendicularly, to calculate the average diameter. The spreading factor was calculated by dividing the average width by the thickness. Colour of flour and cookies were measured with a PCE-CSM 2 colourimeter  using the D65 illuminant with a 2° Standard Observer. Flour was placed into a cylindrical plastic recipient with 30 mm diameter and 15 mm height so that the samples could be considered as an infinite solid. Five samples of each flour were analyzed at the central point. Six cookies of each formulation were measured at the centre of the surface in two different batches (6 × 1 × 2). The results were expressed in the CIE ur space .Texture was analysed using a TA-XT2 texture analyser  and a sounding probe HDP/3PB with a test speed of 2.0 mm/s. The hardness of the cookie was measured by the maximum force required to break the sample. The analysis of dimensions, colour and texture were carried out on six cookies of each elaboration.A consumer test was performed in a hedonic sensory evaluation with a sample of 97 volunteer cookie consumers aged from 16 to 60 years old. This evaluation was carried out in individual booths at the Sensory Science Laboratory of the Agricultural Engineering College at the University of Valladolid, Palencia, Spain. These analyses were carried out according to the protocol previously approved by the Committee of Tests and Research from the Hospital Rio Carrión . Cookies were evaluated using a 9-point hedonic scale, ranging from score 1 for extreme dislike to score 9 for extreme like. Appearance, texture, taste, odour and the overall acceptability of five cookies were evaluated . Samples were presented on white plastic dishes coded with four-digit random numbers and served in random order. Water was available for drinking between samples. Cookies were elaborated 24 h before and stored in plastic bags at 20 °C.p < 0.05. A multivariate analysis was used to determine whether there are correlations between some of the different parameters studied.To perform the statistical analysis, the Statgraphics Centurion XVI software  was used. The data for the different variables were processed through an analysis of variance (simple ANOVA), using the Fisher LSD test to describe significant differences between averages at a significance level of As shown in In bread making, starch gelatinisation occurs ,14, whenThere is a clear correlation between rheology results and cookie size. Thus, the lower the value of G’, the larger the cookie diameter (r = −0.77) and spread ratio (r = −0.82), and the greater the cookie thickness (r = 0.81). Similarly, the lower the G” value, the greater the cookie diameter (r = −0.84) and spreading factor (r = −0.88), and the greater the cookie thickness (r = 0.81). Also, the highest correlation was observed between the tan δ and the spread ratio (r = 0.93). This correlation is logical since cookie dough expands in the oven according to its rheological properties; the greater the expansion, the lower the resistance of the dough to flow ,21,22,23Regarding texture, cookies elaborated with bread flour were harder than those made with wheat flour. However, it should be noted that cookies with 50% mixtures did not differ significantly from those elaborated with wheat flour. In the case of TBF, there were no significant differences between the hardness of cookies elaborated with flours of different particle size. However, in the case of WBF, there was a progressive decrease in cookie hardness as the particle size of the flours increased.In this study, cookie hardness correlated 99% with both diameter (r = −0.87) and spreading factor (r = −0.80). It seems logical, therefore, that cookies that expanded less and were more compact were the hardest ones. This correlation has been observed previously ,23. On ta* value presented lower values, that means less red tone in cookies with wheat flour than in the other cookies. Regarding lightness, control presented the highest values, but they only showed significant differences in cookies made with WBF and intermediate particle size TBF.In terms of colour , there wa*, b* values than the control, and lower lightness values. These results might be related to the presence of the crust in the flours that are darker due to the Maillard reactions that occur during baking. Therefore, the colour of the flours , the elastic and viscous components of the dough increase, the expansion during baking is reduced, and the hardness of the cookie increases. However, with 50% flour replacements the results obtained are very similar to the control. Furthermore, there are few differences between using a traditional bread flour or wholemeal bread flour in terms of oven expansion, colour, and texture of the cookies, although there may be differences in the sensory and nutritional aspects. It should also be noted that for the cookie making process the product does not need to have an excessively thin milling, and it may even be better if it is not; a 1000 μm sieve should be sufficient.


In spite of being a significantly growing segment, there are still problems regarding the nutritional, technological and sensory profiles of gluten-free products. Thus, the combination of a variety of functional ingredients is required in order to achieve the desired product quality.Three types of flour, chestnut, buckwheat and potato, were chosen in this study because they are all gluten-free, nutritionally richer and technologically more advantageous than wheat flour. They are combined with chia seeds, which are also functional ingredients as they are rich in dietary fibre and unsaturated fatty acids. Therefore, this study aims to evaluate the utilization of chia seeds with chestnut, buckwheat and potato flour in biscuits as overall quality enhancers in gluten-free products. The proximate composition, total phenolic content, antioxidant activity, some biscuit quality parameters and the sensory properties of the samples were investigated, and some changes in these products during storage were monitored and evaluated.According to the results, biscuits with chestnut flour had the highest phenolic content  per 100 g dry sample) and total antioxidant activity  per 100 g dry sample). Biscuits with chestnut and chia seeds had the hardness of 30.1 N (p<0.05). In addition, the use of chia seeds significantly increased the overall acceptability and flavour scores according to the sensory analysis results. During storage, chia seeds affected the oxidation stability; however, the fatty acid profile remained almost unchanged, except for the losses in lauric, stearic and α-linolenic acids (p<0.05). In conclusion, the biscuits with chestnut and chia seeds were more attractive than those made with other types of flour, with remarkably better nutritional characteristics and sensory attributes.The study fulfils a need for the growing gluten-free market by combining together the functional nutrients of chia seeds, chestnut flour and buckwheat flour to achieve the nutritionally improved and organoleptically acceptable gluten-free biscuits. Furthermore, this study makes an overall evaluation of the changes in product quality during storage to provide new ideas for an overall innovation in the gluten-free food market. Coeliac disease is defined as the abnormal response of the immune system to wheat gluten and related prolamins of rye and barley  flour with buckwheat (Fagopyrum esculentum) flour and chestnut (Castanea sativa Mill.) flour were used together with chia  (Lamiaceae) seeds in gluten-free biscuit formulations to enhance both nutritional and technological drawbacks.Nutritional properties of gluten-free products should be more in focus, as traditional gluten-free ingredients/flour are high in carbohydrates but low in proteins and antioxidants. Therefore, the use of dietary fibre-rich ingredients is proposed to improve the nutritional quality of these products (Solanum tuberosum) flour is commonly used in gluten-free products because it is rich in essential amino acids , protein dietary fibre and it contains several phytochemicals  in addition to its high carbohydrate content (Potato (Fagopyrum esculentum) is a pseudocereal. Buckwheat flour, which is rich in catechins serving as antioxidants, also includes specific amino acids , as well as minerals. Buckwheat proteins are mainly composed of albumins and globulins, with only a low amount of prolamins. In this respect, they are similar to leguminous proteins and considered as gluten-free (Buckwheat (Castanea sativa M.) flour is popular in gluten-free formulations, due to its high protein quality with essential amino acids, high dietary fibre content and vitamins  and minerals like potassium, phosphorous and magnesium. Recent studies have evaluated the effects of supplementation of gluten-free biscuits and bread with different levels of chestnut flour  originates from Central and South America , and particularly its seeds possess a technically unique property by means of which they can absorb large amounts of water to immediately form a transparent gel referred to as ’chia mucilage‘  buckwheat flour, (ii) chestnut flour, (iii) buckwheat flour and chia seeds, and (iv) chestnut flour and chia seeds were used for the production of gluten-free biscuits. For these formulations, buckwheat flour , chestnut flour , potato flour , chia seeds  lactose-free milk  were provided by the respective companies. In addition, margarine, sugar and whole eggs were purchased from a local grocery store in Turkey.Four different formulations of dough prepared with: (Ethanol (≥99.8%), acetone (≥99%) and copper were from Delta Agricultural Chemicals Industry and Trade Inc. , sulfuric acid, sodium hydride, petroleum ether, hydrochloric acid, boric acid, potassium chloride, sodium bicarbonate, aluminium chloride and trifluoroacetic acid were from Merck . Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) used in this work were from Sigma-Aldrich Co. .In this study, four different types of gluten-free biscuit formulations were produced according to a modified method by Öksüz and Karakaş  , based on AOAC method 985.29 (enzymatic-gravimetric method) (g), sonicated  for 15 min and centrifuged  at 4000×g for 20 min. The extraction was repeated until 30 mL of extract were collected in two replicates for each sample.A slightly modified extraction procedure was applied to prepare the biscuit extracts for measuring the total phenolic content and antioxidant activity  of the biscuit extracts was measured using the DPPH radical scavenging activity method  for 10 min. The percentage of absorbed water was measured by weighing the sediment.The diameter and thickness of the biscuit samples were measured using a digital caliper. Bulk density was measured by dividing the mass of each sample by its volume. The spread ratio was measured by dividing the diameter (D) by the thickness (T). All measurements were made in triplicates. The volume of the biscuits was measured using the rapeseed displacement method by comparing the change in the known volume of rapeseed with each biscuit sample, as described in AACC method 10-05.01 , using the Hunter scale of L* values), redness  and yellowness  of the biscuit samples and the standard white plane parameters given above were calculated to measure the total colour change (ΔE) according to the equation below:The difference between the colour parameters of darkness/lightness , fitted with a 50-kg load cell and coupled with Texture Expert Software, v. 1.22  in locked sterile bags for a period of 45 days. Moisture content of the samples was measured as described above, peroxide value (PV) was determined by iodometric method ISO 3960:2017  protein mass fraction (5.2%) than the other ingredients. This finding is commensurate with previous findings reported in the literature, since chestnut flour reported by Šoronja‐Simović Total mass fractions of protein (6.7%), ash (2.8%) and carbohydrates (81.1%) in potato flour were slightly different with respect to the other flour sources; although the fat mass fraction was significantly higher than the other ingredients (except for the chia seeds) and the moisture mass fraction was lower (p<0.05). In a recent study (Chia seeds had significantly higher protein (23.1%), ash (4.58%) and dietary fibre (34.4%), but lower carbohydrate (36.2%) content than the flour sources (p<0.05) used in this study. Comparable to the current findings, chia seeds were reported to have 9.3% moisture, 25.1% protein, 5.5% ash and 26.2% fat  were detected in the total ash and moisture mass fractions in different biscuit samples . On the The use of ingredients with relatively high amount of phenolic compounds in gluten-free products has a significant benefit for coeliac patients due to their ability to bind to fibre moieties, thus making them readily absorbable in the colon. In addition to the direct antioxidant activity of phenolic compounds, this binding ability was proposed as a beneficial mechanism for coeliac patients by modifying the composition and immune function of the gut microbiota  on dry mass basis) of the biscuit samples changed from 248.2 to 400.2 mg/100 g . BiscuitChia seed supplementation to the biscuit formulation, on the other hand, significantly (p<0.05) decreased the total phenolics . The amoTotal antioxidant activity (TAA), expressed as Trolox equivalents (TE) on dry mass basis, of the biscuits varied between 78.2 and 155.5 mg/100 g . In paraSpread ratio (diameter/thickness ratio) is among the most important characteristics of biscuits in determining their quality. Spread ratio values were higher (p>0.05) in the biscuits with buckwheat flour than in its chestnut flour-containing counterparts . MoreoveHardness of biscuits is among the distinctive textural parameters for consumer acceptability and general textural properties of biscuits are highly related to the starch gelatinization and sugar content, rather than the protein/starch structure . Effect According to the results in L* values), redness  and yellowness  in addition to total colour change (ΔE) were determined and the results are given in L* values than the biscuits with chestnut flour (p<0.05). Addition of chia seeds did not seem to make any significant changes (p>0.05) in the lightness. In the biscuits with chestnut flour, redness values were slightly higher , while the addition of chia seeds had a lowering effect on redness. Although yellowness was slightly higher in the samples with buckwheat flour, the difference was not statistically significant (p>0.05). ΔE data revealed that chestnut flour had a higher effect on the colour of biscuits (p<0.05). Similar to our findings, the use of chestnut flour as a substitute for wheat flour (increasing from 20 to 60% and 0 to 100%) resulted in the increase of the redness of the cookies and bread samples, respectively  in terms of the evaluated sensory parameters. Studies indicating that the level of buckwheat flour substitution in biscuits affected their sensorial acceptance could be found , chia seeds seemed to positively affect the flavour of buckwheat biscuits . Due to According to the results in et al. . This is expected, since the total amount of chia seeds in the biscuit formulation is around 10% of the total flour content, with mostly unsaturated fatty acids. In the literature, the PV of cookies with added chia seeds (10%) was reported as 2.81 mmol/kg at the end of 30 days of storage  is the most common indicator for measuring the oxidation during storage. Significant increase of PV values during storage was detected in all samples (p<0.05). Oxidation stability of biscuits with chestnut flour and chia seeds was found to be lower than the remaining samples at the end of storage, with relatively high PV expressed as OThe fatty acid composition of the biscuit samples (after the 1st day and after 6 months of storage) is presented in etc.), with significant protein and dietary fibre contents, superior product quality and better sensory acceptability.Recent research on gluten-free products has led to the improvement of nutritional profile of these products as the primary point of interest. The results presented here confirm the importance of using functional ingredients in gluten-free biscuit formulations as well as monitoring their storage stability. The results confirm the relative stability of biscuits during storage, particularly of those containing chestnut flour and chia seeds. Therefore, it was concluded that these functional ingredients have the potential to be used in different gluten-free product formulations (biscuits, crackers, snacks,


Plants utilize cell-surface localized and intracellular leucine-rich repeat (LRR) immune receptors to detect pathogens and to activate defense responses, including transcriptional reprogramming and the initiation of a form of programmed cell death of infected cells. Cell death initiation is mainly associated with the activation of nucleotide-binding LRR receptors (NLRs). NLRs recognize the presence or cellular activity of pathogen-derived virulence proteins, so-called effectors. Effector-dependent NLR activation leads to the formation of higher order oligomeric complexes, termed resistosomes. Resistosomes can either form potential calcium-permeable cation channels at cellular membranes and initiate calcium influxes resulting in activation of immunity and cell death or function as NADases whose activity is needed for the activation of downstream immune signaling components, depending on the N-terminal domain of the NLR protein. In this mini-review, the current knowledge on the mechanisms of NLR-mediated cell death and resistance pathways during plant immunity is discussed. At the g others ,4. This g others ,6.However, in resistant plants, effectors can be perceived either directly or indirectly by a second class of immune receptors, the intracellular nucleotide-binding (NB) leucine-rich repeat (LRR) receptor (NLR) family. NLR activation through effector recognition initiates a robust immune response referred to as effector-triggered immunity (ETI) ,8. ETI rNLR genes were cloned in the early 1990s from tobacco  and Araidopsis; ), and siidopsis; . EspeciaThis mini-review aims at summarizing some milestones in NLR biology. It further provides an overview of the recent achievements in determining how NLRs might mobilize antimicrobial defenses, while simultaneously it outlines the still unanswered questions of NLR-mediated immunity.Apaf-1), Resistance (R)-protein, and cell death abnormal (CED) protein) is subdivided into three subdomains: the NB domain, an ARC1 or helical domain 1 (HD1) and an ARC2 or winged-helix domain (WHD), and belongs to the AAA+ ATPase superfamily [R) domain [R domain). The CC and CCR domains share structural homology with the N-terminal 4-helical bundle of mammalian and plant mixed-linage kinase like (MLKL) proteins and pore-forming fungal HET-s/Helo domain containing proteins [Most plant NLRs, as well as the metazoan NLRs, have a modular and conserved tripartite domain architecture, with a variable N-terminal domain, a central nucleotide-binding and oligomerization domain (NB or NOD), and a C-terminal leucine-rich repeat (LRR) domain. The plant NB domain  domain . The difproteins . TIR domproteins ,31, wherproteins . Plant Nproteins .Peronospora parasitica 1A (RPP1) [Brachypodium distachyon lines confirmed that LRR domains of many NLRs show high allelic diversity especially in residues or regions predicted to be specificity determining [R and TIR domains mediate NLR signaling and are required or at least participate in NLR oligomerization, and can therefore be considered as the signaling domains. A number of studies demonstrated that overexpression of isolated CC/CCR or TIR domains was sufficient to induce cell death or auto-immunity [Intramolecular interactions between the different domains keep NLR proteins in an inactive, but signaling competent state, in which the NB-ARC domain binds adenosine diphosphate (ADP) via conserved and functionally important motifs. The NB-ARC domain likely functions as a molecular switch, transitioning the NLR from an inactive ADP-bound to an active adenosine triphosphate (ATP)-bound state ,36,37. HA (RPP1) . The C-tA (RPP1) . DeletioA (RPP1) . In addiA (RPP1) ,43. CryoA (RPP1) ,43. Furtermining . This suermining . The N-timmunity ,39,45–48N. benthamiana TNL ROQ1[Within the last two years the publication of cryo-EM structures of three full length plant NLRs, the Arabidopsis CNL ZAR1 ,49 and T TNL ROQ1, have be TNL ROQ1. Direct  TNL ROQ1,43,51–53 TNL ROQ1. This clIndirect recognition of effectors is mediated by NLRs that guard effector targets or that interact inducibly with effector-modified host proteins. Often these host proteins have a function in immunity, and one of the best-studied examples is the small Arabidopsis protein RPM1-interacting protein 4 (RIN4), which is guarded by two CNLs RPM1 and RPS2 . RIN4 isHow effector-recognition can mechanistically induce NLR activation was impressively demonstrated by recent cryo-EM studies that presented the structure of inactive and effector-activated Arabidopsis CNL ZAR1 ,49,61. ZAn activation mechanism similarly depending on effector recognition and oligomerization  was recently postulated for the two plant TNLs RPP1A and ROQ1 ,43. CryoR-NLR) oligomerization is most likely required to bring the N-terminal domains into close proximity so that these can facilitate their signaling function or the recruitment of downstream components. This is further supported by many reports demonstrating that the expression of TIR or CC/CCR domains alone is sufficient to induce cell death and cause (auto-) immunity [Plant NLR activation by effectors or by mutations affecting nucleotide binding leads to oligomerization and the formation of large NLR complexes, termed resistosomes ,38,43,61immunity ,39,45–48The mechanism(s) of initiating immunity is however very different between the two major NLR classes, the TNLs and CNLs. Intriguingly, all tested TNLs require the presence of members of the RNL family, a CC-NLR subfamily, for proper cell death and resistance activation, and therefore RNLs are also considered as helper NLRs ,71–77. I2+ permeable cation channel at the plant plasma membrane [2+) fluxes and the subsequent initiation of cell death and immunity. How ZAR1 activation exactly triggers cell death during an ETI response is still unclear. However, cell biological time-lapse analyses have shown that ZAR1 activation leads to intracellular calcium influx and reactive oxygen species (ROS) production, changes in morphology of cellular compartments/organelles and ultimately to rupture of the cell, most likely due to loss of plasma membrane integrity. All these responses were lost when the conserved glutamate (E11) was mutated, indicating that channel activity and therefore most likely Ca2+ influxes are upstream of these responses [The cryo-EM structure of activated full-length Arabidopsis CNL ZAR1 suggested that the CC domains of the five ZAR1 proteins in the resistosome complex form a funnel-like structure that may penetrate the plasma membrane to either build a pore or channel or to disrupt membrane integrity . A recenmembrane . Formatiesponses .2+ permeable cation channels at the plasma membrane  channel activity is also required for CNL and RNL-mediated signaling leading to disease resistance needs to be determined. There are some examples where NLR-mediated disease resistance does not require cell death or where cell death can occur even in the absence of resistance — thus, cell death and resistance are not necessarily coupled [2+ fluxes by lanthanum blocks the Arabidopsis RPM1 (CNL)-triggered cell death response, but not disease resistance, may indicate that Ca2+ fluxes are, at least in case of RPM1 mediated immunity, not required for disease resistance [in planta is also not really studied, but this would be important to know, since determining resistance to infections in plants is normally a matter of days.It was previously shown that the Arabidopsis CNL (RPM1) and the ADR1 RNL-subfamily self-associate and localize to and function at the plasma membrane to induce a cell death response ,65. Selfmembrane , and thaR domain . The impR domain . It is vR domain ,64,83, sR domain ,63,65,84 coupled ,85,86. Tsistance ,65. HoweTNL-triggered immune responses require the presence of the helper NLRs (in Arabidopsis the RNL members of the ADR1 and NRG1 subfamilies) and the lipase-like protein family members Enhanced Disease Susceptibility 1 (EDS1), Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101) ,81,87–89CNL-mediated ETI and also TNL-mediated ETI, which is essentially activation of RNLs ,73–76 F, lead toThe mechanisms leading to the loss of cell integrity during ETI are not well studied. There are a few reports showing that NLR activation or the expression of viral, bacterial and fungal effectors can induce severe morphological cellular phenotypes, like the fusion of the vacuole with the plasma membrane , alteratNLR activation leads to calcium influxes and probably initiation of other ion fluxes, and this in turn can activate other immune regulatory proteins, including cyclic nucleotide-gated cation channels (CNGCs)  Figure , CPKs anImportance of the field: Intracellular NLR receptors are important components of a plants defense system against host-adapted and non-adapted pathogens. NLR-triggered immunity leads to strong and robust defense responses and thus understanding their mechanism(s) of action is of high importance for agricultural research to generate better and durable resistant crop plants.Current thinking: Recognition of pathogen-derived effectors activates NLR proteins and induces their self-association, which in turn leads to oligomerization of their N-termini and the production of signaling molecules or the formation of a cation channel at cellular membranes, depending on the architecture of the N-terminal domain. NLR activity generally, but not always, leads to a strong immune response in the whole organism and an induced cell death of the infected cell.Future directions: Identifying NLR binding partners that might contribute to their specific functions and regulate their activities, will be crucial in enhancing our understanding of how the different types of NLRs mediate immunity against a wide variety of pathogens. Determining the exact site of NLR action and the mechanism of cell death induction is also one of the fields next important and interesting endeavors.


The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis. The divisome is a multiprotein dynamic complex driving bacterial cell division. The best known division mechanism is binary fission, for which the following processes need to proceed: chromosomal DNA replication and segregation, constriction of the inner membrane (IM), septal peptidoglycan (sPG) synthesis, and invagination of the outer membrane (OM) in Gram-negative bacteria . The divDivision is a costly process for the bacterial cell, as it needs to increase in mass, double its genome, and produce a PG septum, which will be significantly degraded during daughter cell separation ,8. TheseN-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues, is of utmost importance for the structural integrity of the bacterial cell. However, peptidoglycan also has an additional role during bacterial fission, forming a physical septum between two future daughter cells . Th. Th66]. Thus, it was long assumed that the pedestal domain was primarily involved in FtsN recruitment. However, a recent study found the mutation K211I next to the division-defective R210C mutation could bypass FtsN, indicating that this region is involved in FtsWI activation . G57D, SAs with K211I, the more distant R167, which may interact with E193, can partly bypass FtsN when replaced with a serine residue (S) . ResidueOverall, the FtsI pedestal domain appears to be involved in the activation of sPG synthesis, likely by regulating the GTase activity of FtsW. The fact that in vitro FtsW GTase activity can be activated by a catalytic inactive FtsI variant indicates that the TPase domain is not implicated in FtsW activation . In vitrA figurative tug-of-war for the putative catalytic D297 residue between the region around M269 and the region around E289 could be a possible mode of regulation for FtsW . An inteRegulators may modulate the stability of this interaction, as it is determinative for FtsW GTase activity ,25. ThisBacillus, Escherichia and Pseudomonas genera, and the ovoid-shaped Staphylococcus and Enterococcus bacteria . ThL86F and FtsLE87K) cannot be rescued by FtsN overexpression or active FtsLG92D/E88K mutants, unlike other FtsL DN mutants [M269I and FtsWE289G), indicating that especially L86 and E87 are directly involved in the transmission of an activating signal to FtsWI. Moreover, a mutant lacking the cytoplasmic domain, FtsLΔ1–30, combined with an E88K mutation can still rescue and bind FtsWI; however, additional L86F or E87K DN mutations cancel this effect. This may seem counterintuitive, as it was just mentioned that the cytoplasmic interaction is essential for FtsWI midcell recruitment. Notably, recruitment by a periplasmic FtsL occurs only with additional SF mutations, showing that recruitment is mainly controlled by the cytoplasmic domain [The main activating allosteric interaction appears to be the one between the AWI domain of FtsL and the periplasmic domain of FtsI. Certain mutants in the AWI domain  may interact with the FtsL AWI domain through residue L86. FtsIV86E and FtsIG57D, which are defective in FtsW activation, can be rescued by SF mutant FtsLG92D/E88K [M269I. This supports the FtsW dependance on the FtsI pedestal domain and suggests that the FtsL AWI domain is involved in the stabilization of the FtsWI complex. Overall, an interaction between the FtsL AWI domain and the distal part of the FtsI pedestal domain seems to stabilize the active FtsWI complex, leading to an accelerated sPG synthesis state.The periplasmic interaction is most likely between the AWI domain of FtsL and the pedestal domain of FtsI. A stronger interaction has been reported between FtsI and FtsLL mutant . Based o92D/E88K . HoweverL24 is in the cytoplasmic region that recruits FtsW and is predicted to interact with hydrophobic residues in transmembrane domain 1 (TM1), TM2, and TM10 of FtsW  conformation, as charge substitution on FtsLR61 leads to a mild DN phenotype. Overall, these studies indicate that FtsL stabilizes FtsWI, where especially the AWI domain is involved in the stabilization of the FtsI pedestal domain, thus increasing FtsWI activity.The other two DN mutants outside the AWI domain, L24 and R61, are also involved in the stabilization of FtsW, though they seem less involved in the hyperactivation of FtsWI . FtsLL24 of FtsW d. A FtsWFtsWG282 c. IntereA major missing aspect is the in vitro measured inhibition of the TPase activity of FtsI by FtsQ, in contrast to FtsBL, which did not affect FtsI activity . While F1−57 with FtsI was confirmed by co-IP, and an earlier study reported a potential interaction between FtsI region 51–70 and FtsQ [51−70 consists of an α-helix and a β-sheet  increases the thermal stability of the coiled coil and leads to elongated cells. Interestingly, the adjacent FtsLR74E mutant produces a SF phenotype, indicating a polar cluster that provides dynamic regulation of the stability of the coiled coil. Concurrently, simulations of FtsBL interactions indicate that the coiled coil regions of FtsB and FtsL only tightly interact in the periplasm around a hydrophobic pocket consisting of residue between the TM and CCD domains , while polar clusters in the putative coiled coil destabilize the structure [FtsBL is most likely in a tetrameric (2:2) Y-conformation, where each branch of the Y forms a FtsBL coiled coil . It has tructure . Thus, ttructure .EFtsN unconditionally when mutated, as mentioned earlier. This would suggest that the CCD of FtsB would be a key suppressing region in the context of sPG synthesis. However, there are no indications that residue E56 interacts with either FtsL, FtsQ, FtsI or PBP1b, as would be expected if the region allosterically suppresses sPG synthesis [1−55) can still recruit both [The evidence for a stabilizing role of the FtsB CCD domain is mostly indirect. CCD domain residue E56 is the only known residue that can bypass ynthesis . Furtheruit both . The struit both ,100. Toguit both . OverallThe inherent instability of the coiled coil domain seems to be stabilized by interactions in the C-terminal region of FtsBLQ. The helical structures of both FtsB and FtsL are indicated to break in or close to the CCD domains . EspeciaFurthermore, it is predicted that FtsBL also interacts past the CCD domain at the C-terminus . The intHere, a working model is produced describing the regulation of the FtsWI–PBP1b by FtsBLQ, based on the discussed data described above . The ratFtsBLQ stabilizes FtsWI–PBP1b by interacting with certain domains . Some ofEFtsN domain for PBP1b, which leads to the accelerated activity of PBP1b , and is heavily based on mutagenesis studies, which need to be validated by direct protein–protein interactions, subcellular localization, and in vitro mode of actions.From here, we can decipher a new model for septal peptidoglycan synthesis in In the model, the recruitment of the divisome is completed before the start of constriction. This includes the proto-ring , FtsEX, and a large synthesis complex consisting of FtsK, FtsBLQ, FtsWI, and PBP1b. FtsA is then still in an oligomerized state and thus delays the septum formation. Septation can start when FtsN recruits to the division site and FtsA converts to a monomeric conformation . At thisBased on the role of FtsK as a cell division DNA segregation checkpoint, we expect the whole synthesis machinery, including FtsK, to translocate. Unfortunately, only the dynamics of FtsN and FtsWI have been thoroughly examined for now . If the E. coli is predicted to be mostly single-layered based on atomic-scale simulations, with around 25% being three-layered, equivalent to the surface of the cell poles [Interestingly, peptidoglycan in ll poles ,121,122 ll poles ,122. FroThis model is a starting point and thus still very speculative. Most of the review is focused on the direct regulation of the septal peptidoglycan synthesis machinery through allosteric interactions with regulators. In this review, the combination of protein–protein interaction predictions and extensive literature research uncovered several putative interactions within FtsWI and with regulators that may stabilize the synthesis complex in active or inactive conformations. The next step is the validation of these interactions, as this would elucidate their nature. It is a fascinating model, based on a balance of multiple positive and negative allosteric interactions, of which the sum leads to the activation or inhibition of septal peptidoglycan synthesis.EFtsN, and FtsB and FtsQ [EFtsN–PBP1b interaction, could confirm the abovementioned interactions, which are key parts of the proposed model.Validation of the proposed interactions is needed, as a large part of the genetic analyses conducted with the DN and SF mutants of divisome proteins are heavily reliant on spot assays. These analyses rely on the principle that the (over)expression of certain mutants, or the overexpression/deletion/depletion of certain proteins, may result in varying bacterial growth and viability. These methods provide a quick and straightforward determination of growth and viability in response to mutagenic changes. However, these spot assays, which are in essence ‘blackbox system’ experiments, lack validation of interactions and the assessment of FtsWI–PBP1b activity. Fortunately, in vitro activity and interaction assays have been conducted in the past. The most detailed interactions are those between PBP1b and and FtsQ ,98,105. and FtsQ ,26. SimiEFtsN on PBP1b GTase activity, and will likely be used in future studies of other activating or suppressing interactions [Furthermore, not many in vitro studies have been published in the last few years. The main papers concerning the in vitro effect of FtsBLQ/FtsN on FtsWI–PBP1b activity are the papers of Boes et al., and Marmont and Bernhardt ,26. The ractions .EFtsN is essential for cell division, combined with the partial redundancy between FtsN and DedD, indeed indicates alternative activation factors. DedD was not included in the model, as the direct position of DedD in the divisome is not yet discovered. This will most likely happen in the coming years, as DedD might represent another activation pathway, parallel to FtsN and perhaps through FtsWI. Luckily, these additional factors can easily be incorporated in the proposed model, as these would further contribute to an inactive or actively synthesizing state of FtsWI–PBP1b.Interesting years lie ahead, as this is only a glimpse into the world of the regulation of bacterial cell division. So, what to think about the role of peptidoglycan hydrolases, especially linked to the semi-essential FtsEX subcomplex? While only briefly discussed in this review, the activity of these proteins seems to contribute to the spatial distribution of sPG synthesis . Though Overall, an interesting model is provided that integrates multiple aspects of septal peptidoglycan synthesis. This includes a spatial model for the synthesis complex, multiple regulatory mechanisms, and a compelling organization model for newly synthesized PG strands. While still a pipe dream for now, the model provides new approaches for the development of new antibiotic compounds. Consider destabilizing the FtsWI regulatory site with small-molecule inhibitors, and the development of a compound that binds to the FtsL AWI domain or prevents synthesis complex recruitment with compounds mimicking the FtsQ domain. Altogether, the complexity of septal peptidoglycan synthesis is challenging, but simultaneously provides many opportunities for the development of new antibiotic compounds. This may provide us with new tools to tackle the emergence of antibiotic resistance, which is one of most significant challenges humanity will face in the coming few decades.