Significant intraband states were found in theunit cell of the engineered vacancies, proposing a meaningful domain-wallconductivity for miniaturized data-storage applications. Reductionof the Ti ion as well as enhanced charging and electric-field concentrationwere demonstrated near the vacancy. A 3–5% tensile strain wasobserved at the immediate surrounding unit cells of the vacancies.Engineering individual oxygen vacancies and topological solitons thusoffers a platform for predetermining both atomic-scale and globalfunctional properties of device miniaturization in metal oxides.Nanoscale devices that utilize oxygenvacancies in two-dimensionalmetal-oxide structures garner much attention due to conductive, magnetic,and even superconductive functionalities they exhibit. Ferroelectricdomain walls have been a prominent recent example because they serveas a hub for topological defects and hence are attractive for next-generationdata technologies. However, owing to the light weight of oxygen atomsand localized effects of their vacancies, the atomic-scale electricaland mechanical influence of individual oxygen vacancies has remainedelusive. Here, stable individual oxygen vacancies were engineered  Recent focus is put on 2D structures. The interface betweentwo metal oxides, such as LaAlO3/SrTiO3, isa common example.9 A prominent platform for such 2D structures is ferroelectricdomain walls, which separate neighboring regions with different dipole-momentand polarization orientations.10 Oxygenvacancies have been found lately serving as a hub for topologicaldefects, such as vortices and skyrmions as well as altering significantlythe properties of the encapsulating high-dielectric parent material,14 for instance by introducing 2D magnetism (Hf0.5Zr0.5O215) and 2D superconductivity(WOx16) atthe domain walls. Substantial effort has been given though to domain-wallconductivity in large-bandgap ferroelectrics, vastly because domainwalls are movable by will with external electric fields, giving riseto miniaturized memristive cells.19 While the success ofpresenting domain-wall conductivity in traditional perovskite ferroelectrics,such as BaTiO320 and Pb(Zr0.2Ti0.8)O3, has remained limited,23 attention has been given mainly to BiFeO3,27 ErMnO3,29 and LiNbO3,32 which show high and reproducible conductivity.Oxygen vacancies inmetal oxides are the basis for modern nanoscaleelectronics because they can modify the functional properties significantlywithout affecting the structure and the mechanical properties of thesematerials.37 Changes in the material electronic band structure and charge-carrierconcentration, both contributing to conductivity, have been observedas coupled with an increase in oxygen-vacancy concentration.39 At the mesoscopic scale, enhanced conductivity is frequently observedat walls that separate head-to-head (h–h)or tail-to-tail (t–t) polarization domainswith scanning probe microscopy.40 Theoretical41 and modeling42 analysesof these structures predict that such charged h–h (t–t) domain walls repel (attract) oxygenvacancies, which in turn help stabilize these charged domain wallsglobally. Recent atomic-scale scanning transmission electron microscopy(STEM) studies of bismuth ferrite demonstrated clusters of chargedpoint defects at domain walls.44 Initially, indirectobservations attributed these defects to oxygen vacancies.46 However, very lately, electron energy loss spectroscopy (EELS) studiesdetermined that charged defects at the domain walls are most likelydue to bismuth vacancies, questioning the existence and role of oxygenvacancies at the domain walls.47 A majorreason for the unresolved role of individual oxygen vacancies is thatmodeling point defects at domain walls within a collectively-interactingdipole matrix is complex. Likewise, observing these light atoms isa nontrivial task, and spotting individual vacant sites is even alarger hurdle, especially at the domain wall.50 That is, directobservations of the basic individual oxygen vacancies and the electricfield around it as well as the ability to manipulate these basic buildingblocks have remained elusive, limiting the development of advancedmetal-oxide-based nanoscale devices.The originof oxygen-vacancy-based functionality is at the atomicscale. Yet, observing, controlling, and even modeling individual vacancieshave remained a major challenge. Macroscopic-scale effects of oxygenvacancies are observed readily as an averaged behavior with spectroscopicmethods.in situ manipulation techniquestogether with density functional theory(DFT) modeling to demonstrate intentional formation of oxygen vacanciesat domain walls in the seminal ferroelectric perovskite BaTiO3. Complementary calculations expanded the work to other perovskiteferroelectrics and not only supported the stability of oxygen vacanciesat the domain walls, but also illustrated that they give rise to intragapstates, which are significant for conductance. Individual vacancieswere found to be inducing mechanical strain and electrical chargingat a distance of up to two unit cells, which is much smaller thanprevious predictions.53 Careful analysis of the dipole-moment, electric-charge,electric-field, and strain distribution around the vacancies showeda quasi-linear quadrupole that comprises a pair of h–h and t–t dipole moments. The contributionof this nonconventional topological structure to the local electric-fielddistribution at the domain wall was demonstrated independently.Here, we combined variousatomic-scale imaging and 3 50 nm crystalliteswere used,providing strain-free bulk-like behavior, while yet allowing sufficientelectron transparency to aid the TEM analyses. Recently, it has beenshown that ferroic domain walls can be formed, moved, and switchedcontactless in such materials in situ during atomic-scaleTEM imaging.55 Here, we used this method toform the domain-wall structure, which served as a template for theoxygen vacancies. The guiding hypothesis was that oxygen vacanciesare formed at a window dosage above the value that forms the domainwalls and before the material is damaged. Simultaneous STEM-baseddifferential phase contrast (DPC), integrated differential phase contrast(iDPC), high-angle annular dark field (HAADF), and EELS were usedto characterize the atomic-scale chemical-element, strain, charge,and electric-field distribution, exact atomic location of the ions,and the ions’ oxidation state, respectively .A detailed discussion regarding the oxygen vacancy within the 3D matrixgeometry is presented in Figure S2.in situ formation of an oxygen vacancy within a domain wall.56Figures S1 and S2.Localizationof the oxygen vacancies was demonstrated with theaid of high sensitivity to focal depth of the iDPC method.Figure S3). Two oxygen vacancies wereobserved here along the wall, and the dipole-moment distribution aroundthem was identical. In both cases, the dipole moments were aligned t–t at the site with the missing oxygen atom, accompaniedby an h–h alignment at the immediate neighboringunit cells from each side. Beyond the distance of already one unitcell, the dipole moments returned to their unperturbed h–t orientation. Previous studies proposedthat charged vertices of dipole moments (non-h–t) are accompanied by strain release.3958 In comparison to this value, Figure S4.S5 reveal that the oxidation state of the titanium ion changesfrom Ti4+ within the domains to a lower oxidation stateat the domain wall,62 where the oxygen vacancies are. Using the Kröger–Vinknotation, the vO formation can be summarized:OO× → vO•• + 2e′ + 1/2O2, so that the Ti ion transfers due to the vacancy from +4 to +2 tomaintain charge neutrality, whereas it can also transfer to +3 toallow charging.To further confirm the charging at the vacant site, atomic-resolutionEELS measurements were done across the domain wall. h–h/t–t/h–h structure.Typically, in perovskite ferroelectrics, dipole moments arrange in h–t to maintain charge neutrality,whereas charged domain walls comprise an h–h or t–t structure.In both cases, although the charge distribution is asymmetric, thedipole-moment distribution itself is centrosymmetric. Thus, in thesecases, the system can be described by means of dipole moments only.Nevertheless, in the current scenario of an h–h/t–t/h–h structure, the dipole moment distribution is not centrosymmetric,requiring a higher-order analysis of the system in terms of the multipoleexpansion. That is, this alternating dipole moment organization representsan individual unit of a quadrupole. The alternating dipole momentsorient nearly on one common line, with the exception that the dipolemoments near one of the h–h vertices has anadditional orthogonal component (within the image plane). Thus, thisstructure is a quasi-linear quadrupole. Traditionally, dipole momentsdominate the behavior of ferroelectrics and other dielectric materials.Higher orders of the multipole expansion, such as quadrupoles, areconsidered typically as negligible for the macroscopic behavior ofthe material because their contribution is significant only at theshort-range.63 Nevertheless, in the contextof domain walls, and in particular oxygen vacancies and other pointdefects at domain walls, short-range interactions become prominent.64Summing up the dipole-moment distribution aroundthe oxygen vacancyshows an 3 crystallite with an oxygen vacancyat the domain wall. The simultaneous iDPC mapping in h–h charged vertex . This local electric-field enhancement is typical for such a quasi-linearquadrupole.66 Following Gauss’slaw, the local charge distribution can be extracted from the electricfield; however, this should be done carefully. Mostly, the local fieldthat is mapped with DPC is the electric field, E,which corresponds to the free charge density, ρf.Because in ferroelectrics, bound charge (ρb) andthe resultant polarization dominate ρf and E, the entire displacement field (D) mustbe taken into account67 . That is,the charge density around the oxygen vacancy can be extracted fromthe divergence of the DPC mapping .The short-range electric-field distributionaround oxygen vacancieswas therefore examined at the subatomic scale by means of DPC.  mapping 3B:1where68 the latter is in agreement withthe above EELS results. Note that the oxidation state of the titaniumion was changed only in the charged h–h vertex closest to the vacancy. This asymmetric chargedistribution is accompanied by an off-axis diagonal displacement ofthe reduced Ti ion (d Ti ion 3C.SI for further details regardingthe DFT conditions). 3 that comprises an oxygen vacancy. We note that modeling a90° domain wall in the tetragonal phase of BaTiO3 withDFT turns out to be a nontrivial task. First, DFT calculations aretypically done at 0 K, at which BaTiO3 adopts the rhombohedralground-state phase, whereas a tetragonal structure of BaTiO3 is stable only at finite temperatures. Second, the energy of a 90°domain wall in BaTiO3 is expected to be even smaller thanthat of a 180° domain wall (7.84 mJ/m2).70 In comparison, the energies of 180° and 90° walls in PbTiO3 are 170 and 90 mJ/m2, respectively.72 Modeling a low-energy interface demands a high accuracy in energyand atomic forces. Moreover, to maintain a reasonable oxygen vacancyconcentration at the wall, a relatively large supercell is needed,further increasing the computational cost. These subtle issues likelyexplain the lack of DFT studies on 90° domain walls in BaTiO3 in the literature.74 Here we followed aprotocol developed to study highly unstable charged domain walls.42 A large 10√2 × 2√2 ×1 supercell of 200 atoms was adopted to minimize artificial interactionsbetween a defect and its mirror images. The supercell was dividedinto three zones: atoms in the two end zones were fixed to the bulkvalues of the tetragonal phase, while atoms in the middle region wereallowed to relax. This allowed for structural optimization of a low-energy90° wall sandwiched by two bulk tetragonal domains (fixed endzones). First, the optimized structure of defect-free 90° wallswas obtained .The DFT simulations agree withthe experimental data in severalimportant aspects. First, the simulations show an asymmetric dipolardistribution of the charged e Ti ion2C and 3C3. Three different types of oxygen vacancies at the wall werestudied, all leading to meaningful intragap states at the interface. This suggests that the defectlevels due to oxygen vacancies are promising for enhanced conductivity.To examine the effectof oxygen vacancies on the domain-wall conductivity,layer-resolved local density of states (LDOS) was computed. h–h or t–t dipole-moment organization at the domain wall, which in turn canserve as a hub for oxygen vacancies (and other point defects). Theabove results show that h–h and t–t can be local effects, giving rise to substantial changes in thelocal electric-field, charge, mechanical strain, dipole and quadrupolemoment distribution at the domain wall, and the oxidation state ofnearby ions. It is possible that local effects are more prominentin the case of 90° ferroelastic domain walls than in 180°domain walls because of the lower symmetry of the former. However,we believe that future work is required to examine this hypothesis.Most studies propose that domain-wall conductivity arises froma coherent –4 nA/nm2, the dominant interaction of the sample and theelectron beam is electromagnetic, rather than, say, local heating,resulting in domain-wall formation and switching.55 Moreover, it has been shown that for dosage values greater thanor equal to 270 nA/nm2, the electron beam interacts locallywith the sample, giving rise to cation escape and even to atomic-scaleetching of the material.59 In the currentwork, it was shown that individual oxygen-vacancy engineering is possibleat the dosage window between these two values. That is, the electronbeam has already formed domain walls, while it induces local chargingand isolated point defects, without yet damaging the material. Figure S10 shows the emergence of an oxygen vacancyin a switched domain wall, supporting this conclusion. Note that followingthe experimental results and the DFT calculations, we expect a similarbehavior for the oxygen-vacancy formation also in systems in whichthe domain walls were formed differently.The oxygen-vacancy manipulation presented in this work was obtainedwith the electron beam at a certain dosage window. Previous worksshowed that for dosage values less than 4 × 1053 Moreover, on many occasions  release in the currentwork stems from the high domain-wall periodicity in comparison tothe typical periodicity.The contributionof an atomic-scale topological structure was foundto be important not only to local properties but also for macroscopicproperties, such as electric conductivity. The above results highlightthe significance of local topological and structural effects, includingquadrupoles, which have been considered negligible with respect tothe macroscopic and device-relevant properties. Likewise, the aboveoxygen-vacancy engineering methodologies can be implemented for manipulatingexotic structures and functional properties of ferroelectrics andother metal-oxide materials.3 nanoparticlesof 50 nm size76 were purchased from US Research Nanomaterials, Inc.(99.9% pure). To evenly spread the particles on the TEM grid, theparticles were suspended in ethanol and sprayed on the amorphous carbon-coatedholey Cu grid using nitrogen gas.Commercially available single-crystal BaTiO–4 and 270 nA/nm2 . Lastly,the high accuracy of atom location obtained with iDPC56 was used to map the dipole-moment distribution with confidence.A key factor that helped with the characterization is that whilethe contrast in traditional STEM and HAADF methods is quadratic withthe atomic number—decreasing the signal-to-noise ratio of lightelements—the DPC methods are sensitive also to lighter elements,such as oxygen.77 with generalizedgradient approximation of the Perdew–Burke–Ernzerhoffor solids (PBEsol) type. Given the large supercell used to modeldomain walls, we used the GBRV ultrasoft pseudopotential78 and a 1 × 1 × 4 Monkhorst–Pack k-point grid for structural optimization and a 2 ×2 × 8 k-point grid for electronic structurecalculation. The plane-wave cutoff is set to 40 Ry, and the chargedensity cutoff is set to 200 Ry, respectively.79All first-principles DFT calculations were performedusing QUANTUM ESPRESSO


Aims: Gliomas are the most common malignant brain neoplasms with high recurrence and lethality rates. Recently, studies have reported that cyclin-dependent kinase 5 (CDK5) is involved in tumorigenesis. Herein, we applied bioinformatics analysis to determine the clinical value of CDK5 in patients with glioma and examined the effects of CDK5 on glioblastoma cell proliferation, apoptosis, and cell cycle in vitro.Methods: Gene expression profiles containing clinical data of low-grade glioma (LGG) and glioblastoma cohorts were obtained from The Cancer Genome Atlas database and analyzed to determine the association between CDK5 expression and glioma clinicopathological characteristics. Kaplan-Meier survival analysis was performed for prognosis analysis. Gene set enrichment analysis (GSEA) was used to identify the biological pathways involved in differential CDK5 expression. In vitro experiments were performed to explore the effects of CDK5 on glioma cell functions.Results: CDK5 expression was substantially higher in glioblastoma than in LGG. GSEA showed that some metabolism-related pathways were associated with the high CDK5 expression phenotype. In vitro experiments showed that CDK5 knockdown impaired cell proliferation and colony formation ability, and induced apoptosis and cell cycle arrest.Conclusion: CDK5 may act as a potential biomarker of glioma progression and a valid target for glioma therapy. Gliomas are the most common malignant brain neoplasms Cyclin-dependent kinase 5 (CDK5), a proline-directed serine/threonine kinase and a member of the CDK family, is abundantly present in the central nervous system Recent studies have reported that CDK5 is dysregulated in various tumor cells, and it participates in tumorigenesis In this study, we explored the prognostic value of CDK5 expression in patients with glioma based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Experiments were conducted to validate bioinformatics data reliability and the connection between CDK5 expression and clinicopathological features of gliomas.http://cancergenome.nih.gov) https://www.ncbi.nlm.nih. gov/geo) High-throughput sequencing data (HTSeq-FPKM) were directly downloaded from TCGA  and U87MG, U251MG, and U138MG human glioblastoma cell lines were obtained from our laboratory. NHAs were cultured in astrocyte medium. U251MG and U87MG were cultured in Dulbecco's Modified Eagle Medium  supplemented with 2 mM L-glutamine, penicillin (100 U/mL), and 10% fetal bovine serum .4 cells per well) were grown in 6-well plates  24 h before infection. Lentiviruses containing CDK5 shRNA (sh-CDK5) and scrambled shRNA (sh-NC) were individually transfected into U251MG cells, according to the manufacturer's instructions. The infection efficiency was determined by the intensity of green fluorescence protein after 72 h. The knockdown efficiency was examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting 96 h after transfection.A lentivirus expressing short hairpin RNA (shRNA) that targeted CDK5 was designed and synthesized by Shanghai GeneChem Co., Ltd. . U251MG cells  were as follows: CDK5 up: GGAAGGCACCTACGGAACTG and CDK5 down: GGCACACCCTCATCATCGT and glyceraldehyde-5-phosphate dehydrogenase (GAPDH) up: AGCCACATCGCTCAGACAC and GAPDH down: CTCGCTCCTGGAAGATGGT. The PCR reaction conditions were: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 59 °C for 40 s. Relative quantitative analysis was performed using the 2U251MG cells were homogenized in RIPA lysis buffer, which contained a protease inhibitor mixture , on ice for 30 min, removed from the 6-well plates using a cell scraper, and centrifuged at 12,000 rpm in a 4 °C Eppendorf microfuge (Germany) for 10 min. Loading buffer was added to the protein lysate and boiled for 10 min. Equal amounts of protein were loaded, separated via 10% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane  via electroblotting for 90 min. The membranes were blocked with 5% skim milk for 1 h and then incubated overnight with primary antibodies to CDK5  and β-Actin  at 4 °C along with appropriate secondary antibodies. The enhanced chemiluminescence system  was used to identify the reactive bands, according to the manufacturer's instructions.Transfected and control cells (n = 500) were seeded in 6-well plates and cultured in DMEM (high glucose)  supplemented with 10% FBS (GIBCO) and 1% antibiotics  for 10 d. The colonies were fixed with 75% ethanol, stained with 0.1% crystal violet solution, imaged, and counted.7/mL. Next, Annexin V-APC was added to the cell suspensions (100 µL) and incubated at room temperature for 10-15 min. Signals were detected with a flow cytometer (Millipore Guava easyCyte HT).An Annexin V-APC Apoptosis Detection Kit  was used for cell apoptosis analysis. First, U251MG cells were infected with lentiviruses expressing sh-CDK5 or sh-NC. After 4 d, cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in staining buffer to obtain a final density of 1×10U251MG cells infected with lentivirus expressing sh-CDK5 or sh-NC were cultivated in 6-cm dishes. When the density reached 80% coverage, cells were collected and fixed with pre-cooled 70% alcohol for 1 h. Cells were then washed with PBS and stained with a buffer containing 40×propidium iodide (PI) stock (2 mg/mL), 100×RNase stock (10 mg/mL), and 1×PBS at a dilution of 25:10:1,000. Flow cytometry was performed using the Millipore Guava easyCyte HT flow cytometer for cell cycle analysis.3 cells per well) transfected with sh-CDK5 or sh-NC were cultured in 96-well plates at 37 °C for 72 h and then exposed to 5-ethynyl-20-deoxyuridine  for an additional 2 h. The cells were fixed with 4% formaldehyde for 30 min and treated with 0.5% Triton X-100 at room temperature for 20 min. After washing with PBS thrice, 1×Apollo® reaction cocktail (100 µL) was added to each well and incubated for 30 min. Subsequently, the DNA contents of the cells in each well were incubated with 5 µg/mL Hoechst 33342 (100 µL) for 30 min, washed with PBS thrice, and imaged under a fluorescent microscope.U251MG cells  of glioma patients. All experiments were performed at least thrice. Data are presented as the mean ± standard deviation. Student's t-test was used to determine statistical differences between two groups. One-way analysis of variance (ANOVA) was used to compare three groups. P <0.05 was used to determine the significance level.To investigate the role of CDK5 in gliomas, the gene expression profiles and clinical data of patients with glioma were obtained from TCGA and GSE16011 datasets. A total of 1114 and 276 glioma cases with clinical data were available from TCGA and GSE16011 dataset respectively. The number of patients with CDK5 transcript data is 697 . All clinical characteristics were summarized in To explore the CDK5 expression pattern in gliomas, data on the clinical features of patients with glioma obtained from TCGA and GSE16011 were analyzed. As shown in Figure Logistic regression analysis showed that CDK5 expression was associated with poor prognostic clinicopathological characteristics Table . When stTo investigate the predictive value of CDK5 in glioma prognosis, we analyzed CDK5 expression and the OS of glioma patients. For patients with complete OS data, Kaplan-Meier analysis was performed. As shown in Figure GSEA was conducted between the low and high CDK5 expression phenotype groups to identify the signaling pathways involved in glioma progression based on TCGA. Significant differences (FDR <0.05) were observed in the enrichment analysis. Several signaling pathways, especially metabolism-related pathways, including amino sugar and nucleotide sugar metabolism, galactose metabolism, proteasome, glyoxylate, and dicarboxylate metabolism, oxidative phosphorylation, pentose phosphate pathway, and glutathione metabolism, were enriched in the high CDK5 expression cohort Figure , Table 2in vitro. CDK5 in glioma cell lines  and NHAs was subjected to immunoblotting. As shown in Figure Next, we sought to elucidate the biological functions of CDK5 in glioma cells In this study, cell proliferation was evaluated using the EdU and colony forming assays. EdU assay showed that sh-CDK5 cells, compared to sh-NC cells, inhibited cell proliferation significantly (P <0.01) (Figure Tumor cells show strong resistance to cell death. In this study, Annexin V-APC assay and flow cytometry were performed to evaluate apoptosis of U251MG cells infected with lentivirus expressing sh-CDK5. Apoptosis was detected in 3.16% of cells treated with sh-NC and 14.93% of cells treated with sh-CDK5 (Figure Subsequently, flow cytometry and PI staining were performed to determine the alteration in the cell cycle Figure F, G. It in vitro experiments were performed. EdU incorporation and colony forming assays showed that CDK5 promoted glioma cell proliferation and colony formation capacity. Moreover, CDK5 knockdown promoted cell apoptosis and altered the cell cycle distribution. These results indicated that targeting CDK5 could be a therapeutic strategy to inhibit glioma progression.Malignant evolution, a hallmark of tumors, is essential for cancer progression. Without timely and effective therapy, a glioma may progress from low-grade to high-grade, exacerbating the prognosis Although the role of CDK5 in gliomas has been previously investigated, relevant studies remain limited. CDK5, as a neuron-specific kinase, has been known to be abundantly present in the brain Our analysis has some limitations. First, the number of normal brain tissues used as controls in TCGA was small, therefore, additional studies with balanced sample sizes must be performed. Second, we adopted only one glioblastoma cell line for the cell biological function assays. Finally, GSEA showed that several metabolism-related pathways were enriched in the high CDK5 expression phenotype group. To validate this and fill the knowledge gap in the oncogenic mechanism of CDK5 in glioblastoma, more cell and animal experiments must be performed.Our study revealed that CDK5 expression levels were higher in glioblastoma than in LGG. We also found that CDK5 was related to adverse outcomes and tumor metabolism. Taken together, CDK5 might act as a potential prognostic biomarker and a tumor metabolism target for glioma. We further explored the role of CDK5 in human U251MG glioma cells via CDK5 knockdown. CDK5 knockdown impaired cell proliferation and colony formation ability and induced apoptosis and cell cycle arrest. Overall, CDK5 might prove to be a valid target for glioma therapy.Supplementary table S1.Click here for additional data file.


Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites. In general, several modules are fused into a single polypeptide. Polyketide synthases (PKSs) that are also modular megaenzymes, adopt the same assembly line logic as NRPSs but they use small carbon chain substrates instead of amino acid substrates [NRPSs are classified into two categories, type I and type II. In type II NRPSs, the incorporation of an amino acid into the metabolite necessitates the involvement of several domains carried by distinct proteins . By contfigure 1bstrates . This sibstrates .a); each NRPS module also incorporates the non-catalytic, but essential, peptidyl carrier protein (PCP) domain [a). PCP domains, whose masses are in the range of 10 kDa, have been mostly studied in isolated form by NMR [Bacillus subtilis [Most modules are composed of two catalytic domains, the adenylation (A) and the condensation (C) domains a; each N) domain . In thism by NMR –15. Theym by NMR . The 20 subtilis , for thesubtilis –22.a). Finally, each assembly line ends with a domain, such as a thioesterase (TE) or a reductase (Re) (module 7 in figure 1a), that releases the final product. This final stage can introduce further diversity in the peptide as the release can occur either by hydrolysis or cyclization. Interestingly, since surfactin release occurs via macrolactonization [figure 1b) [The PCP domain delivers substrates to the catalytic domains. First, the adenylation (A) domain activates the incoming acid monomer. A domains select very diverse monomers including α-L- or α-D-amino acids, β-amino acids or aryl acids . Subsequfigure 1nization ,26, surfgure 1b) .NRPS flexibility allows the conformational changes required for the interactions between the PCP and the catalytic domains. However, this flexibility is a drawback to structural characterizations of large NRPS fragments and, accordingly, successful structural studies have often required the employment of chemical tools to reduce conformational heterogeneity –22. Neve. 2a and 2a,b) [Acinetobacter baumanii, the enterobactin termination module EntF and ObiF1 from the obafluorin assembly line (c–e) [f,g) [2h–j and The field of NRPS structural biology achieved major breakthroughs in the last five years with the publication of several crystal structures of multi-domain NRPSs, largely due to the work of the Schmeing and Gulick groups  19–22,2,29. Thes1nd 2a,b) . Since tbly line c–e [20,2ne (c–e) . In 2017e) [f,g) . Lastly,. 3core, consists of around 400 amino acids, while the C-terminal subdomain, Asub, comprises around 100 amino acids [figure 3b, thiolation conformation) [Pseudomonas aeruginosa, was obtained both in its apo form and in a loaded form locked in the thiolation conformation through the use of the inhibitor valyl-adenosine vinylsulfonamide (AVS) [in vivo but, more likely, a conformation that favours crystallization.The adenylation (A) domain is divided into two subdomains: the N-terminal subdomain, Ano acids . The A dno acids ,20,22,31rmation) –20,22. Fde (AVS) . In the b, open conformation) [core subdomain contains the monomer binding pocket that accommodates ATP and the acid monomer. Upon substrate binding, a 30° rotation of the Asub subdomain leads to the closed conformation, which is then suitable for adenylation  [sub loop into the Acore subdomain; this loop contains a conserved catalytic lysine that stabilizes the acid substrate and ATP [sub subdomain allows the conversion between the closed and the thiolation conformations, the latter being able to catalyse thioesterification  [The cycle starts when the A domain, in an open conformation, is available for substrate binding figure 3rmation) . The Acormation .rmation) . This co and ATP . Subsequrmation) .sub subdomain are facilitated by a flexible hinge region containing a conserved aspartic acid or a lysine, located in the Acore-Asub linker [core-Asub linker is essential to allow the movement of the Asub subdomain relative to the Acore, which is necessary to allow conformational changes of the whole module. Indeed, rotations of the Asub subdomain drive movement of the PCP owing to the linker connecting the two domains. Analysis of the A-PCP linker region reveals that it contains multiple prolines, absent in standalone A domains [sub subdomain [The rotations of the Ab linker . The strb linker . The impb linker . Therefo domains . These pubdomain .. 4b, formylation conformation) [figure 3a), a substantial conformational change must occur to allow the PCP domain to position its PPant arm in the F active site. The Asub subdomain rotates 180° from its position in the thiolation conformation while the PCP domain rotates 75°, thus moving 60 Å away from its thiolation position . The interaction surface between the F and PCP domains is very limited. Unfortunately, there is no structure of the full dimodular LgrA in the formylation conformation. It is worth noting that the structure of dimodular LgrA  reveals that the position occupied by the PCP1 domain in the formylation conformation is occupied by the C domain at a later stage of the catalytic cycle [The vast diversity of NRPs arises in part from the action of tailoring domains, such as cyclization (Cy), epimerization (E), formylation (F), ketoreductase (KR), methyltransferase (Met) and oxidase (Ox) domains, that modify the peptide under construction ,36–40. Ifigure 3rmation) . Since tic cycle .. 5b, condensation conformation). If they belong to different polypeptides, docking domains facilitate the interaction between the upstream PCP and the downstream C domain [n catalyses peptide bond formation between the growing chain carried by the upstream PCP domain (donor PCP), that belongs to module n-1, and the activated amino acid carried by the downstream PCP domain (acceptor PCP) located on the same module n  [Elongation is the only reaction that necessitates interactions between domains belonging to different modules. This reaction is catalysed by a condensation (C) domain or, more rarely, by a cyclization (Cy) also called heterocyclization (HC) domain, that catalyses cyclization after condensation . If the figure 3C domain . The C drmation) . The groa–d) [figure 2b) [figure 2c,d) [The C domain adopts the V-shaped pseudo-dimeric fold seen in the chloramphenicol acyltransferase family . It is dfigure 2a–d) ,29,30. Ire 2c,d) ,30. It ib, condensation conformation) [1 domain is docked in the C2 donor site, presenting its conserved Ser towards the catalytic His of the C domain, the two residues being separated by less than 20 Å. The donor PCP1 domain globally has the same orientation in these structures and three of them show electron density for the PPant arm in the donor tunnel of the C domain. However, the structure of F1-A1-PCP1-C2 with f-Val loaded onto the PPant arm of PCP1 reveals that the reactive thioester group must slightly modify its position in order to be properly positioned for attack by the acceptor amino acid. The authors hypothesized that the donor substrate could only be correctly positioned in the presence of the PCP-bound-acceptor substrate in the active site of the C domain [Several dimodular LgrA structures have revealed for the first time the productive interaction between a donor PCP domain and its corresponding C domain (figure 3rmation) . Indeed,C domain . The impC domain ,45. Inde1-A1-PCP1-C2-A2-PCP2 reveals both donor and acceptor PCPs docked onto a single C domain  [2 is located 15 Å away from the catalytic His of the C domain while the Ser from the donor PCP1 is 18 Å away from it. These distances are compatible with the proximity between substrates required for nucleophilic attack. Unfortunately, there is no electron density for the PCP PPant arms so the detailed interaction of donor and acceptor substrates cannot be deduced from this structure.A detailed comprehension of the condensation reaction requires structural information on a NRPS including at least a donor PCP, an acceptor PCP and a condensation domain. The structure of holo-LgrA Frmation) . 29 Å se1-A1-PCP1-C2-A2-PCP2 shows that an inhibitor is not necessary to lock a dimodular NRPS in the condensation conformation, this new chemical probe stabilized the interaction between the donor PCP, the acceptor PCP and the C domains. The enterobactin assembly line served as a model to prove the functionality of this probe [The detailed view of PCP-bound-substrates in the condensation conformation can be obtained using a mechanism-based probe, recently designed by the Gulick and Aldrich groups . Althougis probe . The aut1 domain has been solved in association with its three catalytic partners ; therefore, the comparison of the three crystal structures provides insights into the conformational changes that allow the PCP1 domain to shuttle between its three partners [sub subdomain that are transferred to the PCP domain by the Asub-PCP rigid linker. Shifting from the formylation to the condensation conformation, the PCP1 domain must cross 30 Å, achieved through a rotation of 40° of the Asub subdomain . Similarly, after condensation, the PCP1 domain must detach from the C domain and travel back 50 Å to return to the A1 active site , achieved by a rotation of 150° of the Asub subdomain. Even in the absence of structures that show the second LgrA module in the thiolation conformation, we can easily extrapolate that the movements seen in module 1 could be similar in module 2.The structure of the LgrA PCPpartners ,22. As db) shows the PCP1 domain in the peptide donation conformation while the A1 domain is in the closed conformation and can thus catalyse adenylation [1 domain can catalyse thiolation as soon as the C2 domain has catalysed condensation which will liberate the PCP1 domain. This decoupling between different domain activities likely increases the synthesis rate of NRPSs.Interestingly, a NRPS module can start a second catalytic cycle before the first one is complete . For exafigure 3nylation . After anylation ,48. Subs. 6a–e) [a-d), suggesting that the TE domain is most probably a mobile element. Negative staining EM images of EntF showed the TE domain in various positions compared to the other domains and no density was observable for the TE domain in the corresponding crystal structure (e), confirming that the EntF TE domain can adopt multiple conformations [d). These elements suggest that the high mobility of the TE domain is due to the flexibility of the short PCP-TE linker and to the fact that, in general, no successive domain imposes structural restraints on the final TE domain. As none of the structures of these four termination modules revealed the interaction between the PCP and the TE domains, it was characterized through the crystal structure of the EntF PCP-TE didomain [The structures of four termination modules harbouring a TE domain (C-A-PCP-TE) are now available, i.e. SrfA-C, AB3403, EntF and ObiF1 a–e [20,2a–e) –30. In atructure e, confirdidomain . The autdidomain , thus pr. 7b) with the PCP-C cross-module structure of TycC from the tyrocidine synthetase [As described in the previous sections, NRPS flexibility allows the conformational rearrangements that are required for the PCP domain to interact with its catalytic partners. It is then legitimate to wonder whether NRPS flexibility is only restricted to the movements that shuttle PCP-tethered substrates to the different NRPS active sites or if there is flexibility at the supramodular scale, unrelated to the catalytic cycle. In other words, are there architectural rules governing the relationships between successive modules, or is their relationship random? In addition to the crystal structure of bimodular LgrA, structural models of multimodular NRPSs derive from low resolution techniques or from the combination of crystal structures. Indeed, in 2016, Marahiel and co-workers proposed a helical model for a hypothetical 7-module NRPS assembly by combining the C-A-PCP structure of the SrfA-C termination module b with thnthetase ,50,51. TSeveral EM observations indicated that NRPSs probably adopt a more flexible architecture than the helical model mentioned above. An early negative staining EM observation of a fungal 11-module NRPS, responsible for the synthesis of cyclosporin, pictured this 1.7 MDa machinery as an assembly of globular moieties, most likely modules, that could adopt either very compact or elongated structures . It led 1-A1-PCP1-C2-A2-PCP2 + MLP) was also observed by negative staining EM [1-PCP1-C2 cross-module , there is no density for the PCP1-C2 linker, suggesting that it could be flexible [1-C2 linker combined with the absence of strong intermodule interactions could explain the various conformations adopted by the dimodular DhbF.More recently, the dimodular NRPS DhbF . Within these two molecules, module 1 is identical but module 2 adopts two radically different positions. This behaviour results in two strikingly different LgrA shapes, reminiscent of the two DhbF structures observed by EM, one that is elongated and the other L-shaped. Therefore, it seems that locking one module in a specific conformation does not impose a unique conformation on the adjacent module. The most convincing evidence that NRPSs adopt a flexible architecture was obtained from the SAXS analyses of the LgrA F1-A1-PCP1-C2-A2 construct [The six recent dimodular LgrA crystal structures provided further evidence that NRPSs do not adopt a unique stable architecture but rather a few conformations among a myriad of possibilities . One strric unit h-j. Withonstruct . They inonstruct . The ensonstruct  that cou. 8Klebsiella pneumoniae uses several siderophores for iron acquisition, including the NRPs enterobactin and yersiniabactin [The structural and functional studies of NRPS multienzymes are not limited to providing details regarding the production of complex metabolites but can also be applied to the discovery of new antibiotics. Indeed, the products of these machineries are often essential for bacterial virulence, hence targeting their biosynthesis is a promising strategy to fight microbial pathogens . For insiabactin –57. Straiabactin , suggestAcc-A-PCP-CDon (XUs) [Moreover, the re-engineering of NRPS megaenzymes in order to produce new medically relevant molecules is of particular interest ,60. Thison (XUs) . This st


The aim of this paper is to characterize the mechanical behavior of corrugated cardboard boxes using simple models that allow an approach to the load capacity and the deformation of the boxes. This is very interesting during a box design stage, in which the box does not exist yet. On the one hand, a mathematical model of strength and deformation of boxes with different geometry is obtained from experiments according to the Box Compression Test and Edge Crush Test standards. On the second hand, a finite element simulation is proposed in which only the material elastic modulus in the compression direction is needed. For that, corrugated cardboard sheets are glued to build billets for testing, and an equivalent elastic modulus is obtained. This idea arises from the fact that the collapse of the box is given by the local bucking of the corrugated cardboard panels, due to the slenderness itself, and the properties in the compression direction are predominant. As a result, the numerical models show satisfactory agreement with experiments, concluding that it is an adequate methodology to simulate in a simple and efficient way this type of boxes built with corrugated cardboard. Corrugated cardboard boxes are used in the transport of goods, housing loads of more than one ton of various products, such as frozen food in bags, bulk substances, rigid pieces and so on. In recent years, its use has increased with the emerge of e-commerce, making the analysis of these boxes a subject of interest ,2. AccorThese boxes are usually mounted on pallets in order to be manipulated. Due to the need to optimize warehouses, the containers are stacked one on top of the other, so that the lower container can support a load of several tons, besides the specific actions that the pallet base does on the edge of the ring. Initially, a low range of standardized sizes was common in the industry, but nowadays, companies like Amazon have set what is called the “science of packaging”, by increasing the diversity of sizes, and therefore minimizing the usage of raw materials and optimizing transport. Cardboard is key in this configuration, due to its versatility, price compared with other alternatives and environmental footprint ,7. TheseBCT load, by a safety factor. This safety factor is determined by taking into account several variables, such as the humidity, the type of pallet, the load eccentricity, the effect of punctual loads, the vibration that occurs during handling and transport, fatigue and operational life of the container. The safety factor can be obtained by tables in standards as ASTM D4169. Usually, safety factors greater than two are adopted.The load capacity of the boxes is determined by a compression test called Box Compression Test (BCT), which is carried out with standard humidity and temperature conditions (50% and 23 °C). The maximum applied load is determined by dividing the result of this test, named the BCT load or the deformation that the box will undergo. Numerous empirical studies that propose a way to determine the BCT load have been published, [BCT from the Edge Crush Test (ECT) (that gives the load per unit of length ECT) of the cardboard box, perimeter p and wall thickness h as follows,m result is 5.876 for the tested boxes. Regarding deformation, force-deformation curves have been widely studied by several authors. Urbanik analyzed corrugated cardboard extensively, from plates made in the laboratory to box specimens [Since corrugated cardboard is a highly deformable material, the limit of its use may be delimited by the deformation of the box, so that the upper pallet does not touch the content of the lower box. For design purposes, it is interesting to have a model that allows obtaining an approximation of the blished, ,14,15,16pecimens . His appFor other authors ,21,22,23Some studies ,25,26 si2. It is an analysis developed over thick cardboard, such as the ones analyzed in this article.The ECT test, according to DIN EN ISO 3037 standard, is the most economical way to characterize the properties of a material. The ECT test is performed in a sample of 100 × 25 mm cut from the box material, in such a way that it can represent the entire box if a strength correction factor that is a function of some geometrical scale factor is applied. In this context, in , authorsm for the tested samples, and a useful mathematical relationship to predict the deformation of the boxes is proposed. Finally, a methodology to simulate the behavior of the boxes by finite elements is proposed, in which an equivalent isotropic elastic modulus is considered for the cardboard, although the material is actually orthotropic. This is given as the assumption that the collapse of these kinds of containers occurs by local buckling of the vertical cardboard panels, so the material properties in the compression direction are predominant over the others. In conclusion, it should be pointed out that the mechanical behavior of this type of boxes built with corrugated cardboard can be easily and efficiently simulated from a practical and engineering point of view.Concluding, in this paper, a characterization of the mechanical behavior of corrugated cardboard applied to containers and octabins subjected to vertical stacking loads is presented. More than thirty commercial-use packaging boxes are tested aimed at obtaining the force-displacement curve. From the results, McKee Equation (1) is applied in order to obtain the coefficient m of McKee’s model given by Equation (1) for three sets of boxes of different geometries. The second one is to obtain a model for the vertical deformation and for the deformation modulus of these boxes. The third objective is to obtain an equivalent Young modulus of corrugated cardboard blocks from compression tests performed on billets built of glued corrugated cardboard sheets, aimed at using this modulus in numerical simulations.In this section, the experimental program is presented. Specifically, BCT, ECT and compression tests are performed for three different purposes. The first objective is to determine the coefficient BCT. From this force, the BCT stress p represents the perimeter of the box and h wall thickness. Besides, a force-displacement curve can be reached. For example, BCT. In zone C, the material of the ring is under excessive stress, or the deformation is so high that cracks occur in the ring, weakening it. Once the BCT force is exceeded, the box deforms excessively until it collapses. From The BCT test was carried out in accordance with the UNE 131000 standard. This test is performed on box samples, and the maximum load before the sample collapses is measured, namely z is the height of the sample. The deformation Deformation h are the same as the corresponding box. From the test, the load per unit of length ECT is obtained , from which the stress ECT The ECT standard is performed according to DIN EN ISO 3037. All specimens have dimensions of 100 × 25 mm, the material and thickness m coefficient of McKee’s model yieldsm result is 5.876 for McKee’s model, and f is the shape factor of the box, given byTaking into account Equations (1), (2) and (5), the All the tests were performed with the same hydraulic press. The laboratory environment was air-conditioned.For the BCT test, industrial use packaging of rectangular and octagonal geometry is tested. The footprint perimeter of the packages ranges between 1855 and 5856 mm, and the thickness ranges between 6.6 and 20 mm. Boxes of thickness greater than 13 mm have been manufactured using the ECOBOX technology patented by “Cartonajes Lantegi S.L.”, which allows lamination of single, double or triple layers of cardboard before shaping the box in a die-cutter adapted to the thickness see a. The heh, height z, footprint perimeter p, larger side a, shorter side b, for octabins the length of the corner side x . The samples for the ECT test are obtained by cutting 100 × 25 mm samples from lateral panels of the boxes.de x see b, shape All the samples were maintained in a climatic chamber for at least 72 h, in an environment at 20 °C and a relative humidity of 50%. However, the testing machine was not inside the climate chamber, given its enormous size. Nevertheless, less than 4 min elapsed between sample extraction and testing, so it is not assumed that there is a significant variation in humidity in the sample. This is important due to the significant variation in load capacity that containers experience when the humidity of the cardboard increases.2 to 500 × 500 mm2. The plates are made of conventional double-wave cardboard . Between 6 and 30 of these plates are glued together, resulting in corrugated cardboard billets, with dimensions indicated in num, thickness of a sheet H, total thickness HA num × H, width of the sheet B and the height Z.Simple compression tests are carried out on solid blocks formed by successive sheets of corrugated cardboard see  in orderm coefficient to obtain a model for the BCT-ECT relationship, a model for the vertical deformation For boxes, the E as a function of the stress For corrugated cardboard billet samples, the equivalent elastic modulus The results derived from the tests are:BCT, the deformation ECT and the stress The obtained results from the BCT and ECT tests performed on the samples are shown in BCT load and the ECT load per unit of length of As previously mentioned, it is very usual to find no repeatability in the tests due to the fabrication processes of materials and boxes. Hence, the results of the In order to illustrate the mechanical behavior of the boxes and the ECT samples, some force-displacement curves are shown. As examples, the case of an octabin and an ECOBOX are considered.BCT force, and it was discarded for the analysis. Then, the BCT results were comprised between 14.3 kN and 18.4 kN: the BCT mean value resulting from the remaining nine curves was 16.2 kN, and the mean absolute deviation 0.9 kN (5.6%). In relation to ECT results, the minimum value was 12.3 N/mm and the maximum one 14.2 N/mm. The average value was 13.2 N/mm, and the deviation was 0.5 N/mm, i.e., 3.4%.First, BCT load was comprised between 20.8 kN and 28.8 kN: the mean vale was 24.9 kN, and the deviation was 1.5 kN (6.0%). Concerning ECT results, the minimum value was 18.9 N/mm and the maximum one 26.4 N/mm. The mean result was 24.1 N/mm, and the deviation was 1.6 N/mm (6.6%).Then, In summary, for some cases, the deviation is lesser than 1% , but in most cases the deviation is between 5–10%, as the previously illustrated examples. Although it should be noted that in some cases it reaches between 15–20% . This dispersion is unavoidable when working with this type of material.m from Equation (6). In m = 7.63 for octabins, m = 5.97 for boxes made of ECOBOX cardboard, and m = 5.61 for rectangular boxes made of double or triple corrugated cardboard. The mean value is m = 6.24. These values are not very far from the McKee’s result, m = 5.876.The first objective is to obtain the coefficient To characterize the stress-deformation relationship, in Therefore, according to Equation (4), a model for the deformation From Finally, a relationship between the deformation modulus R and the deformation The results from the compression test performed on corrugated cardboard billets are shown in R of the specimens B-01 and B-03, they are 8.87 kN and 61.9 kN, respectively. The ratio between them is 7, while the ratio between the cross-sectional area of both specimens is 5. This can signify that the load capacity does not depend only on the cross-sectional area, because the greater the thickness, the greater the stability.Comparing the load capacity E be obtained fromThen, the stress The relationship between oxes see  and its From these results, it can be pointed out that deformation modulus increases with stress. The higher the resistance, the higher the stiffness. The dispersion is very significant, but an average linear model can be fitted, yieldingIt can be concluded that the modulus for blocks Generally, it should be pointed out that a significant dispersion has been found in experimental results. This is because the manufacturing process of these products does not provide significant repeatability, and this is the reason because in practical applications safety factors greater than two are employed if the load capacity is obtained from numerical models.From the modeling point of view, Equations (1) and (9) are useful to define the range of use of packages. Therefore, if it must be determined the limit of boxes that can be stacked during transport or storage in a logistics center, the load would have to be divided by a safety factor. The deformation of the frame makes it possible to estimate whether the distance between the content and the cover plates is sufficient so that they do not come into contact, absorbing part of the load transmitted by the pallet overhead. These equations also allow us to obtain a simplified stress-deformation curve for engineering practical applications.Equation (13) allows obtainment of a value for the modulus of elasticity of the material as if it were a homogeneous and isotropic solid. This simplifies the modeling of the boxes using a finite element model in industrial practical applications, since it is possible to consider the material as an elastic solid, as mentioned throughout the text. The lateral deformations that occur in boxes are considered by the numerical method itself, this is the reason for which the modulus In this section, two CAE (Computer Aided Engineering) finite element models are developed aimed at verifying that simplifications about the elastic modulus of the material allows us to obtain simple models that provide results with enough accuracy. The results of the CAE models are compared with experimental results and the given by the mathematical models developed in ECT as a property of the material to be known from experimentation. With this, the stress E is estimated by Equation (13). For the mathematical models, the ultimate stress f and m parameters, and the displacement at the maximum load δmax is determined by Equation (9).The models are developed by means of ABAQUS software. Two models are analyzed for the octabin O-04 and the rectangular R-04 items, respectively. The properties of these items are taken from p is much larger than the thickness h, hence S4R quadrilateral shell-type finite element is used. The O-04 model has 2200 elements and 2288 nodes. The R-04 model has 8232 elements and 8036 nodes.It can be assumed that the footprint perimeter F, which is uniformly distributed over its perimeter. The material model is assumed to be elastic and homogeneous with a uniform modulus of elasticity E. This is due to the hypothesis of this paper that the properties in the compression direction are the predominant ones. The Poisson ratio is taken 0.40 from [The box is subjected to a vertical load .40 from .The analysis is performed by means of the Riks method. Although the analysis could be carried out with the standard structural modulus of ABAQUS, the Riks method has been chosen because it performs arc-length control instead of force or displacement control, so it can even predict post-buckling behavior, such as the one shown in E, and to take into account the dispersion of the results, three curves will be computed for each model: one for the reference value of E = 104 N/mm2 and E = 132 N/mm2, and those of the R-04 model are E = 95 N/mm2 and E = 121 N/mm2. These curves are compared with the corresponding experimental ones and with the results of the mathematical models.As a result, force-displacement curves are obtained for O-04 and R-04 models. To investigate the dependence of the mechanical behavior on the modulus BCT and BCT force and the lower deformation.b of In the same way as in the previous section, a qualitative comparison of the CAE deformation model and the experimental one is presented in BCT and only BCT is similar to experimental and to the stiffest CAE ones.In this paper, a characterization of the mechanical behavior of corrugated cardboard applied to containers and octabins subjected to vertical stacking loads has been presented. Experiments have been completed in order to obtain mathematical models for load capacity of the boxes, for vertical deformation and for deformation modulus. From the experimental results, it can be pointed out the significant existing dispersion, inherent to the characteristics of this kind of products. Thus, the obtained mathematical models are functional in practical engineering applications design stages, but safety factors have to be applied for the final application.In order to employ simple finite element models to simulate the corrugated cardboard boxes behavior, an equivalent Young modulus has been drawn from experimental tests on corrugated cardboard billets. This allows modeling of boxes by means of shell finite elements with a homogeneous equivalent material. Simulations have been performed with different properties to take into account the dispersion. Simulation results have been compared with experimental and mathematical model results. Taking into account the existing dispersion in experimental results, it can be concluded that CAE models based on homogeneous and isotropic materials is an effective way to easily simulate the behavior of containers made of corrugated cardboard, although the material is actually orthotropic. This is due to the fact that these types of containers fail due to buckling of the vertical panels, so the properties in the compression direction are the most significant.


The effects of 10%–30% of wheat flour substitution with potato flour (PF) and sweet potato flour (SPF) on the flour and dough properties, the total polyphenol (TPC), and carotenoid contents (TCC) of bread, as well as their correlation with bread texture and starch digestibility, were investigated. With PF and SPF addition, the peak, breakdown, and setback viscosity of the flour decreased. The addition of PF and SPF reduced the dough formation and stabilization duration, as well as the hardness of the bread. The specific volume of the bread depended on the addition amount of PF and SPF. When the addition of PF and SPF was 15%, the bread had the lowest hardness and highest specific volume. The TPC and TCC in the bread depended on the added flour variety, and negatively influenced specific volume and positively influenced the content of resistant starch (RS). The decreased extent of the peak, breakdown, setback viscosity of the mixed flour depended on the flour variety. Specific volume of bread depended on the variety and amount of added flour. TPC and TCC negatively influenced specific volume, elasticity, and RDS content. Meng et al.  replacedThe aim of this study was to investigate the addition of PF and SPF, including OFSPF and purple‐fleshed sweet potato flour (PFSPF) on the characteristics of mixed flour and the responding dough, as well as the quality of bread, including texture, bioactive components, and digestive property. Based on this, the relationship between the TPC, TCC, and the texture and digestibility of bread was explored. Our study outcomes provide a theoretical and practical basis to produce potato‐wheat bread (PWB) and SPWB, and help accelerate the development of potato and sweet potato‐based resources in the staple food industry.22.1The commonly consumed Chinese varieties of fresh potato (eshu 10), OFSPF (Yanshu 25), and PFSPF (Ziluolan) were purchased from the market of Shenyang. The potatoes were washed, peeled, sliced, steamed for 20 min, and then dried using a heat pump dryer  at 60°C for 12 h. Thereafter, they were crushed using a grinder (XM‐600‐Yongkang Boou Hardware Products Co. Ltd.) and passed through a 50‐mesh sieve, sealed, and stored at 4℃ for use.2.2To prepare the bread, an egg, 15 g of butter , 90 g of water, 40 g of white sugar , 0.5 g of salt , 200 g of mixed flour, 7 g of milk powder (Nestle S. A.), and 3 g of yeast  were put into a bread maker . The WF  was replaced at ratios of 0%, 10%, 15%, 20%, 25%, and 30% with PF, OFSPF, and PFSPF to make the bread. The operation conditions of the bread maker were set as sweet bread, light color, and 750 g.2.3The pasting characteristics of the WF with different substitutions of PF, OFSPF, and PFSPF were measured using a rapid viscosimeter  using the methods described by Zhu and Sun . Briefly2.4The thermomechanical characteristics of the dough were measured using the MixolabⅡenzyme rheometer . Using the method described by Azeem et al. , with so2.53, and the test parameters were set as follows: TA4 probe, the pre‐ and post‐test speed of 2 and 1 mm/s, respectively, the test speed of 1 mm/s, the strain of 50%, and the trigger force 5 g. The specific volume of the bread was measured using the rapeseed displacement method described by AACC  according to the method described by Liu et al. , with so by AACC .2.6The bread was cut into 1‐cm‐thick slices, dried in an oven at 40℃ for 24 h, and then ground into bread powder. Phenols were extracted from the bread flour with 60% ethanol (v/v) at the ratio of 1 g/55 ml at 50℃ for 2 h by shaking (100 rpm), then centrifuged at 10,000 g for 20 min. The supernatant was used for the determination of TPC. The TPC was determined using the Folin–Ciocalteu method described by Singleton and Rossi . TPC is 2.7A is the absorbance and TCC is expressed as mg carotene/100 g bread weight.According to the methods described by Tang et al. , 0.5 g o2.8The in vitro digestion characteristics of the bread were measured according to the method described by Huang et al. , with sl2.9p < .05) were determined using variance and Duncan multiple comparison.Data analysis was conducted using SPSS 20.0 software. All data were measured in triplicate and expressed as mean ± standard deviation (SD). The significant differences between the samples . When the added amount reached 15%, the hardness of the bread reached the lowest. Among them, the hardness of the bread with added OFSPF was the lowest (35.23 ± 0.47 g), and that with PFSPF was the highest. Good‐quality bread means bread with low hardness, as well as high elasticity , which is consistent with the result of Cao et al. ; when the addition of flour reached 30%, the elasticity decreased significantly, which is consistent with the result of Cao et al. , which is consistent with the result of Han and Koh  and slowly digestible starch (SDS) in the bread decreased, while the content of resistant starch (RS) increased Table . Moreove4In this study, the addition of 10%–30% PF, OFSPF, and PFSPF to WF influenced the pasting properties of flour, thermomechanical properties of dough, texture, and TPC, TCC, as well as starch digestibility of bread. Except for the pasting temperature, with the addition of PF and SPF, the peak, breakdown, and setback viscosity decreased. However, the extent of the decrease depended on the flour variety; this was most evident for the flour with added SPF in the peak and breakdown viscosity, and for the flour with added OFSPF in the setback viscosity. The addition of PF and SPF reduced the formation and stabilization time of the dough, as well as the hardness of the bread. However, the specific volume of bread depended on the amount of different flour added. When the proportion was 10%–15% for SP and OFSPF and 10%–20% for PFSPF, the specific volume of the bread was higher than that of WF bread. When the proportion was 15%, the hardness was the lowest while the specific volume was the highest. The bread with added OFSPF and PFSPF had the lowest hardness and highest specific volume, respectively. The TPC and TCC in the bread depended on the flour variety added, and were negatively correlated with the specific volume, elasticity, and RDS content and positively correlated with the RS content.None.Hongwei Meng: dataCuration ; formalAnalysis ; investigation ; methodology ; validation ; visualization ; writingOriginalDraft . Chong Xu: dataCuration ; formalAnalysis ; methodology ; resources ; visualization . Meiying Wu: dataCuration ; formalAnalysis ; visualization . Ying Feng: conceptualization ; formalAnalysis ; fundingAcquisition ; methodology ; projectAdministration ; resources ; supervision ; validation ; writingReviewEditing .


Background: Protein-energy malnutrition and vitamin A deficiency (VAD) are the most important public health issues, and a food-based strategy is crucial to combat those health problems among the vulnerable group of people.Methods: Composite biscuits were made with 100:0:0, 90:5:5, 80:10:10, 70:15:15, 60:20:20, and 50:25:25 percent wheat, haricot bean, and orange-fleshed sweet potato (OFSP) flours.Standard methods were used to evaluate the proximate compositions, β-carotene, physical properties, functional properties, and sensory acceptability. A one-way analysis of variance model was used to statistically evaluate the data using the statistical analysis system software package, version 9.0 standard methods.Results: The results showed that partially replacing wheat with haricot beans and OFSP increased the β-carotene and proximate compositions significantly. When wheat was replaced with haricot beans and OFSP, the physical characteristics of the biscuits did not vary significantly from those of biscuits made entirely of wheat flour. Sensory acceptability  was higher in the composite biscuits with up to 40% wheat substitution than in the 100% wheat flour biscuits.Conclusion: Based on the findings of this report, replacing wheat with OFSP and haricot beans in biscuit formulation appears to be promising in improving nutritional quality, sensory acceptability, and beta carotene. It is proposed that these products can mitigate food insecurity and deficiency of vitamin A. The aim was to produce a balanced, low-cost, and healthy snack product. Considering the potential health benefits of OFSP and haricot bean flour and the increasing consumption of healthy foods, the objective of the present study was to prepare nutritious biscuits to deliver a nutritious and healthy product.Compared to animal-based foods, pulses, in general, an important food category for humans, have high protein content, and thus, can help to improve the protein content of meals, especially for low-income households.2.2.1An orange-fleshed sweet potato of the Alamura variety was collected from Hawassa International Potato Center, Ethiopia. Soft wheat flour was obtained from the Hawassa flour factory, and haricot bean seed (Nasir) was obtained from the Hawassa Agricultural Research Center’s Southern Agricultural Research Institute.2.2The method described by Nshimiyimana2.3The formulation and development of biscuits are clearly presented in the current study. Accordingly, orange-fleshed sweet potato flour and haricot bean flour were blended into wheat flour at varying percentages of 5%, 10 %, 15%, 20%, 25%, and 30% .The biscuit dough was made following a commercial recipe and baking procedures.2.4Functional properties of flours2.4.1Bulk densityThe bulk density of composite flour was determined using the method described by Oladele and Aina.Dispersibilityet al.The method described by KulkarniWater absorption capacity (WAC)et al.The water absorption capacity of the flour samples was determined according to AremuWhere w1 = weight of the sample, w2 = weight of the tube, w3 = weight of the sample after centrifugation.Oil absorption capacity (OAC)et al.The oil absorption capacity of the flour samples was determined according to AremuWhere w1 = weight of the sample, w2 = weight of the tube, w3 = weight of the sample after centrifugation.Proximate and beta-carotene analyses of flour and biscuits2.4.2et al.The standard methods of the Association of Official Analytical ChemistsWhere A = Absorbance; V = Total extract volume (ml); P = Sample weight; A1 = 2592*β carotene extinction coefficient in petroleum ether.Determination of physical properties of biscuit2.4.3The spread factor (SF), diameter (D), and thickness (T) of the biscuit were calculated using different methods.Sensory acceptability2.4.4Panelists measured the sensory acceptability of biscuits based on their willingness to engage in the study.Statistical data analysis2.4.5The experimental data were subjected to one-way analysis of variance, and Duncan’s multiple range tests were used to detect the difference (p ≤ 0.05) between the mean values. Statistical analyses were performed with the statistical analysis system software package, version 9.0 standard methods (RRID: SCR_008567), and the data were presented as mean and standard deviation.3.3.1The bulk density, water absorption capacity, oil absorption capacity, and dispersibility of OFSP, wheat, and haricot bean flours are shown inet al.In this analysis, the bulk density of OFSP (Alamura variety) flour is higher than that recorded by Tirunehet al.The water absorption capacity of haricot bean flour was higher than the value (17.3-16.8%) recorded by Shimeliset al.et al.,et al.et al.Both haricot bean flour and OFSP flour have a higher WAC than wheat flour, so adding haricot bean and OFSP to the composite flour would improve the WAC of the composite flour compared with wheat flour. Increased water absorption weakens the dough and causes it to lose its development and stability. The ability to absorb water is essential for product consistency and bulking, as well as in baking applications.Due to the lipophilic quality of its constituents, wheat flour has a higher oil absorption capacity than OFSP flour and haricot bean flou.3.2et al.,In terms of protein, ash , fiber, and fat content, haricot bean flour appears to have an advantage over wheat flour. The addition of OFSP flour appears to improve the fiber and ash content as well. According to Biniyam,3.3et al.Since OFSP and haricot bean flour have a higher water absorption ability and fiber content than wheat flour, the increase in moisture content in this study may be due to increasing the percentage of OFSP and haricot bean flour to wheat flour. This study confirmed the results of Biniyam,et al., the hydroxyl group present in the fiber structure allows the higher total fiber in non-wheat flour to interact reasonably well with a large amount of water.et al.The results of this study agreed with those of Vieira,et al.According to Ndifeet al.The current study’s findings are consistent with those of Ndifeet al.et al.The result of the current study is consistent with Ndifeet al.et al.et al.Since haricot bean flour has a higher fat content than wheat flour, the rise in crude fat content in this study may be attributed to increasing the ratio of haricot bean flour to wheat flour. The current results are consistent with those of Abayomiet al.et al.The decrease in carbohydrate content of the cookie may be due to a rise in moisture, fat, ash, and fiber content as the proportion of OFSP and haricot bean flour in the formulation was increased, resulting in a decrease in carbohydrate content because carbohydrate is measured by difference. The current result was consistent with the results of Biniyam,et al.et al.,,Consumption of β-carotene-rich foods, such as OFSP, which are also readily available in the home garden, can help to boost VA intake and alleviate VAD.3.4,,,,With the addition of OFSP, different studies recorded a decrease in the diameter,3.5In comparison with 100% wheat flour biscuits, the appearance, color, flavor, taste, and overall acceptability scores of the biscuits improved with the addition of haricot bean and OFSP flour up to H20%, OF20%. The crispiness of the biscuits, on the other hand, decreased as haricot bean and OFSP flour were added. Despite this, the crispiness of the composite flour biscuits is not substantially different from the control up to the addition of 15% haricot and 15% OFSP. The most critical quality attributes that can affect the acceptability of a food product and a consumer’s buying decision are its appearance and color.et al.The current study’s taste results are consistent with the findings of Afework4.Based on the findings of the current study, blending wheat with OFSP and haricot bean during biscuit formulation appears to be promising in improving nutritional quality, sensory acceptability, and physical and chemical properties. Biscuits from wheat, haricot bean, and OFSP composite flours had increased protein content where their compositions could alleviate protein-energy malnutrition. The inclusion of OFSP in the composite biscuit also significantly increased the amount of beta carotene. It is anticipated that these foodstuffs can lessen food insecurity and VAD. Furthermore, the composite flour biscuits had better sensory acceptability than biscuits produced from 100% wheat.https://doi.org/10.17605/OSF.IO/Y3ANX.OSF: Underlying data for “Effect of blending ratio of wheat, orange fleshed sweet potato and haricot bean flour on proximate compositions, β-carotene, physicochemical properties and sensory acceptability of biscuits”;Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data is available under the terms of theThe study was reviewed and approved by the Institutional Review Board of the College of Medicine and Health Sciences of Hawassa University. Written permission was also obtained from the School of Nutrition, Food Science and Technology. Informed written consent was obtained from the panelists before the actual sensory data was collected. The purposes and importance of this study were explained to all panelists. The responses of each panelist were kept confidential by coding. The data was collected and analyzed anonymously. The author has made revisions in accordance with the input and recommendations given previously so that this paper can be indexed.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Food process technologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In general, the manuscript does fit consideration for indexing, provided that some improvement is made by the authors based on comments/suggestions given by reviewers. It requires both technical and mainly typographical editorials/corrections. I have tried to go through it and thus made the following points:TitleThe title is too long and also it needs some modification; I suggest the following: "Effect of blending ratio on the physicochemical properties and sensory acceptability of biscuits prepared from blends of wheat-orange-fleshed sweet potato-haricot bean flours".AbstractThe background information mentioned only protein-energy malnutrition as the main problem; what about the micronutrient problem prevailing in the country? For example, the conclusion mentioned vitamin A deficiency.GeneralThere are a number of grammatical errors  that need to be edited/corrected throughout the manuscript.There are lots of missing references (citations) almost in all sections. That should be revised/corrected. IntroductionTheIntroduction section needs revisions; it should specifically focus on issues of malnutrition in Ethiopia and also its coherence/logical flow needs revisiting.The rationale of the work is not clearly indicated. As to me, the rationale can still be made stronger by adding other ideas such as the contribution of OSFP to fight vitamin A deficiency. MethodsThe Methods section also needs a thorough revision: repetition of ideas and also re-ordering of the sub-sections and editorial things on these sections is required.It is not clear from where the OFSP was collected.Sections 2.2 and 2.4 should be merged as they refer to the same thing.Results and DiscussionThe Results and Discussion section is relatively well written. However, tables (Results) should normally appear immediately after the first mention; this is not the case in this manuscript. Kindly edit accordingly.Values of both results of the research and those of cited reports (for comparison purposes) are not shown. This should be made so as to make things clear to the reader and also improve the quality of the article.Statistical significance has not been shown in most of the reports of the results. Only mere reports were made. To my knowledge, this should be corrected; otherwise, the possibility of indexing would be reduced.ConclusionIn the conclusion section, the phrase ".....with more OFSP supplementation....." is not clear. In the first place, the work was not supplementation.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Complementary food processing, food quality and safetyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The title has been modified to make it shorter and more precise. The background information and abstract conclusion are narrated while taking into account both protein energy malnutrition and vitamin A deficiency. The contribution of OFSP clearly indicates the logical flow of the idea concerning malnutrition and the rationality of the problem. Nutritional and food science perspectives are also discussed. OFSP's origin is stated in the study setting. The merging of related sub-headings and re-ordering of subsections were done carefully. The enhancement to increase the impressiveness of the result has been completed. Only statistically significant values were indicated in the discussion section. A concerted effort was made to ensure that the comparison point was both impressive and adequate. To bring the study's work to a close, paraphrasing was used in the conclusion section. Grammatical errors, referencing, and citation correction were all considered. Order and coherence were also maintained adequately throughout the document. The authors used different terms for OFSP and haricot bean (powder for OFSP and flour for haricot bean), please make a justification about these terms.In the introduction, the authors state that from the results of the study it is expected that a good blend of wheat, OFSP, and haricot bean flours will produce nutritionally enriched biscuits, while in the methodology, the observations are only made on the proximate composition of biscuits. No observations on micronutrients.There was no observation of the color and texture of the biscuit. The addition of OFSP and haricot bean flour must have a great effect on these two parameters. Observation of these two parameters would highly improve the credibility of the manuscript. This can also justify the assessment results from the panelists because the consumer acceptance test is only carried out by 30 panelists.In Table 3, the unit of energy should be kcal/100g.Does it cite the current literature? Partly, as only about 33% of the references are within the last 10 years.The paper investigates the proximate composition, physical properties and sensory acceptability of biscuits made from wheat, orange-fleshed sweet potato (OFSP) powder and haricot bean flour. The authors explain the societal and economic value by substituting imported wheat flour with domestic crops in the production of food products such as biscuits. The content of the paper is in line with the scope of the journal and addresses an important research question. No doubt about that. However, in my point of view, the paper does need some revision before indexing. Please find my specific comments below:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Food process technologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The consistency of OFSP and haricot bean (powder for OFSP and flour for haricot bean) is taken into account. Methodological observations on beta carotene content are combined. The sensory test was carried out in accordance with the guidelines of Hawassa University's school of nutrition, food science, and technology, with a panel of 30 panelists. Texture was never intended to be a study variable from the start. Furthermore, a more detailed explanation of the color is being considered. In Table 3, Kcal/100g is used as a correction. To update the manuscript, recently published papers are reviewed and comparisons was done accordingly. This manuscript studied the blending ratio of different types of flour substituted in wheat flour for biscuit preparation.The information in the Introduction section was explained in the nutrition field while the Methods were not determined by any nutrition factors after changing the ingredients of the fortified biscuits.This manuscript was more like in the food science field. The authors need to rewrite the whole Introduction by adding some new studies of biscuit production, the gap of recent works, and the use of beans and pulses in the food industry. The biscuit preparation can be easily understood by adding all recipes to a separate table. The sensory test was inappropriate to interpret the results due to the use of a small group of panellists that were involved with this study (only 30 people). Normally, the sensory test was acceptable at 100 untrained panellists. The researchers also used only a 5-point hedonic scale which is too rough. It is quite hard to believe the results from this part. The results were unclear and must be improved. It is better to take a photo of OFSF and haricot bean flour and also the composited flour. I do believe that the colour of biscuits should interfere with orange sweet potato flour colour. Moreover, varying the composition of flour in the biscuit formula normally changes the texture of biscuits affecting consumers liking scores. This parameter should be evaluated. That's why this manuscript can't ensure reproducibility.The Conclusion was inadequate to summarise the whole work. The recommendations are listed below. Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Development of biscuits, utilisation of agricultural waste, chitosan extractionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The information in the introduction section describes the nutritional concepts and their application from an ingredients' perspective in the methodology part as well. Additionally, nutritional and food science perspectives are discussed, with an emphasis on biscuit production, recent discrepancies, and the use of beans and pulses in the food industry. A separate table is used for presenting the recipes. The manuscript includes photographs of the raw materials and blended biscuits. The sensory test was carried out in accordance with the guidelines of Hawassa University's school of nutrition, food science, and technology, with a panel of 30 panelists. Texture was never intended to be a study variable from the start. Corrections were made as much as possible in the conclusion section to summarize the work of the study.