Distinct from other LAR-RPTP ligands, SALM5 mainly exists as a dimer with LRR domains from two protomers packed in an antiparallel fashion. In the 2:2 heterotetrameric SALM5/PTPδ complex, a SALM5 dimer bridges two separate PTPδ molecules. Structure-guided mutations and heterologous synapse formation assays demonstrate that dimerization of SALM5 is prerequisite for its functionality in inducing synaptic differentiation. This study presents a structural template for the SALM family and reveals a mechanism for how a synaptic adhesion molecule directly induces cis-dimerization of LAR-RPTPs into higher-order signaling assembly.SALM5, a synaptic adhesion molecule implicated in autism, induces presynaptic differentiation through binding to the LAR family receptor protein tyrosine phosphatases (LAR-RPTPs) that have been highlighted as presynaptic hubs for synapse formation. The mechanisms underlying SALM5/LAR-RPTP interaction remain unsolved. Here we report crystal structures of human SALM5 LRR-Ig alone and in complex with human PTPδ Ig1–3  PTPδ, which reveals how SALM5 dimerization facilitates higher-order signaling assembly of LAR-RPTPs. Synaptic adhesion molecules (SAMs) are precisely positioned to sculpt synaptic protein interaction networks for mediating synaptic early differentiation, formation, maturation, terminal elimination, and plasticity4. Disruption of synaptic protein networks involved by SAMs may contribute to the pathology of many neurological disorders5.The human brain consists of billions of neurons that exploit a myriad of proteins to establish synaptic connections in precise patterns for processing information, shaping neural circuits, and maintaining proper brain functions7. Members of the SALM family are predominantly expressed in the central nervous system (CNS) and known for their engagement in neurite outgrowth, synapse formation, and synapse maturation15. All five SALMs may form heteromeric and homomeric complexes in cis- or trans- configurations, suggesting that SALMs exhibit both pre- and post-synaptic functions through protein interactions16. Notably, SALM3 and SALM5 may induce both excitatory and inhibitory presynaptic differentiation in contacting axons via trans-synaptic interactions with LAR-RPTPs15. Interestingly, SALM4 may suppress excitatory synapse development by cis-inhibiting trans-synaptic SALM3-LAR adhesion14. SALM5 has recently been associated with familial schizophrenia in which inherited copy number variation of SALM5 has been observed17, and with severe progressive autism, where expression levels of SALM5 are dramatically reduced by tenfold due to chromosomal translocation and intragenic multiple-exon deletion19. These reports not only point to the clinical importance of SALMs, but also suggest that SALM5-dependent presynaptic induction may play critical roles in brain development and function15.SALMs, also known as leucine-rich and fibronectin III domain-containing proteins, are a family of newly characterized SAMs that belong to the type I transmembrane proteins. The SALM family includes five members, SALM1–5, that share a similar domain organization: a leucine-rich repeat (LRR) domain composed of LRRs flanked by cysteine-rich regions at its amino terminus (LRRNT) and carboxy terminus (LRRCT), an Ig-like domain, and a fibronectin type III (Fn) domain, a transmembrane region and a cytoplasmic tail that binds to PSD-95 for SALM1–3, but not for SALM4–522. Distinct from the other subtype of RPTPs, LAR-RPTPs interact trans-synaptically with a growing list of post-SAMs including NGL-324, TrkC25, IL1RAPL127, IL-1RAcP28, the Slitrks30, and the newly found SALM family members, SALM313 and SALM515. LAR-RPTPs may also bind to presynaptic molecules, netrin-G131 and glypicans32, in a cis manner. In particular, the MeA and MeB insertion sites within the N-terminal Ig domain of the LAR-RPTPs have received extensive attention since they are involved in regulating interactions with various post-synaptic ligands, like TrkC, IL-1RAcP, IL1RAPL1, and SALM522. Through interacting with such a large collection of protein ligands, LAR-RPTPs exhibit a wide repertoire of cellular signaling functions and have recently been highlighted as hubs for extracellular interactions in neurons, regulating neurite outgrowth, axon guidance, synaptic organization, and CNS regeneration34. In humans, PTPδ has been implicated in correlation with neurological disorders such as ASDs, ADHD, bipolar disorder, schizophrenia, and restless legs syndrome39.LAR-RPTPs, short for leukocyte common antigen-related receptor protein tyrosine phosphatases, are classified as type IIa RPTPs that, in vertebrates, include three members, LAR, PTPδ, and PTPσ. LAR-RPTPs are type I transmembrane proteins and share a common domain architecture of an ectodomain containing up to three immunoglobulin-like (Ig) domains and up to nine fibronectin III (Fn) repeats, followed by a transmembrane domain and tandem intracellular phosphatase domains. Multiple LAR-RPTP isoforms are generated by alternative splicing of whole domains or four short peptides encoding mini exon inserts, termed MeA to MeD40, whereas the architectures and functions of the extracellular regions lack complete understanding22. Recently, structural work on the ectodomains of LAR-RPTPs has demonstrated their surprising flexibility as a valuable feature that allows these receptors to serve as a synaptic signaling nexus for interacting with a broad spectrum of different protein ligands in the narrow synaptic cleft41. Also, structural analyses of type IIa RPTPs in complexes with Slitrk42, TrkC41, IL1RAPL1, or IL-1RAcP43 and Slitrk244 have revealed the molecular basis for their interaction specificity as well as minimal units and 1:1 stoichiometry for receptor–ligand recognition. However, how extracellular ligands mediate receptor dimerization or clustering for downstream signaling has remained outstanding questions in the RPTP field45.Early structural analysis has focused on elucidating catalytic mechanism and substrate specificity of the intracellular phosphatase domains of RPTPs46, which could confer itself a unique mechanism for RPTP signaling. Therefore, deciphering the codes for SALM5 dimerization and its interaction with LAR-RPTPs at the structural level will yield novel insights into the molecular mechanisms underlying RPTP transmembrane signal transduction. Here we report crystal structures of human SALM5 LRR-Ig alone and in complex with human PTPδ Ig1–3 (MeA−). Together with other biophysical, biochemical, and cellular evidence, the structures reveal that dimerization of SALM5 is prerequisite for its functionality in inducing synaptic differentiation through presynaptic LAR-RPTP receptors. Also, this study provides a structural template for the SALM family and shed light on how a SAM directly induces oligomerization of type IIa RPTPs into higher-order signaling assembly.In contrast to other known ligands of the type IIa RPTPs, SALM5 forms a dimer in solution15, we focused on the N-terminal three Ig domains of human PTPδ with or without MeA or MeB splice inserts . The crystal structure of human SALM5 LRR-Ig domains was solved with an iodine derivative using the single isomorphous replacement and anomalous scattering (SIRAS) method, while the structure of SALM5/PTPδ complex was solved using the molecular replacement method , flanked by an N-terminal LRRNT (residues 14–49) and a C-terminal LRRCT (residues 240–285) subdomain. The LRRNT subdomain is mainly composed of an β-hairpin plus two loops cross-linked by two pairs of disulfide bonds C20–C26 and C24–C37. The LRRCT subdomain folds into loops interwoven with short helices that are bridged to loops by disulfide bonds C244–C263 and C246–C308. Interspaced between the LRR repeats and LRRCT is the tenth long β-strand plus a long loop, which is termed “R-loop” as in platelet glycoprotein Ibα47. Both LRRNT and LRRCT serve as a cap to shelter the LRR repeats from solvent environment through hydrophobic residue contacts  to the first LRR repeat because (1) T53 projects its Cγ atom to the hydrophobic core of this segment involved by residue L56; (2) this segment adopts a configuration similar to that of a canonical LRR repeat  with a long list of residues involved  migrated as a dimer on a SDS-PAGE gel. In comparison, the R110N/E160S mutant moved as a monomer in the presence or absence of BS3, further indicating that the SALM5 R110N/E160S mutant is a monomer in solution Please be noted that a serious error was introduced here during your production process. In our manuscript, BS3 represents a compound as defined above, and the superscript "3" does NOT mean a reference. Please delete the introduced reference here.Since in line with our crystal structures, SALM5 has recently been reported to mainly exist in solution as a dimerion Fig. . The monlls Fig. . This me2 SASA is buried in the SALM5/PTPδ interface that can be divided into three principal regions, sequentially named site I, II, and III along the primary structure of SALM5  and PTPδ M273 and M296.In site II, the β-strands βA and βB of SALM5 Ig domain interact with the βC strand and the loop connecting strands βC and βD of PTPδ Ig3 domain, burying a total of ~890 Å2 SASA) with the β-strands A and B of PTPδ Ig2 domain  of PTPδ. Adjacent to this hydrophobic interaction spot, K326 extends its side chain out to form a salt bridge with PTPδ D154. Noteworthy, a double-alanine mutation of S329 and S360 was reported to weaken SALM5 binding to LAR, PTPδ, and PTPσ15. Although both S329 and S360 are hardly involved in direct interaction with PTPδ, their mutation to alanine would impair PTPδ binding in an allosteric manner since they are in proximity to Site III , whereas mutants E365N and K309N/R311T could not  with approximately close affinities (281.8 vs 145.3 nM). However, mutation of E365N or K309N/R311T abolished SALM5 binding to immobilized PTPδ  displayed on the surface of HEK293T cells, whereas the mutant L327N bound to PTPδ-expressing cells as the wild-type SALM5 did  and tested PTPδ-binding capacity of these mutants by pull-down, surface plasmon resonance (SPR) and protein–cell interaction assays. The presence of the glycan wedge, GlcNAc2Man5, in each mutant was confirmed using mass spectrometry 49. Consequently, the intracellular tandem phosphatase domains (D1D2) of PTPδ could be precisely located at a proper position, although the precise distance between two phosphatase domains is not known  on pre- or post-synaptic membrane might competitively regulate SALM5-mediated synaptic differentiation.Since the key residues in PTPδ for SALM5 binding are highly conserved among the LAR-RPTP family that may share a similar topology, the above mechanism model would be suitable for SALM5 activating other LAR-RPTP family members. Universality of such a model may explain the recent report that a double mutation of S329 and S360 not only impaired SALM5 binding to the whole LAR-RPTP family, but also weakened presynaptic differentiation governed by themcis-dimerization or oligomerization of which negatively regulates their catalytic activity55, dimerization of LAR-RPTPs may couple with phosphatase activity for an activating regulation56. Crystallographic studies have suggested that LAR-RPTP tandem phosphatase domains are monomeric in solution58. This means that extracellular ligand binding would be critical for LAR-RPTP oligomerization and activation. Therefore, our SALM5-mediated PTPδ dimerization model sets a point for unraveling LAR-RPTP transmembrane signaling mechanism. A caveat about this model is that PTPσ was suggested to exist as a dimer on the cell59. Due to lack of direct biochemical evidence, we could not rule out the possibility that PTPδ is a dimer on the cell membrane as PTPσ. In case of this possibility, binding to a dimeric SALM5 would facilitate remodeling of LAR-RPTPs or seduce a conformational modulation so that they could be activated for downstream signaling in synaptogenesis.Different from other RPTPs, 22. A good example for this point is HSPGs (heparan sulfate proteoglycans), which induce tetrameric clustering of PTPσ that leads to the promotion of neurite outgrowth60. The NT3 neurotrophin may also induce formation of dimeric or higher-order RPTPσ arrays through binding to the TrkC/PTPσ complex, thus enhancing the synaptic organizing function of TrkC/PTPσ in rat hippocampal neurons61. From this point, the SALM5-induced PTPδ dimerization still has biological significance.HSPGs (heparan sulfate proteoglycans).Another extreme possibility is that SALM5-mediated PTPδ dimerization would not couple with intracellular phosphatase activation. For this possibility, it is still conceivable that SALM5-induced dimeric clustering of PTPδ would result in enrichment of PTPδ in local milieu for enhancing PTPδ signaling efficiency. Indeed, RPTP clustering would lead to a redistribution of tyrosine phosphatase activity at the plasma membranecis-dimerization of PTPδ for their function in presynaptic differentiation. No matter which mechanism will underlie SALM5-mediated LAR-RPTP transmembrane signaling, it is conclusive that SALM5-induced dimerization or precise re-positioning of LAR-RPTPs is vital for the functionality of SALM5 in synaptogenesis. Future structural, biochemical, and functional studies on the full-length LAR-RPTPs in the absence or presence of SALM5 will be crucial to further explain how dimerization of SALM5 is coupled with their intracellular tyrosine phosphatase activity for synaptogenesis.In conclusion, we have determined the crystal structure of a 2:2 SALM5/PTPδ heterotetramer that yields insights into how a SALM5 dimer induces 62. cDNAs encoding human SALM5 LRR-Ig (residues 18–374) or its mutants , human SALM5 ectodomain (residues 18–526), human PTPδ Ig1–3 (MeA−) , human PTPδ Ig1–3 (MeB−) , human PTPδ Ig1–3 (MeA−/MeB−) (residues 21–320 with both residues 181–189 and 227–230 deleted) and human PTPδ Ig1–3 WT  were amplified by a single-step PCR or a two-step overlapping PCR  gene was also cloned into a BacMam vector with C-terminal human IgG1 Fc plus 7× His-tag. For each construct, the resulted transfer plasmid was used to transfect Sf9 insect cells in the presence of BacVector-3000 baculovirus DNA (EMD Biosciences). Seven days after transfection, low-titer baculovirus stock was harvested and sequentially used to infect insect cells for amplification. For protein expression, the amplified high-titer baculoviruses were used to transduce HEK293 GnTI− cells63 at a density of 2 × 106 cells per ml. The cell culture was further maintained in suspension for 72 h at 37 °C in a humidified incubator supplemented with 5% CO2. Cells were removed by centrifugation at 3000 × g for 15 min, and the conditioned medium was concentrated and completely buffer exchanged into high-salt HEPES-buffered saline . Interest proteins were captured using Ni2+-NTA agarose (Invitrogen) and in gradient eluted with HBS supplemented with different concentrations of imidazole. The eluate was confirmed using SDS-PAGE and then subjected to a gel-filtration column  pre-equilibrated and washed with HBS. The fractions were pooled and concentrated to 8 – 10 mg ml−1 for crystallization or to a proper concentration for biochemical and biophysical assays. For detecting complex assembly, the purified SALM5 and PTPδ proteins were mixed for 1-h incubation at 4 °C and then subjected to gel-filtration analysis performed on the same column. Samples from peak fractions were analyzed using SDS-PAGE and visualized by Coomassie blue staining. All the recombinant proteins were purified sequentially using Ni2+-NTA affinity and gel-filtration chromatography. Except that used for crystallization, all the purified recombinant proteins were not applied to glycan trimming.Proteins used for crystallization, gel filtration assay, mass spectrometry, AUC study, pull-down assay, surface plasma resonance analysis, cell surface-binding assay, and polyantibody generation were expressed in the BacMam systemresidues 8–374 or −1 or its complex with human PTPδ Ig1–3 (MeA−) at 10 mg ml−1 was trimmed using Endo H (New England Labs) for glycan removal. Protein crystallization was performed at 20 °C using the vapor diffusion sitting-drop method. The drops were set up with 0.5 μl protein in mixture with 0.5 μl reservoir solution. Diamond-like SALM5 LRR-Ig crystals grew from drops equilibrated against reservoir solution containing 2% (w/v) polyethylene glycol (PEG) 3000, 0.1 M sodium acetate (pH 4.5), and 0.2 M Li2SO4. The complex crystals with a cubic shape were observed after 3 weeks from reservoir solution containing 0.76 M potassium sodium tartrate and 0.1 M MES (pH 6.0). Crystals were flash-frozen in liquid N2, followed by soaking in the reservoir solutions supplemented with 26% (v/v) ethylene glycol for SALM5 LRR-Ig crystals and 20% (v/v) ethylene glycol for complex crystals. To prepare a heavy atom derivative, SALM5 LRR-Ig crystals were soaked in the cryoprotectant buffer containing 0.2 M NaI for 3 min before being flash cooled.The purified human SALM5 LRR-Ig alone at 8 mg ml64. On the BL17U1 beamline, two sets of high-redundancy data were collected at the wavelength of 1.54719 Å for native and NaI-derivative SALM5 LRR-Ig crystals. The two processed data sets were subjected to the AutoSol program in PHENIX65 for locating iodine atoms and calculating initial phases using the single isomorphous replacement with anomalous scattering (SIRAS) method. Some LRRs of SALM5 could be discerned in the electron density map calculated from initial SIRAS phases. The initial model was improved by the AutoBuild program in PHENIX that generated a model with 60% of SALM5 LRR domain residues assigned. The model was then completed by iterative manual building in COOT66 and refinement in PHENIX against a 2.8 Å resolution data collected on the BL19U1 beamline with a wavelength of 0.97853 Å. Diffraction data for crystal of human SALM5 LRR-Ig in complex with PTPδ Ig1–3 (MeA−) were also collected on the BL19U1 beamline with the same wavelength. The complex structure was solved by the molecular replacement (MR) method using the PHASER program in the CCP4 suite67. The N-terminal two Ig domains of PTPδ from IL1RAPL1/PTPδ complex (PDB code 4YH7)43, together with the structure of SALM5 LRR domain, was used as the search model. A sigma-A weighted 2Fo−Fc electron density map calculated from the MR solution was clear enough to manually locate the third Ig domain of PTPδ and the Ig domain of SALM5. The resulting complex model was iteratively refined in PHENIX and manually built in COOT. Guided by a sigma-A weighted Fo−Fc electron density map, waters, glycans, ions, and buffer molecules were cautiously integrated into the final models for SALM5 LRR-Ig alone and its complex with PTPδ Ig1–3 (MeA−). The stereochemistry of the final model was assessed by the program Molprobity in PHENIX. Percentages of residues in the favored regions of the Ramachandran plot are 98.56 and 97.76% for free SALM5 and SALM5/PTPδ structures, and no residues are distributed in the outlier region. Data collection, phasing, and refinement statistics are summarized in Table All the diffraction data sets were collected at Shanghai Synchrotron Radiation Facility (SSRF) and processed with HKL2000q of 0.25 and activation time of 30 ms were applied in MS2 acquisitions. The mass spectrometer was operated in positive ion mode, and a data-dependent automatic switch was employed between MS and MS2 acquisitions. For each cycle, one full MS scan in the Orbitrap was followed by 15 MS2 in the LTQ on the ten most intense ions. Each precursor ion selected for MS2 was dynamically excluded for subsequent LC-MS runs with 30 s exclusion duration. The LC-MS/MS data were submitted to database searching against the human SALM5 sequences with mutations R110N/E160S, K309N/R311T, L327N, and E365N using the software ByonicTM. Mass tolerances were 10 ppm and 0.6 Da for the precursor and fragment ions, respectively. Enzyme specificity was set as KR/P, and a maximum of two missed cleavages was allowed. Asparagine glycosylation, cysteine carbamidomethylation, and methionine oxidation were set as variable modifications.The nano-LC-MS/MS experiments were performed using a LTQ orbitrap velos pro mass spectrometer (Thermo). Tryptic peptides were applied onto an EASY nano-LC system following the manufacturer’s instructions. Each elute was then subjected into a C18 reverse phase column . The peptide mixtures were eluted with a 0–40% gradient from buffer A (0.1% formic acid) to buffer B (0.1% formic acid in acetonitrile) over 30 min and were then online detected in mass spectrometer using a data-dependent TOP15 method. The general mass spectrometric conditions were spray voltage, 2.2 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 250 °C. For MS/MS (MS2), normalized collision energy using wide-band activation mode was 35%, and ion selection threshold was 1000 counts. An activation Sedimentation velocity experiments were performed on a Beckman XL-I analytical ultracentrifuge at 20 °C. Protein samples were diluted using HBS buffer to 400 μl at an A280 nm absorption of about 0.8. Samples were loaded into a conventional double-sector quartz cell and mounted in a Beckman four-hole An-60 Ti rotor. Data were collected at 50,000 rpm at a wavelength of 280 nm for 7 h. Interference sedimentation coefficient distributions were calculated from the sedimentation velocity data using the SEDFIT software.−). The Ni2+-NTA affinity-purified SALM5 LRR-Ig and its mutants, K309N/R311T, L327N, and E365N, were respectively mixed with PTPδ Ig1–3 (MeA−)-Fc at a molar ratio of 1.5:1, and then immobilized with Protein G Sepharose resin  by rotation at room temperature for 2 h. After intensively washing with phosphate-buffered saline (PBS), the resin-bound protein complexes were eluted by 0.1 M glycine (pH 2.7) and immediately resolved by SDS-PAGE and Coomassie blue staining.Qualitative analysis of SALM5/PTPδ interaction was performed by pull-down assay with the C-terminally Fc-tagged PTPδ Ig1–3 (MeA−) was immobilized to the surface of a CM5 sensor chip using the amine-coupling method at a density of about 500 resonance units (RU), while the wild-type SALM5 LRR-Ig and its variants K309N/R311T, L327N, and E365N were, respectively, injected as the analytes in serial twofold dilutions with the concentrations ranging from 23.43 to 1500 nM. For determining binding affinities of SALM5 with different splice code variants of PTPδ, the His-tagged SALM5 LRR-Ig was immobilized on a CM5 chip, while the wild-type His-tagged PTPδ Ig1–3 and its variants MeA−, MeB−, and MeA−/MeB− were run as the analytes in serial threefold dilutions with concentrations ranging from 2.74 to 6000 nM. Generally, each analyte sample was injected over the chip at a flowrate of 30 μl min−1 for a 120 s or 60 s association and then the running buffer was switched over the chip to allow the analytes to undergo a 180 s or 90 s dissociation and the signal to return back to baseline. No regeneration of the chip surface was required between different analyte injections, since injection of the running buffer could bring the signal back to baseline. All SPR data were analyzed assuming the Langmuir model with a ligand to analyte molar ratio of 1:1.All the SPR experiments were carried out on BiacoreT200  instrument at 25 °C in a running buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20. For measuring binding affinities for PTPδ interacting with SALM5 and its mutants, the purified Fc-tagged PTPδ Ig1–3 (MeA−) coding sequence was amplified PCR  paraformaldehyde and incubated with rabbit anti-Myc tag (Proteintech) and mouse anti-His-tag  antibodies in the presence of 3% (w/v) BSA. After removing the primary antibodies, cells were washed with PBS twice, and the secondary antibodies, PE-conjugated goat anti-rabbit IgG  and Alexa Fluro 488-conjugated goat anti-mouse IgG (Jackson) were added for immunostaining PTPδ and SALM5.Human PTPδ Ig1–3 (MeAbis (sulfosuccinimidyl) suberate (BS3) at a molar ratio of 1:100. After incubation at room temperature for 30 min, 50 mM Tris-HCl (pH 7.5) was used to quench the reaction. The cross-linking mixture was applied to SDS-PAGE analysis, followed by Coomassie blue staining.Human SALM5 LRR-Ig and its mutant, R110N/E160S, were respectively incubated with Approval for use of animals was provided by the Animal Care and Use Committee of Peking University Health Science Center . Mouse experiments were conducted in conformity with institutional guide for the care and use of laboratory animals. Three 8-week-old female BALB/c mice were immunized subcutaneously with 200 μg of purified human SALM5 LRR-Ig protein per injection with complete Freund’s adjuvant for the first dose and incomplete Freund’s adjuvant for subsequent boosts. The mice received two booster injections at 10-day intervals. Blood was taken for sera analysis from mice harvested 4 days after the final boost. Sera were obtained from blood samples by centrifugation and applied to ELISA evaluation of anti-SALM5 polyclonal antibodies.68. Briefly, embryonic rat hippocampi were dissected out of E18 embryos, trypsinized and triturated to single-cell suspension. The resulted cells were seeded on a 96-well plate coated with poly-L-lysine and cultured in Neurobasal A (Invitrogen) supplemented with 2% B-27 supplements, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. At DIV9/10, wild or mutant SALM5 ectodomain-expressing HEK293T cells were harvested by centrifugation, dispersed in Neurobasal A medium, and then plated into cultured hippocampal neurons at a density of ~5000 HEK293T cells per well. After 48 h of co-culture, cells were fixed with 4% paraformaldehyde for 1 h at 37 °C, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 30 min at 4 °C and blocked with 5% (w/v) BSA for 2 h at 37 °C. For immunostaining SALM5, fixed cells were incubated with mouse anti-HA monoclonal antibody  for 2 h at 37 °C, washed thrice with PBS and then incubated with Alexa Fluro 488-conjugated goat anti-mouse IgG antibody for 40 min at 37 °C. For detecting synapsin-I, cells were sequentially incubated with rabbit anti-synapsin-I  and PE-conjugated goat anti-rabbit IgG antibodies following the procedure as used for SALM5 immunostaining. Prior to image acquisition, cells were washed thrice with PBS to remove non-specifically bound antibodies.cDNAs encoding human SALM5 ectodomain residues 18–526) and its variants, K309N/R311T, L327N, and E365N, were amplified using one-step or two-step overlapping PCR  and emission (λem) filter sets to detect Alexa Fluro 488  and PE . Fields of view were randomly selected for each well of fixed culture. All quantitative measurements on fluorescence intensity were performed with the web-based software Columbus. Data were represented as mean ± standard error of the mean (S.E.M.). Statistical comparisons were evaluated with Microsoft Excel using two-tailed Student’s t test. A value of p < 0.05 was considered statistically significant.All images were acquired with Operetta High-Content Imaging System (PerkinElmer), and collected from at least two separate cell cultures. For cell surface binding and heterologous synapse formation assays, images were respectively taken under a set of constant conditions including −) have been deposited in the Protein Data Bank (PDB) under accession numbers 5XNQ and 5XNP, respectively.The authors declare that the main data supporting the findings of this study are available within the article and its Supplementray Information file. Uncropped images of gels and blots that include the marker lanes are available in Supplementray Fig. Supplementary InformationPeer Review File


International Journal of Environmental Research and Public Health [The authors wish to make the following corrections to their paper published in the c Health .Social Risk Evaluation, the Greek letter “π” was missed out of Equation (4), thus, the correct version of this equation should be:In Section 3.1 Notations, in the definition of In Section 4.1 The authors would like to apologize for any inconvenience caused to the readers by these changes. The changes do not affect the scientific results. The manuscript will be updated and the original will remain available on the article webpage.


Balance training has been shown to be effective in preventing ankle sprain recurrences in subjects with chronic ankle instability (CAI) but the biomechanical pathways underlying the clinical outcomes are still unknown. This study was conducted to determine if a 4-week balance training intervention can alter the mechanical characteristics in ankles with CAI.Twenty-two recreationally active subjects with unilateral CAI were randomized to either a control  or intervention  group. Subjects in the intervention group were trained on the affected limb with static and dynamic components using a Biodex balance stability system for 4-weeks. The ankle joint stiffness and neutral zone in inversion and eversion directions on the involved and uninvolved limbs was measured at baseline and post-intervention using a dynamometer.p < 0.011, 95% CI ) than that of uninvolved contralateral ankle (0.99 ± 0.41 Nm/degree). With the available sample size, the eversion stiffness, inversion neutral zone, and eversion neutral zone were not found to be significantly different between the involved and uninvolved contralateral ankles. The 4-week balance training intervention failed to show any significant effect on the passive ankle stiffness and neutral zones in inversion and eversion.At baseline, the mean values of the inversion stiffness (0.69 ± 0.37 Nm/degree) in the involved ankle was significantly lower (Decreased inversion stiffness in the involved chronic unstable ankle was found that of uninvolved contralateral ankle. The 4-week balance training program intervention was ineffective in altering the mechanical characteristics of ankles with CAI.Randomized controlled clinical trial; Level of evidence, 1. Similarly, neutral zone data was normalized to calculate the normalized neutral zone of the involved ankle in inversion (INNZ %) and eversion (EVNZ %) using the formula [(Dinvolved − Duninvolved) / Duninvolved] × 100, where D is the neutral zone in degrees. The difference in the normalized values for each dependent variable between the baseline and post-intervention was also calculated to assess the effect of balance training.Stiffness data was further normalized to calculate the normalized stiffness of the involved ankle in inversion (INS %) and eversion (EVS %) using the formula ) than that of uninvolved contralateral sides (0.99 ± 0.41 Nm/degree) (p = 0.69) from that of uninvolved contralateral sides (17.5 ± 7.6 degrees), even though the mean value was slightly lower. In addition, no significant difference was observed for eversion stiffness and eversion neutral zone between the involved ankles and uninvolved contralateral side. We further examined the distribution of normalized inversion stiffness and inversion neutral zone in all subjects at baseline. Approximately 77% of individuals in this study presented with decreased inversion stiffness. The inversion stiffness decreased in the majority of the involved ankles compared to the uninvolved ankles within subjects, shown as the negative values in the percentage difference  on the involved ankle was significantly lower (/degree) . With thfference . Howeverfference .For group comparisons, the normalized values for each dependent variable were calculated and compared. At baseline, the normalized values for each dependent variable between groups were not statistically different (p > 0.05). Following 4-week balance training, the changes in the normalized values for each dependent variable between the baseline and post-intervention was calculated. No significant differences were observed between two groups in changes of the normalized values of the inversion stiffness, eversion stiffness, inversion neutral zone, and eversion neutral zone .The primary finding of this study was that the involved ankle with CAI demonstrated decreased inversion stiffness when compared to the contralateral uninvolved ankle. No difference in the neutral zones between the involved and contralateral uninvolved ankles was found. The 4-week balance training intervention failed to show any significant effect on the passive stiffness and neutral zone measured in this study. This study was unique in that it examined the effect of balance training on the neutral zone in addition to stiffness in the ankles with CAI. The results presented in this paper are a portion of the study results and the results on other measurements will be reported later.Several investigators have examined ankle joint laxity in patients with CAI, and conflicting results have been reported in the literature . The resThe inversion stiffness in the involved ankles of subjects in the present study showed a large variability , which mTo the best of our knowledge, only one study reported laxity values using a similar device under comparable testing conditions . Direct In the present study, the 4-week balance training failed to show any significant effect on the passive stiffness or neutral zone between groups. This result is consistent with the finding of McKeon et al., which was the only past study that examined the effect of balance training on ligamentous laxity and stiffness of the ankle . McKeon Our study had limitations that should be addressed in future studies. First, the testing of ankle flexibility characteristics in this study was limited to inversion/eversion without anterior drawer testing. It was therefore limited in terms of representing the status of the ATFL in ankles with chronic CAI. Measurements of fibular position, hypo- or hypermobility of the ankle, gender differences, and muscle activation during the testing were not recorded and could have been the limiting factors in this study. Second, the leg and ankle were supported and the subjects were instructed to relax their leg muscles during the testing. Thus based on our testing method, we also cannot determine ligamentous and capsular contributions toward ankle joint stiffness which may be possibly measured with indwelling strain transducers. Third, the balance training program may not have been of sufficient duration to bring changes in the mechanical characteristics of the ankle. In the literature, balance training programs for patients with CAI have been generally prescribed for 3–5 sessions per week for 4–8 weeks. It would be interesting to identify the effect of 6 or 8 weeks balance training program on the mechanical characteristics of the ankle. Lastly, relatively small sample size in this study limited the statistical power of our analyses and could lead to a type 1 statistical error.We evaluated the effect of balance training on mechanical characteristics in the ankles with CAI. Our results were in agreement with previously published studies that have reported decreased inversion stiffness in the involved ankle with CAI when compared to the contralateral uninvolved ankle. In addition, no difference in the neutral zones between the involved and contralateral uninvolved ankles was found. The 4-week balance training program failed to show any significant effect on the passive stiffness and neutral zone measured in the relatively small sample size of this study. Further research with additional stiffness measures along with functional tests, larger sample size, and progressive balance training exercises is needed to identify if the mechanical characteristics of chronic unstable ankles can be altered.


Curcuma longa, has been widely used in medicine for centuries due to its anti-inflammatory, anti-cancer, anti-oxidant and anti-microbial effects. However, its bioavailability during treatments is poor because of its low solubility in water, slow dissolution rate and rapid intestinal metabolism. For these reasons, improving the therapeutic efficiency of curcumin using nanocarriers  has been a research focus, to foster delivery of the curcumin inside cells due to their small size and large surface area. Silk fibroin from the Bombyx mori silkworm is a biopolymer characterized by its biocompatibility, biodegradability, amphiphilic chemistry, and excellent mechanical properties in various material formats. These features make silk fibroin nanoparticles useful vehicles for delivering therapeutic drugs, such as curcumin. Curcumin-loaded silk fibroin nanoparticles were synthesized using two procedures  more scalable than methods previously described using ionic liquids. The results showed that nanoparticle formulations were 155 to 170 nm in diameter with a zeta potential of approximately −45 mV. The curcumin-loaded silk fibroin nanoparticles obtained by both processing methods were cytotoxic to carcinogenic cells, while not decreasing viability of healthy cells. In the case of tumor cells, curcumin-loaded silk fibroin nanoparticles presented higher efficacy in cytotoxicity against neuroblastoma cells than hepatocarcinoma cells. In conclusion, curcumin-loaded silk fibroin nanoparticles constitute a biodegradable and biocompatible delivery system with the potential to treat tumors by local, long-term sustained drug delivery.Curcumin, extracted from the rhizome of  Curcuma longa, which comprises, approximately, 2–5% of turmeric [CH3COO−], by high-power ultrasound, as previously reported [CH3COO−]. The mixture was treated with a 3/8″ tapered horn of a Sonifier Branson 450D , with pulsating ultrasonication steps at 30% amplitude at a controlled temperature below 90 °C until complete dissolution. To this solution freshly prepared, 3 mL of ultrapure water was slowly added to reduce viscosity. The final concentration of the SIL solution after diluting with 3 mL of ultrapure water was 6.66 wt %. After heating to 60 °C, the SIL solution was propelled using a peristaltic pump and then sprayed onto 100 mL of gently stirred methanol at −20 °C by a thermostatically controlled 0.7 mm two-fluid nozzle  which uses compressed N2 to disperse the solution into fine droplets. A milky white suspension appeared and the suspension was allowed to reach room temperature while stirring for 2 h. Then, the nanoparticle suspension was transferred to centrifuge vials and centrifuged at 13,400 rpm for 15 min, at 4 °C . The supernatant, which is free of nanoparticles, was removed and reserved for subsequent recycling of the ionic liquid. An equal volume of fresh methanol was added to the vial, and the white precipitate was suspended by vigorous stirring in a vortex mixer for 2 min and 5 min of ultrasonication with a Branson 450D sonicator . The centrifugation step was repeated under the same conditions. The white precipitate was subjected to successive rinses with ultrapure water to remove the remaining methanol and ionic liquid. The particles were lyophilized in an Edwards Modulyo 4K Freeze Dryer  for 72 h, at −55 °C and 0.5 mbar to obtain dry particles. The methanolic fractions were mixed before recovery of the ionic liquid by removing of the methanol/water on a BÜCHI RE-111 rotary evaporator  at 80 °C and 80 mbar. The ionic liquids were kept in a desiccator until reuse. The preparation of SFNs was based on the method described previously by Lozano-Pérez et al. , with moThe drug was loaded into the SFNs by two different experimental procedures  to obtain Curc-SFNs. The average yield of the process after lyophilization was of 95.8% and 90.3% for Curc-SFNs 2 and Curc-SFNs 1, respectively. All the experiments have been carried out at least in triplicate.For loading of curcumin by physical adsorption, forty milliliters of a 1 mg/mL solution of curcumin in ethanol was used to resuspend 325 mg of SFNs obtained as described in Drug loading content (DLC) and entrapment efficiency (EE) of the Curc-SFNs 1 obtained were calculated according to the following expressions:In the case of coprecipitation, the curcumin was loaded during the nanoparticles synthesis process. Briefly, 25 mg of curcumin was dissolved in 3 mL of 0.1 M NaOH solution and the resulting solution was immediately dissolved in 5 g of SIL solution (10 wt %). The curcumin-SIL solution was heated to 60 °C and sprayed onto 100 mL of ethanol using the same equipment as described in The characteristic parameters (DLC and EE) of the Curc-SFNs 2 obtained were also indirectly calculated by the above expressions from the measurements of UV-Vis absorbance of curcumin.Curcumin release studies were carried out with Curc-SFNs 1 and Curc-SFNs 2. Twenty milligrams of Curc-SFNs were dispersed in 1 mL of PBS 1× (0.5% Tween 80) by ultrasonication. The samples were incubated at 37 °C in a tube rotator for 3 days to study curcumin release. At predetermined time points , the samples were centrifuged at 13,400 rpm and the curcumin in the supernatant was monitored using UV-Vis spectrophotometry at 421 nm. The PBS solution was replaced with the same volume (1 mL) of fresh solution. The release experiments were carried out in triplicate for every sample. Experimental data were fitted using four release kinetic models found in the literature   to know The mean hydrodynamic diameter (Z-average), the Polidispersity Index (PdI), the Zeta Potential and the Electrophoretic Mobility were measured using a Zetasizer Nano ZSP instrument  by DLS. All measurements were performed in purified water at 25 °C, a 173° angle relative to the source and with a nanoparticle concentration of 0.66 mg/mL. The influence of the SFNs concentration on size distribution and Zeta Potential was evaluated and it was checked that both parameters were practically constant in the range from 0.30 to 40 mg/mL and they remained constant up to 3 months. The mean values of the measurements performed in triplicate are reported.TM microscope . The sample was placed on a pedestal as powder and then coated with gold.The morphology of the nanoparticles was examined by FESEM using an FEI SciosTM 12 microscope  operated at an acceleration voltage of 120 kV.Sample preparation for TEM was carried out using 0.06 mg/mL nanoparticle suspensions in purified water and ultrasonification for 3 min with 30% amplitude. A drop of this suspension was placed on a 200 mesh copper grid coated with carbon. Once the drop was dried at room temperature, a drop of uranyl acetate was added and the copper grid was imaged in an FEI TecnaiTM iS5 spectrometer , equipped with an iD3 ATR accessory  controlled by OMNIC Software Ver. 6.1.0.0038 , measuring in absorbance mode with a resolution of 4 cm−1, a spectral range of 4000–550 cm−1, and 64 scans, using N-B strong apodization and mertz phase correction. ATR-FTIR analysis was performed to detect the possible structural changes of SF after loading with curcumin. Each spectrum was acquired on a NicoletAbsorbance spectroscopy was performed on a Synergy MX UV-Vis spectrometer . Samples were scanned from 230 to 700 nm in 96 well non-sterile acrylic Costar UV Plates, (N° 3635) , with UV transparent flat bottom.Fluorescence was studied on a Synergy MX UV-Vis spectrometer . The fluorescence excitation spectra of curcumin samples or Curc-SFNs were obtained using 96 well non-sterile Costar black stripwell plates (N° 3914)  by varying excitation from 300 to 510 nm (5 nm step) and recording emission intensity at 530 nm. Fluorescence emission spectra of curcumin samples or Curc-SFNs were obtained by exciting at 420 nm and monitoring emission from 450 to 700 nm (5 nm steps). ® vials containing a mixture of 800 μL of methanol and 100 μL of a water suspension of the samples at 1 mg/mL of curcumin or Curc-SFNs. It is well known that substances which have radical scavenging activity such as curcumin induce color bleaching of a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) solution, which can be measured spectrophotometrically . To asseg at room temperature) to remove particles in the supernatants that could affect the spectrophotometric measurements. The radical scavenging was evaluated by measuring the absorbance at 515 nm, by using a Synergy MX UV-Vis spectrometer  adding 200 μL/well. The radical scavenging activity was presented as Ascorbic Acid Equivalents per milligram of Curc-SFNs (nmol AAE/mg Curc-SFNs). All tests were done in quintuplicate and results were expressed as mean ± standard deviation (SD).Vials were thoroughly mixed and kept in darkness for 30 min. In the same conditions, ascorbic acid solutions (0–300 nmol in methanol) were tested as positive controls. After 30 min of reaction at 25 °C in darkness, the suspensions were centrifuged in an Eppendorf Centrifuge 5415D , human neuroblastoma (Kelly Cells) and human bone marrow-derived mesenchymal stem cells (hBMSCs). Hep3B cells were obtained from the American Tissue Culture Collection  and Kelly Cells were purchased from Sigma Aldrich . The hBMSCs were obtained as previously described in a paper with silk materials and curcumin of the Kaplan research group . The cul4 cells/well (for Hep3B and hBMSCs) and 2.0 × 104 cells/well (for Kelly Cells) and left for 24 h, after which the growth medium was removed and replaced with the medium containing SFNs, Curc-SFNs 1 or Curc-SFNs 2. In each experiment, growth medium without nanoparticles was used as a control. After 24 h, the culture medium of each well was replaced with fresh medium containing SFNs or Curc-SFNs in a wide concentration range: from 1.62 to 6650 μg/mL. Initial suspensions of nanoparticles in ultrapure water were previously sonicated 3 min with 30% amplitude and then diluted to 10% (v/v) with the medium. After 48 h, the medium was removed and AlamarBlue® assay  was performed following the manufacturer’s protocol. Then, post reaction medium aliquots (90 μL) were transferred to black 96-well plates and quantified for fluorescence intensity within a SpectraMax M2 plate reader  using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The data are presented as mean values ± SD calculated from at least three samples per condition.For cytotoxicity studies, the three cell lines were seeded onto 96-well tissue culture plates at a density of 1.0 × 10The SFNs, Curc-SFNs 1 and Curc-SFNs 2 were characterized by DLS to ascertain their hydrodynamic diameter (expressed as Z-average), PdI, Zeta Potential and Electrophoretic Mobility . Figure The results showed that SFNs had a lower diameter while the PdI was similar (and lower than 0.15) for all of the samples, resulting in near monodisperse size distributions. The Zeta Potential of the SFNs and Curc-SFNs was in the same range and sufficiently negative, which is especially relevant if nanoparticles are to have high colloidal stability. The absolute Zeta Potential values of the SFNs was higher than the values found in the literature for SFNs obtained by other classical methods ,54,69,70To detect any differences in the size and morphology of SFNs, Curc-SFNs 1 and Curc-SFNs 2, TEM was performed. The TEM observations  showed gFESEM is a commonly used technique to study the morphology and size of a variety of nanoparticles. FESEM observation  of these−1 , 1517–1521 cm−1  and 1232 cm−1 , as in the literature . Fo. Fon, are change . The same change  and for e change .The DPPH· assay has been previously used to test the free radical scavenging capacity of curcumin and curcumin-loaded nanocarriers ,82. In iThese results agree with the DLC of curcumin in nanoparticles, previously described in Drug Loading Section, and remarks that curcumin retain the full radical scavenging activity after being adsorbed onto SFNs in the assay conditions. This antioxidant protective effect of SFNs has been previously described with other sensitive phenolic compounds as Resveratrol  or Querc® in three cell lines: Hep3B cells, Kelly cells and hBMSCs. We found similar trends for the tumor cell lines (Hep3B cells and Kelly cells) with regard to nanoparticle cytotoxicity  plays a vital role in the sensitivity of these cell lines to curcumin. Depletion of GSH further sensitized the cells to curcumin effects, and the cell death is caused by the generation of reactive oxygen species. Curcumin also down-regulated the expression of bcl-2 protein in tumor cells, which may be responsible for making them vulnerable to apoptotic death.In cancer cells, metabolic pathways are reprogrammed to satisfy tumor cell proliferation and survival requirements. In these cells, glycolysis and glutaminolysis are strongly increased. These metabolic processes and the role of mitochondria in supporting tumor cell metabolism are probably the reason of the different behavior between Hep3B, Kelly Cells and hBMSCs. Syng-ai et al.  showed tCurc-SFNs were successfully synthesized by two environmentally friendly procedures using ILs and high-power ultrasound to dissolve the SF. High DLC and EE values were obtained in both cases compared with those in the literature, and curcumin release was influenced by the synthesis method of the Curc-SFNs. The SFNs and the Curc-SFNs obtained showed a narrow size distribution, with a hydrodynamic diameter of <175 nm, and high Zeta Potential (in absolute terms), which make them excellent nanocarriers for use in therapeutic treatments. The nanospherical morphology was confirmed by FESEM and TEM, and the curcumin was fluorescent when encapsulated or adsorbed on the SFNs but not in its free state. The antioxidant activity against DPPH· showed that the curcumin loaded in Curc-SFNs retains full antioxidant activity. The Curc-SFNs obtained in this paper enhanced the antitumor activity of curcumin towards the two different tumor cell lines studied, while the viability of the healthy cells did not decrease. This work broadens the possibility of using these SFNs, which have been synthesized by an industrial process, as future systems for other drugs of hydrophilic or hydrophobic nature, such as curcumin.


Asymmetrical shear rolling with velocity asymmetry and geometry asymmetry is beneficial to enlarge deformation and refine grain size at the center of the thick plate compared to conventional symmetrical rolling. Dynamic recrystallization (DRX) plays a vital role in grain refinement during hot deformation. Finite element models (FEM) coupled with microstructure evolution models and cellular automata models (CA) are established to study the microstructure evolution of plate during asymmetrical shear rolling. The results show that a larger DRX fraction and a smaller average grain size can be obtained at the lower layer of the plate. The DRX fraction at the lower part increases with the ascending speed ratio, while that at upper part decreases. With the increase of the offset distance, the DRX fraction slightly decreases for the whole thickness of the plate. The differences in the DRX fraction and average grain size between the upper and lower surfaces increase with the ascending speed ratio; however, it varies little with the change of the speed ratio. Experiments are conducted and the CA models have a higher accuracy than FEM models as the grain morphology, DRX nuclei, and grain growth are taken into consideration in CA models, which are more similar to the actual DRX process during hot deformation. Aluminum alloy thick plates with light weight, high strength, good corrosion resistance, and formability, as a key material for integrated-structure parts, are widely used in the aerospace field . Hot rolDynamic recrystallization (DRX) plays a vital role in grain refinement during hot rolling . THe kinAsymmetrical shear rolling is a rolling method with velocity asymmetry and geometry asymmetry, which is beneficial to deformation permeation and grain refinement, especially at the center point of an ultra-thick plate. Many previous studies have focused on the effect of parameters of symmetrical rolling or different speed rolling on DRX fraction and grain size by microstructure evolution models or experiments. However, the effects of the speed ratio and offset distance on grain refinement or microstructure distribution of the plate in asymmetrical rolling are rarely published. Microstructure evolution models are empirical models and they are in relation to the variation of macro parameters in the rolling process, such as strain, strain rate, and temperature, in which the DRX process can be calculated very quickly. The nucleation and grain growth in the DRX process are considered in CA models and the topology of the microstructure can be obtained; however, the computing efficiency is relatively lower. Metallographic experiments can be conducted after rolling or just at given passes, but they are not able to track the microstructure variation during multi-pass rolling processes. Meanwhile, these experiments tend to damage the plate and cause material loss as well as decrease production efficiency. Therefore, the microstructure evolution in asymmetrical shear rolling as well as the effects of the speed ratio and offset distance on grain size variation need to be studied further in order to regulate and control the microstructure variation. However, the difference between these two methods is rarely studied and the accuracy of them needs to be compared in order to obtain the microstructure evolution efficiently. In this study, coupled FEM-microstructure evolution models and CA models are established to study the DRX behavior and variation of grain size for aluminum alloy plate during asymmetrical shear rolling, especially for the cross shear zone. The effects of the speed ratio and offset distance on the DRX fraction and average grain size are analyzed. Finally, experiments are conducted to compare the accuracy of these two kinds of models.S) of the slow roll in outlet direction, as shown in Asymmetrical shear rolling is a rolling method with velocity asymmetry and geometry asymmetry. The lower roll has a larger velocity than the upper roll and there is also a horizontal offset distance –(7).dX is the DRX fraction, ε0.5 is the strain corresponding to 50% DRX fraction, dD is the recrystallized grain size, D0 is the initial grain size, and The DRX process mainly depends on two parameters: The DRX fraction During the hot rolling process, work hardening and dynamic recovery are common phenomena; DRX will take place when the dislocation density reaches the critical value, which has a significant role in grain refinement. In this study, probabilistic CA models are adopted to predict the variation of the microstructure and grain size during hot rolling. In the CA model, the complex object is divided into cells discrete in time and space and each cell is defined by its dislocation density, crystal orientation, temperature, and strain rate, in which cells with the same crystal orientation belong to a specific grain. The status values of each cell are updated at each CA time step according to the transition rules between the cell and its neighborhood. In the CA model, the temperature, strain, strain rate, and flow stress are derived from FEM results. To acquire equiaxed grain and decrease microsegregation from casting, the homogenization treatment is conducted before hot rolling. The nucleation points are spread uniformly into the simulation zone and they grow into equiaxed grains in all directions with the same probability. ρ is the dislocation density, k1 and k2 are work hardening and dynamic softening coefficients, respectively; α is the dislocation interaction term, μ is the shear modulus, and b is the modulus of Burger’s vector.7055 aluminum alloy is a kind of material with high stacking fault energy, which plays a vital role in its softening mechanism in hot deformation. The softening mechanism during hot rolling consists of two processes: Dnamic recovery and DRX. The flow stress decreases in the macro level and the dislocation density reduces in the micro level. The stacking fault energy of the material significantly affects the variation of dislocation density and thus influences the softening mechanism. The dislocation density in a material provides the relationship between flow stress at the macro level and microstructure characteristics at the micro level, as shown in Equation (8). Therefore, the variation of dislocation density can be obtained by true stress-strain curves . A Kocksv) [p) and the grain boundary (GB) mobility (M), as shown in Equation (11).C is the material constant, m is the sensitivity coefficient of strain rate, and actQ is active energy.Newly dynamically recrystallized nuclei start to appear when the dislocation density exceeds critical value. The nucleation rate   is relatIn the hot rolling process, DRX plays an important role in grain refinement. With velocity asymmetry and geometry asymmetry in asymmetrical shear rolling, the distributions of the DRX fraction and average grain size are asymmetrical, as shown in In the author’s previous studies , the speThe effect of the speed ratio on distributions of the DRX fraction and average grain size is depicted in X, is defined. X, which increases significantly with the ascending speed ratio. A large speed ratio is beneficial to induce strong shear strain and will enlarge the strain gap between the upper and lower surfaces. The offset distance has little effect on the DRX fraction difference. Therefore, a proper speed ratio should be set by comprehensive consideration of a sufficient DRX fraction at the center point as well as a small DRX fraction difference between the upper and lower surfaces.As discussed above, the speed ratio and offset distance have an effect on the asymmetrical distribution of the DRX fraction and average grain size between the upper and lower surfaces. In order to quantificationally describe this asymmetry, a parameter of the DRX fraction difference, ΔIn asymmetrical shear rolling, asymmetrical strain distribution and temperature distribution will result in heterogeneous microstructure distribution. The movement asymmetry of the speed ratio and geometry asymmetry of the offset distance during asymmetrical shear rolling are the obvious distinction between symmetrical rolling. The effects of the speed ratio on the average grain size and recrystallization fraction at the center point are depicted in i = 1~4), while the velocity of the lower roll is fixed (0.0785 m/s). Different offset distances are achieved by inserting spacers with different thicknesses  between bearing boxes of rolls and the frame. The material of the plate is 7055 aluminum alloy at casting state, with compositions of (wt %) 6.7Zn-2.6Mg-2.6Cu-0.15Fe-0.13Zr-0.12Si-0.06Ti, that is machined into plates with different sizes. The initial size of the plate is 150 mm (length) × 60 mm (width) × 20 mm (thickness). The initial rolling temperature is 410 °C and the plates are heated to the rolling temperature and then held for 30 min to acquire temperature balance in the heating furnace. The plates are immediately transferred to the rolling mill in less than 10 s and the heat exchange during the transfer period is disregarded. Due to the restriction of the mill capacity, each pass reduction is 2 mm and six rolling passes are conducted. A large speed ratio (i = 1.2) and large offset distance (S = 4 mm) are applied in the previous three rolling passes to acquire strong shear deformation. For the last three passes, the speed ratio turns to 1.1 and offset distance turns to 2 mm. An asymmetrical shear rolling mill is remolded by a symmetrical rolling mill as follows: two work rolls with a diameter of 100 mm are driven by two motors to achieve a speed ratio (3 + 1.5 mL HCl + 1 mL HF + 95 mL H2O). The optical microstructures at different positions of the section plane were examined using a Leica DMI5000M image analyzer . The average single-circle-intercept grain sizes were measured using the method described in the American Society for Testing and Materials (ASTM) standards, which is beneficial for the grain size measurement of the material with obvious grain size difference at different positions. It should be guaranteed that there are at least 35 sections in each circle to improve the accuracy. Metallography experiments results are shown in After multi-pass asymmetrical rolling, the specimens were cut at different positions (at the upper and lower surfaces as well as at the center) from the plates by a wire cutting machine. Then, the sections were polished by different types of abrasive paper and polishing paste, and etched by Keller’s reagent (2.5 mL HNO(1)Microstructure evolution models and CA models are established to study the DRX behavior of the plate in asymmetrical shear rolling. The effects of the speed ratio and offset distance on the DRX fraction as well as grain size during asymmetrical shear rolling are analyzed.(2)The distributions of the DRX fraction and average grain size are asymmetrical in asymmetrical shear rolling and the lower part of the plate has a larger DRX fraction and finer grain than the upper part. The DRX fraction difference between the upper and lower surfaces increases with ascending speed ratio, but the variation of the offset distance has little effect on this difference.(3)More sufficient DRX and finer grain can be obtained with ascending speed ratio, while the increase of the offset distance will slightly decrease the DRX fraction and increase the average grain size.(4)A proper speed ratio should be selected to ensure sufficient DRX on the basis of small bending behavior of the plate, and the existence of the offset distance is mainly used to decrease the bending behavior of the plate during asymmetrical shear rolling.(5)CA models have a higher accuracy than FEM models because they take comprehensive consideration of DRX nuclei, grain growth, and grain morphology.


HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley.The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 ( Starch is the most important storage carbohydrate of higher plants and plays important roles during their life cycle. Our economy depends on starch as primary food source, to exploit its unique physicochemical properties in numerous industrial applications, as feedstock and for the production of bioethanol . Crop plThe enzymes responsible for the biosynthesis of starch granules were amongst the first enzymes studied. An initial hint as to how starch is synthesized was already given by Hanes, Green and Stumpf in the early 1940s. They reported on a “series of reversible chemical reactions by which glucose is transformed” ,3 througIn vitro experiments with potato tubers showed high levels of glucose incorporation into tuber starch using [14C] G1P as a glucosyl donor ..in situ HvPho1 (from a 1.5 mg·ml-1 stock) and 5μl of HvBEIIa (from a 4 mg·ml-1 stock). The concentration of G1P was 50 mM. The reaction buffer was 50 mM MOPS pH 6.5. The reactions were boiled after different incubation times, spun down and diluted 1:10 before analysis (10 μl injected per run) in a DIONEX ICS-3000 system with pulsed amperometric detection and a Carbopac PA-100 column. For analysis, buffer A was 70 mM KOH and buffer B was 70 mM KOH plus 500 mM KOAc. A linear gradient was run going from 100% buffer A at 0 minutes to 40% buffer A, 60% buffer B at 30 minutes, followed by 98% buffer B to 35 minutes and 100% buffer A to 38 minutes. Debranching of an aliquot from the reaction incubated for 4 h was performed by adding CrISA1 to a final concentration of 0.01 mg·ml-1 and incubation overnight at 25°C.Reactions were done in 250 μl including 5μl 2O in 85:20:50:50 proportions. Development was done with a 5% H2SO4 and 0.5% α-naphtol solution in EtOH followed by charring in a hot plate at 250°C.TLC was performed with a protocol adapted from  on a TLCS1 Fig-1 of different enzyme preparations were incubated for 1 hour at 37°C. Reaction and analysis conditions were otherwise the same as indicated in the main text for the reactions including HvBEIIa. The product pattern is qualitatively the same as for the WT and Δ78 constructs of Pho1.Reaction mixtures with 10 mM maltoheptaose, 10 mM glucose-1-phosphate and 0.1 mg·ml(TIF)Click here for additional data file.S2 FigHvPho1ΔL78  was incubated with G1P (25 mM) as sole substrate. (A) The figure shows production of 1,4 glycosidic bonds, usage of G1P and generation of reducing ends recorded over time by proton NMR spectroscopy. (B) Plot of the generation of 1,4 glycosidic linkages over the number of reducing ends indicating approximate lengths of glucans produced over time.(TIF)Click here for additional data file.S3 FigHvSSIIa in the soluble fraction of endosperm extracts of grains from 0 to 24 DAF as indicated above the lanes. The correct bands, marked with a blue ellipse, are identified with the help of a lane loaded with recombinantly produced HvBEIIa (red arrow and “Recomb” label). No HvBEIIa was detected in the insoluble fractions.Immunological detection of (TIF)Click here for additional data file.S4 FigHvAGPPase is highly active and drives the production of G1P from ADP-glucose. HvPho1 is present as active protein able to produce glucans de novo that act as substrates for branching enzyme HvBeIIa, possibly in concert with G1P produced by phosphoglucomutase. (B) During the second step, the branched glucans, produced by HvBeIIa and HvPho1 are substrates for starch synthases and debranching enzymes that produce crystalline precursors for starch and thus initiate starch biosynthesis. (C) The last step is grain filling in which starch granules grow to macroscopic structures. The photo inserts in each developmental step picture the approximate developmental stage of a grain. ADP, adenosine diphosphate; ADPglucose, adenosine diphosphate glucose; AGPase, adenosine diphosphate glucose pyrophosphorylase; AGPPase, adenosine diphosphate glucose pyrophosphatase; ATP, adenosine triphosphate; DBE, starch debranching enzyme; Glucose6-P, glucose-6-phosphate; Glucose1-P, glucose-1-phosphate; PGM, phosphoglucomutase; PPi, pyrophosphate; PPiase, pyrophosphatase; SBE, starch branching enzyme; SS, starch synthase.Shown is a simplified model of a barley endosperm plastid and the principal set of reactions that drive starch biosynthesis during endosperm development. (A) During the initial stage of endosperm development (TIF)Click here for additional data file.S1 FileDescribed is a hypothetical three-step model for the involvement of Pho1 in starch biosynthesis in barley endosperm.(PDF)Click here for additional data file.


Microbial Biotechnology (MBT), which was created as a sister Journal of Environmental Microbiology for publication of the best research in applied microbiology, attained the age of 10 which, in journal lives, probably corresponds to mid‐puberty! Certainly, the Journal has recently been experiencing remarkable spurts in growth of submissions, which is healthy because it enables the Journal Editors, Editorial Board and other Reviewers, to be increasingly selective and the Journal to publish papers of increasing quality and originality.This year, Minireviews, and Thematic and Special Issues. On a biannual basis, it publishes a Crystal Ball Feature in which invited luminaries from both inside and outside the field predict – sometimes wildly – which new ideas, technologies, discoveries or applications from other disciplines, will drive the most important advances in microbial biotechnology . In order to identify bottlenecks in the roads to important medium‐term biotechnology goals and encourage efforts to circumvent such obstacles, MBT recently published the feature Microbial Biotechnology – 2020, in which, leading researchers defined key goals for 2020 and developed road maps to attain these goals. Most recently, the previous issue of MBT was devoted to an assessment of the current and potential contributions of microbial biotechnology to attainment of the Sustainability Development Goals (SDGs) specified by the United Nations , with the goals of not only encouraging younger researchers to appreciate the wider socio‐political context in which their work plays out, but also exposing to policymakers the incredible range of important applications that characterizes work in microbial biotechnology, and their impacts on key issues and problems currently facing the planet and its inhabitants.Quality refers to both individual published works and to new or topical thematic collections that represent initiatives of the Editors, Editorial Board Members and author–readers of the Journal. As can be appreciated from the MBT website, MBT philosophy is not only to publish excellent author‐initiated submissions, but also to stimulate and support the field of microbial biotechnology, by soliciting papers on, and showcasing, (to become) hot topics or technical developments that will catalyse significant advances in the field. It does this with One of numerous global problems facing the biosphere is the accumulating mountain or, more accurately, sea of plastic waste, and the current issue is devoted to the potential of microbial biotechnology to contribute to mitigation strategies and to channel plastic waste into a value chain that includes environmentally friendly bioplastics and valuable synthons.Editorials that identify exciting and rapidly expanding sectors offering excellent employment opportunities for young people. The current sector emphasized is the microbiome  and has thus far featured inter alia microbiome transplants, microbiome applications in agriculture and microbiome‐based forensics. The series is ongoing, and further, highly interesting contributions are in the pipeline.In its efforts to stimulate the field, MBT focuses considerable effort on young researchers and their careers. One initiative it started this year was to publish a series of Microbiome Yarns, that exploits humour and fantasy to draw attention to key papers in the fields featured. While some of the current initiatives will continue beyond 2017, other new ones will be launched, so watch this space!It is a truism that serendipity underpins many important discoveries . However, serendipitous discoveries tend to be made by researchers who are  receptive to the unusual and unexpected, and to the possibilities that current laboratory lore may be incorrect or laboratory paths may be leading to dead ends, and/or are able to make connections between apparently unrelated topics. To both encourage a healthy critical attitude to laboratory lore and directions and to expose young people to important and interesting topics and advances that may not be on their radar screens, MBT publishes a series, Since its inception, MBT has benefitted from an outstanding team of dedicated Editors who devote much time and energy to provide authors with the best possible support and critical, objective advice. The Founding Editors were Willem de Vos, Willy Verstraete, Juan Luis Ramos, Marty Rosenberg and Kenneth Timmis, with Joan Timmis as Journal Administrator. After many years of dedicated service, Marty and Willy rotated off, to be replaced by Siegfried Vlaeminck and Auxi Prieto. Larry Wackett has been Web Alert Editor since the launch of the Journal, and Antoine Danchin joined recently as Genome Update Editor. It has been an incredible personal experience for us to work with such a cohesive, inspiring and fun group of scientists, and we take this opportunity to express our intense and undying gratitude for their exceptional dedication to the Journal and the field in general.But, we should also like to single out Juan Luis Ramos for particular mention: although all Editors have substantial workloads, Juan Luis, who is also Minireview Editor and Special/Thematic Issue Editor, and hence responsible for the recruitment and editorial handling of practically all the minireviews published in the Journal , and for the handling of the majority of SI/TI submissions, has an exceptional workload, far exceeding those of others, which he cheerfully and efficiently manages, on top of all the other demanding responsibilities that come with his day job. Thank you so much Juan Luis!ad hoc reviewers, who represent the best talent in the field and who provide knowledgeable, constructive and supportive critiques, and decision recommendations, on manuscripts they review. Moreover, Editorial Board Members contribute significantly to the development of Journal policy, and some, in particular, Victor de Lorenzo, Sang Yup Lee and Jack Gilbert, play vital roles in many Journal initiatives. Thank you all for your super contributions to the success of MBT!Of course, the task of the Editors can only be accomplished through the reviews we receive from our outstanding Editorial Board and other And, of course, MBT would neither exist nor have experienced the success it enjoys, if it were not for its publisher, Wiley, who has always done a sterling job of production, management and marketing: thank you everyone at Wiley for your constant support and professional handling of the Journal.And finally, a big thanks to the Society for Applied Microbiology who welcomed MBT into its journal stable and has provided a tremendous amount of moral and microbiological support: thank you so much Phil and Lucy!None declared.


Plant storage compounds such as starch and lipids are important for human and animal nutrition as well as industry. There is interest in diverting some of the carbon stored in starch-rich organs  into lipids in order to improve the energy density or nutritional properties of crops as well as providing new sources of feedstocks for food and manufacturing. Previously, we generated transgenic potato plants that accumulate up to 3.3% triacylglycerol (TAG) by dry weight in the tubers, which also led to changes in starch content, starch granule morphology and soluble sugar content. The aim of this study was to investigate how TAG accumulation affects the nutritional and processing properties of high oil potatoes with a particular focus on starch structure, physical and chemical properties. Overall, TAG accumulation was correlated with increased energy density, total nitrogen, amino acids, organic acids and inorganic phosphate, which could be of potential nutritional benefit. However, TAG accumulation had negative effects on starch quality as well as quantity. Starch from high oil potatoes had lower amylose and phosphate content, reduced peak viscosity and higher gelatinization temperature. Interestingly, starch pasting properties were disproportionately affected in lines accumulating the highest levels of TAG (>2.5%) compared to those accumulating only moderate levels (0.2–1.6%). These results indicate that optimized engineering of specialized crops for food, feed, fuel and chemical industries requires careful selection of traits, and an appropriate level of transgene expression, as well as a better understanding of starch structure and carbon partitioning in plant storage organs. Plants are important sources of starch and lipids (oil) for food and industry. Starch is the primary storage compound in leaves, cereal grains, and tubers while lipids are a major component of seeds such as sunflower and oil palm. As well as contributing significantly to human caloric intake, starch granules have a complex molecular structure that has led to many applications in food processing and industry Jobling, . Lipids Increasing the lipid content of starch-rich organs could improve the nutritional value or energy density of crops as well as increase lipid production for food, feed, fuel, and chemical industries. It may even be possible to generate crops that provide both a conventional grain harvest as well as a biomass harvest for oil. Significant progress has already been made in the production of biomass oil crops with TAG accumulating to 2 and 35%  in the vegetative tissue of sugar cane and tobacco, respectively  and transgenic potato plants  were grown in the glasshouse and potatoes were harvested at maturity as described in Liu et al. , diacylglycerol acyltransferase 1  and oleosin (lipid packaging protein from Sesamum indicum). Tissue from potatoes was freeze dried for 72 h prior to lipid, starch, soluble sugar and metabolite analysis. Fresh tuber tissue was used for starch extraction.Wild-type  and quantified by Bradford assay  was determined using a Europa 20–20 isotope ratio mass spectrometer with an ANCA preparation system, comprising a combustion and reduction tube operating at 1,000°C and 600°C, respectively. Potato TAG was quantified by chloroform/methanol/water extraction, thin layer chromatography separation, fatty acid methylation and gas chromatography as described in Liu et al. . Measure−1 ribitol  in H2O was added and samples were again homogenized. After centrifugation at 16,100 × g for 5 min, 30 μl of the supernatant was dried down and derivatized at 37°C for 90 min with 15 μl of 20 mg ml−1 methoxyamine hydrochloride (Sigma-Aldrich) in pyridine . Following derivatization, 15 μl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide  was added to each sample and further incubated at 37°C for 30 min. Aliquots (25 μl) of derivatized sample were transferred to 2 ml GC sample vials along with 5 μl of n-alkane standard mix , and left to equilibrate for 4 h. The samples were analyzed by GC-MS on a DB5MS column with a 10 m integrated guard column  fitted to an Agilent 7890A gas chromatograph with a 5975C inert MSD quadrupole detector (Agilent Technologies). Samples (1 μl) were injected at a 10:1 split into the inlet that was heated to 230°C with helium as the carrier gas with a total flow of 24 ml min−1, septum purge flow of 3 ml min−1, and a split flow of 10 ml min−1. The column gas flow was set at 1 ml min−1. The oven temperature was as follows: the initial temperature of 70°C was held for 1 min, then increased to 320°C at 15°C min−1, and held at 320°C for 3 min. The MSD transfer line was heated to 250°C, MS source at 230°C and MS quadrupole at 150°C. The electron impact ionization energy was 70 eV and the MS detector was run in full-scan mode with an m/z range between 35 and 550 and 2.83 scans s−1. MetabolomeExpress  analysis were conducted based on Roessner et al.  with theStarch was isolated as described in Liu et al. . Starch −1, held at 95°C for 4 min, cooled to 50°C at 11.25°C min−1 and then held for 4 min at 50°C. Peak viscosity, hold viscosity (end of maximum temperature holding phase), final viscosity and pasting temperature were recorded using the Thermocline software provided with the instrument.Pasting properties of isolated starch were determined using a Rapid Visco Analyzer RVA-4SA . Starch samples (2 g) and deionized water (25 ml) were weighed directly into a test container and mixed well using a plastic paddle before loading on the instrument. Starch suspensions were stirred at 960 rpm for 10 s after which stirring was reduced to 160 rpm for the remainder of the experiment. Samples were equilibrated at 50°C for 2 min, heated to 95°C at 7.5°C min−1) were mounted onto StarFrost silane coated slides . The coverslip was sealed with nail polish to prevent streaming of starch granules during heating. Hot stage temperature ramp rate was 4°C per minute to a maximum of 90°C. Images were taken at 15 s intervals over 17 min. Intensity profiles created in LASX software were exported to Microsoft Excel. The initial intensity was set at 100% and profile temperatures  were calculated assuming a linear loss of intensity. Only wild-type and starch from the highest TAG lines were analyzed as these were expected to reveal the greatest differences. Starch swelling power was determined using the method of Konik-Rose et al.  fitted to a Leica DM6 B microscope with CTR6 LED controller and LASX software  was used to analyse loss of birefringence of isolated starch granules under polarized light during heating. Aliquots (2 μl) of homogenous slurries of starch in water . Starch/water mixtures  were vortexed vigorously and incubated at 4°C for 16 h. Triplicate aliquots (50–55 μl) were weighed into stainless steel capsules with rubber O-rings. The program heated from an initial temperature of 20°C to 140°C at 10°C minin situ by light microscopy of fresh, unstained sections (Axio Imager).Isolated starch granules were stained with iodine and imaged using an Axio Imager light microscope . Isolated granules were also sputter-coated with gold and imaged using a Zeiss EVO LS 15 scanning electron microscope at 5 kV. Starch granules were observed t-test, p < 0.01) than wild-type levels (<0.05% DW) in all eight independent transgenic potato lines but varied widely from 0.17% DW to 3.35% DW  compared to wild-type  although the total carbon content was unchanged . Correlation coefficients between TAG and the other carbon- and nitrogen-containing components are shown in Supplemental Table Overall, the increase in total nitrogen but not total carbon in transgenic lines led to a significant negative correlation between tuber TAG content and the carbon-to-nitrogen ratio . Inorganic phosphate was also more abundant in high oil potatoes. The two metabolites that were more abundant in wild-type potatoes consisted of one citric acid cycle intermediate (citrate) and one amino acid (asparagine). Orthogonal partial least squares analysis of metabolite profiles revealed that lines accumulating the highest levels of TAG >2.5%) clustered separately to those accumulating lower levels of TAG (0.2–1.6%) and to wild-type lines  and positively correlated with granule size  and within the variation observed between wild-type replicates  as well as higher hold viscosities  and final viscosities . There was no significant difference in the pasting temperature between wild-type and high oil lines , hold viscosity  and final viscosity  and final viscosities , end temperature  and peak temperature  than wild-type starch but did not have a significant different enthalpy of transition . Correlation analysis identified significant relationships between starch granule size and onset temperature , end temperature  and peak temperature  but no correlation between apparent amylose content or TAG content and any of these chemical properties of starch. DSC profiles of starch from wild-type and medium TAG lines showed a large, sharp peak whereas starch from high TAG lines showed a lower, broader peak.The chemical properties of starch from wild-type and high oil potatoes were also investigated using differential scanning calorimetry Figure . Starch In addition to large reductions in starch content, high oil potatoes had such strong differences in starch chemistry that thermal and pasting properties were severely affected, especially in lines accumulating the highest levels of TAG. Large changes in the levels of other metabolites were also observed in high oil potato lines, which may affect their nutritional and processing properties. While TAG accumulation might be expected to affect potato starch and soluble sugar content through competition for carbon, the dramatic changes to nitrogen partitioning and starch structure are likely indirect effects. The high oil potato lines thus provide an interesting model to investigate different levels of metabolic and biosynthetic regulation as well as the effects of starch structure on gelatinization and pasting profiles. The potential for these potatoes as a novel source of oil or specialized crop will also be discussed.GBSS expression in TAG-accumulating (WRI1-overexpressing) potatoes.The structure and chemistry of starch granules was investigated in detail in order to identify possible causes of the striations, altered granule shape and reduced granule size. High oil potato starch had significantly reduced amylose content. This combination of low amylose content and small, misshapen granules is surprising given that striations and a pocked surface are usually observed in high amylose mutants  and/or plant glycogenin-like starch initiation proteins 1, 2, 5, and 6  which are all upregulated at the transcript level in WRI1 affects only a small number of starch-related genes, it remains unclear how TAG accumulation leads to altered biosynthesis, chemistry and morphology of starch granules. While transcripts for SSIV and the PGSIPs are upregulated in WRI1-overexpressing potatoes, the AGPase small subunit transcript (AT5G48300.1) is downregulated and no other starch-related genes are affected  via the trehalose-6-phosphate pathway and then induce amino acid biosynthesis in an attempt to balance the carbon-to-nitrogen ratio, as occurs in Arabidopsis leaves  to deliver yields comparable to cotton seed, potentially making it a commercially feasible source of oil (Liu et al., An alternative approach would be to use potatoes accumulating medium levels of TAG for novel products or added nutrition. The starch content and quality of these lines is close to wild-type and TAG accumulation correlates with changes to other metabolites that may give nutritional or processing benefits. For example, the high oil potatoes are notably low in asparagine, normally the most common amino acid in potato (Golan-Goldhirsh, In summary, metabolic engineering of potato tubers to accumulate high levels of TAG also resulted in potatoes with altered nutritional profiles, starch structure and quality. Starch granule size, morphology, chemical and physical properties were particularly affected, which may have implications for downstream applications and processing. Future manipulations of carbohydrate pathways as well as source/sink relationships could further improve potato, oil, and/or starch yields. Such strategies have the potential to produce novel crops with benefits to biotechnology and the food industry as well as human nutrition.MM conceived of and performed experiments, analyzed data and wrote the manuscript. JP, SO, OL, DY, and NS performed experiments and analyzed data. QL provided plant material. FP and SS participated in the design and coordination of the study. JR conceived of experiments, analyzed data, contributed to the manuscript and participated in the design and coordination of the study. All authors read and approved the manuscript.This research was funded by CSIRO Agriculture and Food.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SEM of starch from five EAHB cultivars showed a mixture of irregular granule shapes with smooth surfaces. The starches' average diameter size ranged between 16.31 and 21.98 μm. The moisture, protein, and ash content of the starches were: 11.12%–11.84%, 0.1% and 0.23%–0.47% respectively. The amylose content of these starches was between 11% and 13%. The starches peak viscosity ranged between 488.42 and 558.66 RVU. The EAHB starches exhibited relatively low pasting temperatures (<75°C), a high peak viscosity (488.42–558.71 RVU), high level of viscosity breakdown (235.00–311.92 RVU) and low set‐back values (61.21–104.33 RVU). In general, the EAHB starches' WHCs and SPs increased substantially at 80°C with maximum SP (12.43–14.27 g water/g starch) and solubility (12.52%–14.19%) values obtained at 90°C. The starch clarity ranged from 1.7% to 2.3% and followed the same pattern. The starches exhibited poor freeze‐thaw stability as they had high syneresis (68% to 72%) after 10 freeze‐thaw cycles.The  The main botanical sources of starch are wheat, maize, potato, and cassava with only minor quantities from rice and other starches produced  Muranga, . Due to Although, banana starch characterization has been carried out extensively in the last two decades  namely: Nandigobe, Enyeru, Mpologoma, Mbwazirume, and Bukumu were selected from a 24‐acre demonstration plantation of the Presidential initiative on Banana Industrial Development (PIBID) project in Bushenyi district, which is at an altitude of about 1607 m above sea level with bimodal rainfall seasons receiving an average rainfall of about 1233 mm and average temperatures of 19.3°C. The plants were cultivated under normal conditions of cultivation.Five cultivars of edible green (unripe) pre‐climateric EAHB . The EAHB starch powders were placed on the SEM stubs using a two–sided adhesive tape . The specimen stub was subsequently coated with Pt‐Pd using an MSP‐1S magnetron sputter coater (Vacuum Device Inc.). The coated samples were then analyzed using the SEM operated at 10.0 KV.2.5Particle size was analyzed with a Laser scattering particle size analyzer  installed with a batch sample cell using methyl propanol as the solvent. The percentages of the number of granules corresponding to the different sizes were recorded and average granule diameter was obtained directly using the equipment's software.2.6The pasting properties of the EAHB starch samples were measured by a Rapid ViscoAnalyser . The General pasting method STD1 profile ICC,  was used2.7g using a bench top centrifuge . The supernatant was decanted and the volume determined. The sediment was dried in an oven for 2 hr at 110°C and the swelling power (SP) determined by difference.These were determined as described by Waliszewski et al. . Starch 2.8Paste clarity was determined according to the method by Waliszewski et al. . In brie2.9This was evaluated using Bello‐Perez et al.'s  method. 33.1The chemical composition of the starches from the different EAHB cultivars is presented in Table The moisture content (MC) of the starch from the five EAHB cultivars ranged from 11.1% to 11.8% and protein content was 0.1% Table  of 10.8%The amylose content of the starches from the five EAHB cultivars was in the range of 11.96–12.83% Table ; ~17% fo3.2Figure The EAHB starches showed a mixture of irregular granule shapes of elongated, spheroid and oval granule shapes with smooth surfaces devoid of dents Figure . These r3.3Musa paradisiaca) to have an average diameter of 24.31 μm. Waliszewski et al.  with no significant differences, while Mpologoma and Mbwazirume starches (12.52% and 12.8% respectively) exhibited lower solubility values that were not significantly different from each other at 90°C.The starch solubility for the EAHB cultivars was lower than that reported by Coulibaly et al.  for nati3.6The pasting of the EAHB starches exhibited gradual viscosity increase with increasing temperature during the heating stage 50°C–90°C) until peak viscosity was attained  used for determination of their pasting characteristics. However, EAHB starch exhibited lower temperature (<75°C) for paste development in comparison to corn starch (>80°C)  of the starches from the EAHB cultivars ranged from 1.7% to 2.3% Figure . Hoover,3.8Freeze–thaw stability is one of the quality characteristic of starch gels. When a starch gel is subjected to repeated freezing and thawing cycles, it releases water a condition termed as syneresis. The extent of syneresis is a measure for its freeze–thaw stability Goff, .The starMpologoma starch, which had the smallest mean granule diameter and lowest SP, exhibited the lowest freeze–thaw stability while Mbwazirume and Nandigobe starch starches, which had the biggest mean granule diameter and highest SP respectively, exhibited the highest freeze‐thaw stability. Singhal and Kulkarni  attributEarlier findings have demonstrated low freeze‐thaw stability for plantain banana starch (Bello‐Perez et al., We hereby declare that all the authors of this manuscript have no conflict of interest pertaining to this study whatsoever.