Photolysis of nido-1 and nido-2 with group 6 metal carbonyls, M’(CO)5.THF (M’ = Mo or W) were performed that led to the formation of a series of adducts [(Cp*M)2B6S2H4(CS3){M’(CO)5}]  instead of cluster expansion reactions. In these adducts, the S atom of C=S group of dithione {B2CS3} moiety is coordinated to M’(CO)5 (M = Mo or W) in η1-fashion. On the other hand, thermolysis of nido-1 with Ru3(CO)12 yielded one fused metallaheteroborane cluster [{Ru(CO)3}3S{Ru(CO)}{Ru(CO)2}Co2B6SH4(CH2S2){Ru(CO)3}2S], 7. This 20-vertex-fused cluster is composed of two tetrahedral {Ru3S} and {Ru2B2}, a flat butterfly {Ru3S} and one octadecahedron {Co2RuB7S} core with one missing vertex, coordinated to {Ru2SCH2S2} through two boron and one ruthenium atom. On the other hand, the room temperature reaction of nido-2 with Co2(CO)8 produced one 19-vertex fused metallaheteroborane cluster [(Cp*Rh)2B6H4S4{Co(CO)}2{Co(CO)2}2(μ-CO)S{Co(CO)3}2], 8. Cluster 8 contains one nido-decaborane {Rh2B6S2}, one butterfly {Co2S2} and one bicapped square pyramidal {Co6S} unit that exhibits an intercluster fusion with two sulfur atoms in common. Clusters 3–6 have been characterized by multinuclear NMR and IR spectroscopy, mass spectrometry and structurally determined by XRD analyses. Furthermore, the DFT calculations have been carried out to gain insight into electronic, structural and bonding patterns of the synthesized clusters.In an effort to synthesize metallaheteroborane clusters of higher nuclearity, the reactivity of metallaheteroboranes,  I from thermal autofusion of [(Pme2Ph)2PdB8H12]  with [Co2(CO)8] led to the formation of a 6 SEP hypoelectronic cluster [(Cp*Re)2Co2(CO)5B4H4], in which a 4-electron {Co2(CO)5} fragment is coplanar with the B4 fragment [2(CO)6(η5-C5Me5RuCO)(η5-C5Me5Ru)B6H10] from the reaction of nido-[(η5-C5Me5Ru)2B6H12] and [Fe2(CO)9], in which a {Fe2B2} tetrahedron has been fused to ruthenaborane moiety through two common boron atoms [2 B6H6E2{Co2(CO)2}(μ-CO)2{Co4(CO)8}] (E = Se or Te), III which can be described as a fusion of three individual olyhedral such as the two 5-vertex nido-square pyramid {Co3B2} and one 12-vertex icosahedron {Co4B6E2} units having two common triangular faces  . I is a B2} edge . SuccessB2} edge ,41,42,43fragment . Fehlneron atoms . Recentl2)(PPh3)2], the first CS2 metal complex by Wilkinson and co-workers, a range of CS2 complexes containing transition metals have been synthesized [2CS} moiety can act as a ligand to bind to one or two metal centres [2}2-o-C2B10H10] (IV) in which Sme2 groups are coordinated to exo-boron atoms   from the photolysis of {CH2S2} bridged decaborane-14 analogue nido-[(Cp*Co)2B6S2H4(CH2S2)] with {M’(CO)5·THF} (M’ = Mo or W) fragments  was treated with [Fe2(CO)9] at room temperature, it afforded an 11-vertex nido-[(Cp*Co)2B6S2H4(CH2S2){Fe(CO)3}], which is similar to [C2B9H11]2− with a five-membered pentahapto coordinating face [nido-[(Cp*Co)2B6S2H4(CH2S2)], we also have isolated CS3 bridged decaborane-14 analogues, nido-[(Cp*M)2B6S2H4(CS3)]    as violet and red solids, respectively  and δ = 24.6, 26.4. 33.4 and 38.2 ppm (for 4). However, the identity was unclear until an X-ray crystallographic analysis was carried out for one of them. Despite of our several attempts, we could not get good quality crystals for 3. The CH2Cl2-hexane solution of 4 at −5 °C yielded X-ray quality crystals that allowed us to study the X-ray diffraction of 4.As shown in ectively . These c4, shown in 2CS3} moiety is coordinated to W(CO)5 in η1-fashion. The S1-C1 bond distance of 1.648(12) Å in 4 is slightly longer than C=S bond length of nido-1 [1.636(6) Å]. The W-S bond distance of 4 [W1-S1 2.529 (3) Å] is slightly longer compared to [CpW(CO)2{η2-(S2CC6H4Me4)}] (2.472 Å) . The M’-S bond distances of 5 [Mo1-S5 2.539 (15) Å] and 6 [W1–S5 2.293 (7) Å] are slightly longer compared to those of [CpMo(CO)2{η2-(S2CCH2tBu)}] (2.477 Å) and [CpW(CO)2{η2-(S2CC6H4Me4)}] (2.472 Å), respectively. moiety is coordinated to C=S group of di-thione {B2CS3} is more as compared to its tungsten analogue 4. The similar trend has been observed in case of 5 and 6. However, the HOMO-LUMO gap for the Rh systems (5 and 6) is greater than the Co systems (3 and 4). The highest HOMO-LUMO gap is observed for compound 5, which indirectly indicates higher stability than its Co analogue.To provide some insight into the electronic structures and bonding relationship of the adducts nido-1 and nido-2 with group 6 metal carbonyls yielded simple coordination compounds, reactions with both Ru3(CO)12 and Co2(CO)8 led to the formation of condensed clusters [{Ru(CO)3}3S{Ru(CO)}{Ru(CO)2}Co2B6SH4{Ru(CO)3}2 (SCH2S2)] 7 and [(Cp*Rh)2B6H4S4{Co(CO)}2{Co(CO)2}2(μ-CO)S{Co(CO)3}2] 8, respectively (nido-1 and nido-2 while doing thin-layered chromatography (TLC). Although we were not able to purify them, fractional crystallization allowed us to get a few crystals of them for X-ray diffraction studies. Therefore, the characterization of both 7 and 8 are solely based on the molecular structures obtained from the single crystal XRD analysis.Although the reactions of ectively . Note th7 reveals the molecular formula as [{Ru(CO)3}3S{Ru(CO)}{Ru(CO)2}Co2B6SH4{Ru(CO)3}2(SCH2S2)]. This is a 20-vertex macro-polyhedral metallaheteroborane cluster, in which fusion and coordination are observed concurrently } unit bridge to Ru5, B6 and B5 atoms of nido-{Co2B6RuS} unit, respectively in exo fashion. Additionally, the butterfly {Ru3S} unit of 7 is almost flat with a dihedral angle of 172.93° (3S} butterfly core of 7 (avg. 2.489 Å) are considerably longer than the bond length of [(Cp*Ru)22] (2.3733 Å) . Although, this looks like a fusion of three polyhedra, for example, one bicapped square pyramid {Co6S}, one butterfly {Co2S2} and one 10 vertex nido-{Rh2B6S2}, careful evaluation of this cluster shows that one bicapped square pyramidal {Co6S} core is fused with a butterfly {Co2S2} unit via one common {Co2} edge. This entire fused cluster {S2Co6S} is coordinated to B4 and B6 atoms of 10 vertex nido-{Rh2B6S2} through the wingtip S3 and S4 atoms of the butterfly {Co2S2}.As shown in 8 can also be described in a different approach. As shown in 6S} and two boron atoms (B4 and B6) on the 10-vertex nido-{Rh2B6S2} subcluster in µ3-fashion. A similar description has also been proposed for [(PPh3)NiS2B16H12(PPh3)] by Kennedy , [Ru3(CO)12], [Mo(CO)6], [W(CO)6], LiBH4 (2.0 M in THF) were used as purchased from Sigma Aldrich. The starting materials nido-[(Cp*M)2B6S2H4(CS3)]  were synthesized according to the literature procedures  from the photolysis of Mo(CO)6  in THF (5 mL) was added dropwise to the yellow solution of nido-1. The reaction mixture was irradiated for 3 h, and we observed the color change from yellow to reddish brown. The solvent was removed in the vacuum. Then, the residue was extracted with n-hexane and filtered through 3 cm of celite. The reaction mixture was purified on TLC plates. Elution with a CH2Cl2/n-hexane (30:70 v/v) mixture afforded a violet solid 3 . In the same reaction conditions, treatment of nido-1 with [W(CO)5·THF] prepared from the photolysis of W(CO)6  in THF (5 mL), yielded a red solid 4 .In a flame-dried Schlenk tube under an Ar atmosphere, previously reported 3: 11B{1H} NMR : δ (ppm) = 26.5 , 27.6 , 33.0 , 37.7 ; 1H NMR : δ (ppm) = 1.65 , IR : ῡ = 2418 (B-Ht), and 1941 .Spectroscopic data of 4: MS (ESI+): calcd for [C26H34B6Co2O5S5W]+: m/z 953.9741, found: 953.9784; 11B{1H} NMR : δ (ppm) = 24.6 , 26.4 , 33.4 , 38.2 ; 1H NMR : δ (ppm)= 1.69 ; 1H{11B} NMR :3.49 , 3.75 , 4.03 ; IR : ῡ = 2477 (B-Ht), and 1923 .Spectroscopic data of nido-2  was suspended in 5 mL of dry THF at room temperature. A freshly prepared solution of [Mo(CO)5·THF] from the photolysis of Mo(CO)6  in THF (5 mL), was added dropwise to the yellow solution of nido-2. The reaction mixture was irradiated for 3 h, the color changed from yellow to reddish brown. The solvent was removed in the vacuum. Then, the residue was extracted with n-hexane and filtered through 3 cm of celite. The reaction mixture was purified on TLC plates. Elution with a CH2Cl2/n-hexane (30:70 v/v) mixture afforded a orange solid 5 . In the same reaction conditions, treatment of nido-2 with [W(CO)5·THF] prepared from the photolysis of W(CO)6  in THF (5 mL), yielded a red solid 6 .In a flame-dried Schlenk tube under an Ar atmosphere, 5: MS (ESI+): calcd for [C26H34B6Rh2S5MoO5 – (Mo(CO)5) + H]+: m/z 719.0010, found: 719.0036; 11B{1H} NMR : δ (ppm) = 17.3 , 20.3 , 24.6 , 30.4 ; 1H NMR : δ (ppm) = 1.85 ; 1H{11B} NMR :3.50 , 3.75 , 4.06 ; IR : ῡ = 2403 (B-Ht), 1941 .Spectroscopic data of 6: MS (ESI+): calcd for [C26H34B6Rh2S5WO5 + NH4]+: m/z 1059.9531, found: 1059.9754 11B{1H} NMR : δ (ppm) = 16.7 , 20.5 , 23.9 , 24.9 ; 1H NMR : δ (ppm)= 1.80 , 1H{11B} NMR : 4.67 , 4.75 , 5.09 ; IR : ῡ = 2453 (B-Ht), 1925 .Spectroscopic data of nido-1  in 10 mL dry toluene was charged with [Ru3(CO)12]  at room temperature. The reaction mixture was stirred for additional 16 h at 80 °C. The solvent was removed under vacuum and the residue was extracted into hexane/CH2Cl2 (70:30 v/v) mixture and passed through Celite. Note that compound 7 was isolated in very poor yield and it was combined with the unreacted precursor nido-1 in thin-layered chromatography (TLC). As a result, purification of 7 was not possible for spectroscopic characterization. Although we were not able to purify it, the fractional crystallization allowed us to get few crystals of 7 for X-ray diffraction studies.In a flame-dried Schlenk tube, under argon atmosphere, a yellow solution of nido-2  in dry toluene (10 mL) was charged dropwise with a solution of [Co2(CO)8]  in 5 mL dry toluene at room temperature for 16 h. The colour of the reaction mixture was changed from yellow to deep brown. The solvent was removed under vacuum and the residue was extracted into hexane/CH2Cl2 (70:30 v/v) mixture and passed through celite. Compound 8 was isolated in very poor yield that was merged with unreacted precursor nido-2 in thin-layered chromatography (TLC). Thus, the purification of 8 for spectroscopic data was not possible. Although we were not able to purify it, fractional crystallization allowed us to get few crystals of 8 for X-ray diffraction studies.Under argon atmosphere, in a flame-dried Schlenk tube, the yellow solution of 4, 5, 6, 7 and 8 were grown at −5 °C by slow diffusion of a CH2Cl2-hexane solution. The X-ray data were collected and integrated by using D8 VENTURE Bruker AXS for 4, 5, 6 and 7 with a CMOS-PHOTON70 detector having multilayer device monochromated Mo-Kα (λ = 0.71073 Å) radiation at 150(2) K. For 8, the X-ray data were collected and integrated by using Bruker D8 VENTURE diffractometer with PHOTON II detector having graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation at 297(2) K. Using SHELXT-2014, SHELXS-97, , and 417 parameters.Crystal data of 5. C26H34B6MoO5Rh2S5, formula weight, rM.= 953.45, orthorhombic, Pbcn, unit cell, a = 18.0346(10) Å, b = 15.4968(12) Å, c = 27.550(2) Å, α = 90°, β = 90°, γ = 90°; Z = 8; V = 7699.6(9) Å3; μ = 1.471 mm–1; F(000) = 3776; calcdρ = 1.645 g/cm3; 1R = 0.0422; w2R = 0.0969; 8750 independent reflections [2θ ≤ 54.99°], and 428 parameters.Crystal data of 6. C26H34B6O5Rh2S5W, formula weight, rM.= 1041.36, orthorhombic, Pbcn, unit cell, a = 18.0153(15) Å, b = 15.5395(16) Å, c = 27.555(3) Å, α = 90°, β = 90°, γ = 90°; Z = 8; V = 7714.0(13) Å3; μ = 4.124 mm–1; F(000) = 4032; calcdρ = 1.793 g/cm3; 1R = 0.0219; w2R = 0.0494; 8770 independent reflections [2θ ≤ 54.99°], and 428 parameters.Crystal data of 7. C39H36B6Co2O18Ru7S5, 2(CH2Cl2); formula weight, rM.= 2013.04, triclinic, P-1, unit cell, a = 11.6527(14) Å, b = 13.0484(12) Å, c = 21.925(2) Å, α = 94.971(4)°, β = 105.005(5)°, γ = 99.109(5)°; Z = 2; V = 3150.8(6) Å3; μ = 2.539 mm–1; F(000) = 1940; calcdρ = 2.122 g/cm3; 1R = 0.0559; w2R = 0.1293; 14194 independent reflections [2θ ≤ 54.97°], and 758 parameters.Crystal data of 8. C33H34B6Co6O13Rh2S5, formula weight, rM.= 1423.16, Triclinic, P-1, unit cell, a = 12.4725(4) Å, b = 14.4351(5) Å, c = 16.0368(5) Å, α = 88.7070(10)°, β = 74.7360(10)°, γ = 79.9110°; Z = 2; V = 2741.52(16) Å3; μ = 2.594 mm–1; F(000) = 1396; calcdρ = 1.724 g/cm3; 1R = 0.0295; w2R = 0.0764; 13201 independent reflections [2θ ≤ 56.00°], and 596 parameters.Crystal data of All molecules were fully optimized using the BP86 functional ,68, in c3–6 in which the S atom of the {B2CS3} moiety is coordinated to group 6 metal carbonyl fragments in η1-fashion. On the other hand, we have established the structures of clusters 7 and 8 having very characteristic and unusual fusion. The molecular structure of cluster 7 consists of a butterfly {Ru3S} moiety that is almost flat. Cluster 8 shows bicapped square pyramidal {Co6S} unit that is fused with a butterfly {Co2S2} via a common {Co2} edge. Although both the clusters are of condensed types, the electron counts are very complex and they do not follow any of the cluster-counting rules, including Mingos’ fusion formalism and Jemmis’ mno rule.In summary, we have synthesized and structurally characterized numerous exciting clusters


Musa balbisiana genome that would enable carbon flow back to growth and starch synthesis and maintain a high starch content even during ripening. Enzymes associated with the initiation of ripening were involved in ethylene and auxin metabolism, starch breakdown, pulp softening, and ascorbate biosynthesis. The initiation of ripening was cultivar specific, with faster initiation being particularly linked to the 1-aminocyclopropane-1-carboxylate oxidase and 4-alpha glucanotransferase disproportionating enzymes. Information of this kind is fundamental to determining the optimal time for picking the fruit in order to reduce post-harvest losses, and has potential applications for breeding to improve fruit quality.In this study, we aimed to investigate for the first time different fruit development stages in plantain banana in order gain insights into the order of appearance and dominance of specific enzymes and fluxes. We examined fruit development in two plantain banana cultivars during the period between 2–12 weeks after bunch emergence using high-throughput proteomics, quantification of major metabolites, and analyses of metabolic fluxes. Starch synthesis and breakdown are processes that take place simultaneously. During the first 10 weeks fruits accumulated up to 48% of their dry weight as starch, and glucose 6-phosphate and fructose were important precursors. We found a unique amyloplast transporter and hypothesize that it facilitates the import of fructose. We identified an invertase originating from the  Combined analyses of the proteomes and metabolomes of plantain banana cultivars indicate the order of appearance and dominance of specific enzymes/fluxes during fruit development. Fruit development is a complex phenomenon that encompasses several overlapping stages: cell division and enlargement, maturation, ripening, and senescence . During Musa species) are parthenocarpic and hence the ovaries develop into seedless fruits without a pollination stimulus and hence without fecundation. The pulp-initiating cells are situated within the inner epidermis of the fruit pericarp and septa. In parthenocarpic bananas, these cells start to proliferate very rapidly after flowering (bunch emergence) (M. acuminata (A genotype) and M. balbisiana (B genotype) (Edible bananas (ergence) . There aenotype) . AlthougProteomics is a technology that has been successfully applied to study complex biological processes in fruit . A betteAll biological samples were collected from the IITA Experimental Field in Ibadan, Nigeria, during the period from October 2016 to February 2017.3) = [Fruit length × (Fruit circumference)2 × 0.0616] + 0.3537. After determination of fruit length and circumference, the fruits were separated from their peels, cut into pieces and stored at –80 °C until lyophilization. Samples were lyophilized to ensure safe transportation from the growing site in Nigeria to the laboratory in KU Leuven in Belgium, and to facilitate the protein and metabolite extraction process (Five banana plants of each of the Agbagba and Obino l’Ewai cultivars were selected and the same plants were followed throughout the experiment. One fruit per plant was collected from 2 weeks after bunch emergence (WAE) until the fruits reached full maturity. The collected fruits were cleaned and measurements of fruit length (L) and circumference (C) were taken. For the calculation of fruit volume our formula was based on . To deteExtractions were performed following the phenol-extraction/ammonium-acetate precipitation protocol described previously (® software (Nonlinear Dynamics). In this software we used MASCOT v.2.2.06 (Matrix Science) against the Musa V2 database of M. acuminata and M. balbisiana  and purified using Pierce C18 Spin Columns (both ThermoFisher Scientific). The digested samples (0.5 µg per 5 µl) were separated in an Ultimate 3000 UPLC system and then in a Q Exactive Orbitrap mass spectrometer (both ThermoFisher Scientific) as described by lbisiana . Proteinhttps://www.expasy.org/resources/prosite). Subcellular predictions were analysed using the software DeepLoc 1.0 . The supernatants were collected and pooled for measurement of soluble metabolites. Glucose, fructose, and sucrose were measured enzymatically  and the optimization toolbox solver quadprod with interior-point-convex algorithm for the minimization. Flux maps were drawn with the flux visualization tool of VANTED 2.1.0.x matrix) and the time-points, cultivar, and quantified metabolite as dependent variables (y matrix). We applied a two-way ANOVA (P<0.05) to the selected proteins to determine their significance as affected by the time-point and cultivar, or the interaction between both.For proteins, statistical analyses were conducted using the software Statistica 8 (TIBCO) based on the exported protein quantifications from Progenesis. We performed a principal component analysis  to get an overview of the proteome data. We performed a partial least-squares analysis (PLS)  to differentiate proteins with a significant correlation to the time-points, the cultivar, or metabolite using all protein quantifications as continuous predictors  for Pearson correlations in the relationships between the proteins and fluxes.To integrate the different omics data, the protein inference and isoform redundancy issue was tackled by quantifying the proteins at the protein cluster level and EMPAI quantification . To find the protein clusters that correlated to the modelled fluxes, we performed a two-block sparse partial-least-squares discriminant analysis (sPLS-DA) with the mixOmics package of R  (M. acuminata with an AA genome)  . The fir genome) ; Supplem genome) . HistoneFast cell division and growth is also accompanied by high activity of cell-wall building and modifying enzymes , mRNA translation , and protein folding (T-complex proteins) and turnover (proteasome complexes) . The ideBanana pulp is a starch-synthesizing sink tissue that needs to get all its energy from sucrose unloaded from the phloem. From studies in tomato, it is known that the fruit growth consists of two phases: first, a period of rapid growth where sucrose synthase determines the sink strength, and second, a phase after the rapid growth has ceased, where invertase takes over . In the On average across the two cultivars, the pulp at 6 WAE contained three times more fructose than glucose, but the concentration of fructose represented <1 % of that of starch and <15% of that of sucrose . Among tThe accumulation of starch in the pulp cells was initially very rapid but showed a clear linear decline with time . Starch P<0.0001, R=0.99) with SuSy (1)  . PlastidSuSy (1) ; Fig. 6.Following the uptake of glucose 6-phosphate into the pulp amyloplast, starch synthesis starts via the concerted actions of phosphoglucomutase (7), glucose 1-phosphate adenylyltransferase (8), and the starch polymerizing reactions  ; Fig. 6.On average across the two cultivars, the pulp at 12 WAE contained twice as much fructose as glucose, but the concentration of fructose represented <0.5 % of that of starch and <5% of that of sucrose . Among tStarch-to-sucrose metabolism has been extensively studied in model systems in the context of energy sources for plant growth and development ; howeverPhosphorolytic cleavage seems to be one of the first starch breakdown reactions and this was corroborated by the abundance profiles of phosphorylase (20) and the G6P transporter (6) ; Fig. 7.M. acuminata genome (A genotype) is most probably either not expressed or expressed at only a very low level since we did not find a confident specific spectrum. We have previously shown that invertase is more abundant in plantain compared with a Cavendish type banana , alpha-1,4 glucan phosphorylase (20), and chloroplastic phosphoglucomutase (7) . UTP-glue banana . Plantaie banana ; Fig. 7.e banana  would ale banana . Alterate banana ; Fig. 7,e banana  and antie banana . Ascorbi2+) 2-oxoglutarate dioxygenase domain profile. A 2-oxoglutarate-dependent-Fe(2+) dioxygenase in rice has been shown to convert active auxin (indole acetic acid) into biologically inactive 2-oxoindole-3-acetic acid, supporting a key role in auxin catabolism (The increase in sucrose production was significantly correlated with the abundance of 1-aminocyclopropane-1-carboxylate oxidase (ACO) , the enztabolism . We hypoIn plum, it has been shown that ethylene is a crucial factor that affects overall sugar metabolism, stimulating sorbitol breakdown via increased sorbitol dehydrogenase .The concentrations of metabolites in the pulp determined from 2–12 WAE  were fitThe flux analyses at 6 WAE and 12 WAE complemented the proteome data, and good concordance was observed for the major reactions in starch biosynthesis : sucroseBy combining proteomics and flux studies, our study provides unique insights into the order of appearance and dominance of specific enzymes/fluxes involved in starch and sugar synthesis and breakdown in plantain. Our results for fluxes give a broader analysis of metabolism. Although the fluxes were calculated in a non-compartmented network, we have shown that in conjunction with proteome data they can give a satisfactory picture of the dynamics of metabolism during fruit development. Maturation in plantain is completed around 10 weeks after bunch emergence, as indicated by a net breakdown in starch. The import of G6P and possibly fructose into the amyloplast are the main drivers of starch synthesis. Soluble starch synthase probably plays a more important role in starch synthesis during the early fruit development period, while granule-bound starch synthase most likely influences starch at the mature stage. For starch breakdown, DPE and phosphorylase mainly produce the first hexoses for sugar synthesis and amylases come into play at a later stage in ripening. Cytoplasmic invertase in plantain seems to play an important role in the breakdown of sucrose to support further growth and to maintain a high starch content. Our data point towards an interplay between auxins and ethylene controlling the ripening process. Despite the fact that both the plantain cultivars that we studied are extremely close genetically, we still found significant differences in ripening. The earlier ripening in Agbagba might be related to an earlier induction of the second ethylene system and a greater scavenging of auxins. Overall, our results contribute to a better understanding of fruit development and maturation in banana, and more specifically in plantains.JXB online.The following supplementary data are available at Fig. S1. Principal component analysis of the metabolic compounds..Fig. S2. Changes in fruit volume over time for the two plantain varietiesFig. S3. Changes in starch contents over time for the two plantain varieties.Fig. S4: Simplified flux maps at 2, 6, and 12 WAE.Table S1. All proteins identified in fruit pulp.Table S2. Proteins with differential abundance during development.Table S3. Concentrations of soluble sugars and sugar-phosphates, and quantification of starch.Table S4. Flux modelling data.erac187_suppl_Supplementary_Figures_S1-S4Click here for additional data file.erac187_suppl_Supplementary_Tables_S1-S4Click here for additional data file.


In this issue of the journal, Koizumi and colleagues  have demThere is no doubt that the L-shaped incision provides excellent exposure for total arch replacement . This in


Journal of Environmental and Public Health has retracted the article titled “Study on the Impact of Chinese Comedy International Communication on the Health of Older People under Cultural Ecological Environment” [ronment”  due to cFollowing an investigation conducted by the Hindawi Research Integrity team , signifi


A novel strategy for improving wet resistance and bonding properties of starch-based adhesives using enzymatically polymerized lignosulfonates and carboxylic acids as additives was developed. Therefore, lignosulfonates were polymerized by laccase to a molecular weight of 750 kDa. Incorporation of low concentrations (up to 1% of the starch weight) of 1,2,3,4-butanetetracarboxylic acid (BTCA) led to further improvement on the properties of the adhesives, while addition of greater amounts of BTCA led to a decrease in the properties measured due to large viscosity increases. Great improvements in wet-resistance from 22 to 60 min and bonding times (from 30 to 20 s) were observed for an adhesive containing 8% enzymatically polymerized lignin and 1% BTCA. On the other hand, the addition of citric acid (CA) deteriorated the properties of the adhesives, especially when lignosulfonate was present. In conclusion, this study shows that the addition of the appropriate amount of enzymatically polymerized lignosulfonates together with carboxylic acids (namely BTCA) to starch-based adhesives is a robust strategy for improving their wet resistance and bonding times. The increasing interest in substituting fossil-based products with renewable bio-based resources has made starch one of the preferred natural polysaccharides to develop various industrial applications ,2,3. StaLignosulfonates would be a non-toxic and renewable alternative to increase the wet resistance of adhesives . Lignin Polycarboxylic acids are a group of safe and green chemicals whose use as additives for improving the properties of starch materials, such as retrogradation, friction coefficient, wear degree or hydrophobicity are already well known ,29,30,31Here, the combined effect of the addition of enzymatically polymerized lignosulfonates and of said carboxylic acids on starch-based paper-adhesives, in order to enhance its properties, was investigated for the first time.MtL8 was used to polymerize a 20% solid content LS solution, while monitoring the changes in molecular weights (MW) and viscosities. As previously reported, the viscosity of lignin increases while it is being polymerized ,27,36. PLaccases  are multicopper containing enzymes that catalyze the oxidation of various aromatic compounds, reducing molecular oxygen to water. In lignin, one electron abstraction from phenolic hydroxyl groups can lead to cross-linking via formation inter-unit linkages, which include β-O-4, β-5, 5-5, β-β, 5-O-4 ,38.Despite the potential of enzymatically polymerized lignosulfonates to increase wet-resistance of starch based adhesive, this could be on the expense of the bonding properties . On the w/w) led to a steep increase in the viscosity, changing the properties of the adhesive, turning from a viscous liquid to a paste-like adhesive, increasing more than 1150% . Since the difference with the other viscosity values was very high and would make the visual comparison between the other samples difficult, it was not included in When LS, polymerized until a MW of 750 KD, was present in the starch adhesive, the viscosity augmented with increasing concentration of BTCA, while the opposite occurred for the starch-only adhesives A. The hiWhen CA was added, the viscosity of the starch-only adhesive behaved in a similar way as seen with BTCA. In this case, the addition of 10% CA decreased the viscosity of the starch-lignin adhesive, with this effect being higher with increasing amounts of this acid B.The reduction on the viscosity of starch materials in the presence of CA has been previously reported in literature, due to granule surface corrosion, the hydrolysis of lamellar/nonlamellar amorphous starch as well as the degradation of starch molecules ,40.w/w) led to a remarkable increase in the strength of the material, greatly reducing the time needed for a complete bonding. Interestingly, higher concentrations of BTCA had the opposite effect, being more pronounced when no lignin was present . This concentration is, by far, lower than determined in other studies in which the addition of higher concentrations of BTCA (up to 80%) were reported to be optimum . In thisOn the other hand, the addition of CA always lowered the wet resistance of the starch only adhesive B, while,The data shows that the addition of BTCA is an attractive approach for improving the material characteristics of the adhesives, while the addition of CA had a negative effect both on bonding and only a marginal positive effect on wet-resistance. In contrast to previous reports in which high temperatures for crosslinking the acid with the starch were used ,30,31, tMyceliopthera thermophila (MtL8) was obtained from Novozymes . Industrial LS from softwood  and the starch adhesives were provided by SAPPI Austria and AGRANA Austria, respectively.All used chemicals were of analytical grade. Sodium hydroxide (NaOH) was obtained from Merck , 2′azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,2,3,4-butanetetracarboxylic acid (BTCA) were obtained from Sigma-Aldrich , and citric acid was obtained from Carl Roth . Laccase from w/v) and 120 μL MQ-water were added. After 2 h at 21 °C under 800 rpm agitation, 200 μL of the reactions were transferred into a 96-well transparent plate and their absorbance measured in a plate reader (Tecan Infinite M200). A standard curve was constructed using vanillin in concentrations ranging from 0.05 to 1 mg/mL. All samples were measured in triplicate.For determining the content of phenolic groups in the LS, the Folin–Ciocalteu (FC) method was used, as described in . Brieflyw/w, adjusting the pH to 7. The enzymatic polymerization of 250 mL of LS solution was carried out in 500 mL transparent Durham bottles with tight-fitting tube holes on the lid to allow air supply on an IKA RCT standard magnetic stirrer equipped with an IKA ETS-D5 thermometer . The activity of the laccase (1.761 × 104 nkat/mL) was determined following the protocol described in [3 min−1 air under constant stirring at 600 rpm, was started by introducing 233.0 nkat of MtL8 per mL of the lignosulfonate solution, corresponding to 3.300 mL of MtL8.The dried lignosulfonates (LS) were dissolved in ultra-pure water with a solid content of 20% ribed in . The reaIn order to follow the polymerization process of the LS, two processes were carried out.First, the viscosity increase during polymerization was monitored by rheometry. Samples of 0.8 mL were measured in an Anton-Paar  rheometer (MCR 302) with a CP50-1 cone plate at 200 1/s for 10 s with a constant temperature of 20 °C, obtaining the viscosity of the material in mPa·s.3, 3 mM NaN3) to a concentration of 4 mg mL−1 before injecting 100 μL into the system. The SEC system consisted of an auto sampler 1260 series , a quaternary/binary pump, a Diode Array Detector and a Refractive Index detector system (Agilent Technologies 1260 Infinity), and a MALLS (Multi-Angle Light Scattering) HELEOS DAWN II detector . The system was equipped with a pre-column PL aquagel-OH MIXED Guard  and a separation column PL aquagel-OH MIXED H , having a mass range from 6 kDa to 10,000 kDa. The Agilent Software Openlab Chemstation CDS and the ASTRA 7 software (Wyatt technologies), were used for acquiring and processing the data, respectively. Bovine serum albumin (BSA) was used as standard for the normalization, band broadening and alignment of the MALLS detectorSecondly, the molecular weight (MW) distribution was analyzed using size exclusion chromatography (SEC) following the protocol described in . A totalSamples were withdrawn at intervals and used for measurements of MW and changes in viscosity.Starch based adhesives were prepared following the protocol described in . Brieflyw/w polymerized LS and 92% w/w starch-based adhesive.For producing the starch-lignin adhesive, laccase polymerized LS was mixed, as described above during 10 min, with the starch adhesive in a ratio of 8% The carboxylic acids (BTCA and citric acid) were added in order to investigate the effect on the properties of the adhesives. Different amounts of the carboxylic acid, in % solid weight of the starch, were added after the adhesive was produced. The materials were stirred as before for 10 min. The material properties of these adhesives  were measured once the acid was completely mixed and compared with samples without acid added.The viscosity of the adhesives was measured in duplicates, with a standard deviation lower than 552 A and 589For determining the bonding time, a sample of adhesive was applied in a 60 µm thick layer using a film applicator  in a sheet of paper with a surface of 17 cm of length and 7 of width. Another piece of the paper, with the same surface, was put on top, pressed together by hand, and pulled apart. The time needed to produce a complete bond  was measured. The measurements were measured in triplicates, with a standard deviation lower than 11.3 A and 17.Wet resistance was measured gluing two 20 × 3 cm stripes of paper with a 60 µm thick adhesive layer using the same film applicator previously described, overlapping an area of 6 cm of length and 3 cm of width. The samples were maintained under a light weight (660 g) overnight and kept under a controlled atmosphere in a desiccator for one week. The paper stripes were hanged with a 100 g weight in one of their ends in a container with water at 25 °C, measuring the time needed for the separation of the pieces of paper. The measurements were measured in triplicates, with a standard deviation lower than 3.66 A and 1.4The addition of the carboxylic acids to a starch-lignin/lignosulfonate adhesive, was found to have a positive effect on its bonding properties and wet-resistance. However, the effects strongly depended on the acid used. The addition of BTCA improved the bonding time, reducing it by 24% when 1% of the acid was added A and, at


In the interests of more flexible and less stiff bridge constructs to stimulate bone healing, the technique of far cortical locking has been designed to improve locked plating constructs in terms of stress concentration, stress shielding, and inhibition of issues around fracture healing. However, far cortical locking screws currently lack objective designs and anti-fatigue designs. This study investigates an optimization algorithm to form a special locking screw composed of various metals, which can theoretically achieve the maintenance of the excellent mechanical properties of far cortical locking constructs in terms of fracture internal fixation, while maintaining the biomechanical safety and fatigue resistance of the structure. The numerical results of our study indicate that the maximum von Mises stress of the optimized construct is less than the allowable stress of the material under each working condition while still achieving sufficient parallel interfragmentary motion. Numerical analysis of high cycle fatigue indicates that the optimized construct increases the safety factor to five. A high cycle fatigue test and defect analysis indicates that the sandwich locking constructs have better fatigue resistance. We conclude that the sandwich locking construct theoretically maintains its biomechanical safety and fatigue resistance while also maintaining excellent mechanical properties for fracture internal fixation. Implants were evaluated in normal femoral diaphysis surrogates to minimize inter-specimen variability . We adopnstructs . The FCLnstructs . The plaTwo modifications were made to the currently adopted FCL constructs; all other parameters remained the same. First, the 9° staggered arrangement of screws was omitted; the second modification was to optimize the topography of the screw structure, and to design grooves where the screw was close to the near cortex. Six threaded screw-holes (6 mm diameter) were arranged in a colinear pattern . The strThe cross-section of the SWL screw and the optimization variables are shown in Model establishment was based on axial compression tests through a proximal sphere (rigid clamp), replicating the axial loading scenario of the bench-top test . The disThe finite element model of each construct in this study was calculated and analyzed by using the structural mesh C3D8R and the mesh independence was further discussed in the Supplementary material .In our optimization, there were three intuitive optimal objects: controllable two-phase stiffness, nearly parallel interfragmentary motion and comprehensive strength.First, the SWL constructs should reduce the stiffness of a standard locked plating construct by over 50%, and the stiffness of the normal model was 2.9 Second, these should induce nearly parallel motion at the near cortex and far cortex, and the difference of the displacement between the near cortex and far cortex should less than 15%:Third, the strength of the SWL constructs should meet the requirement of the safety assessment based on previous work . The resThe flowchart of the optimization method is shown in There are three steps in the optimization process. First, a random array was established for the optimization parameters . Implants were evaluated in surrogate specimens of the femoral diaphysis to minimize inter-specimen variability  using a biaxial universal material testing system  . For theAll samples were carefully collected, sorted, and cleaned, and then micro-nano tomography was performed to observe the damage location and damage mode of the samples under high cycle fatigue testing  construct stiffness of the fixation models for each load, and 2) the interfragmentary motion results at the near and far cortices. Axial stiffness was calculated by dividing the axial load amplitude by the actuator displacement amplitude. Torsional stiffness was calculated by dividing the torsion amplitude by the amplitude of rotation (α) around the diaphyseal axis. Torsional stiffness was multiplied by the unsupported specimen length to derive torsional rigidity. What’s more, we compared the differences in biomechanical safety in three constructs for both loading mode, included 1) the von Mises stress distribution and peak values of the screws and bone models, 2) the average von Mises stress of all elements of the screws and bone models, and 3) numerical and experimental results of high-cycle fatigue test.α = 0.05 were used to detect significant differences.For statistical analysis, the average von Mises stress of all elements of the screws and bone models and the construct stiffness were compared among three groups individually, for both loading mode using one-way ANOVAs with Bonferroni post-hoc tests. For all statistical analyses, a level of significance of Using the optimization method, the sandwich composite metal locking screw was designed for controllable two-phase stiffness, near parallel interfragmentary motion, and comprehensive safety. The final results of the three variables are shown below. The width of the groove (d) was 0.25 mm, and the thickness of the titanium alloy layer (t) was 0.65 mm. The Young’s Modulus (E) was 98 GPa, so titanium alloy (Ti-13V-11Cr-3Al) was chosen as the core material . For torsion with torque greater than 2 Nm, the secondary stiffness of the SWL structure increased to 0.276 Nm2/deg, which was still 13% lower than that of the FCL construct. The stiffness differences of the three constructs are shown in Under an axial load of 150 N, the axial compressive stiffness of the SWL construct was 0.47p < 0.01).The groove of the sandwich composite screw changes the contact pattern between the screw and the cortical bone, so that the displacement difference between the proximal and distal ends of the fracture was small, and an approximately parallel pattern was achieved. , and the von Mises stress of SWL screws was generally lower than that of the FCL screws under axial compressive load. When the axial compressive load was below 800 N or the torsion was below 3 Nm, the maximum von Mises stress of SWL screws was lower than the allowable stress. In addition, the average von Mises stresses of all elements of the screws and bone models were compared among the three groups individually for each loading mode (p < 0.0001). At 10 Nm torsion, the average von Mises stress of the screw elements of the SWL construct was significantly smaller than that of the FCL construct (p < 0.0001). In this study, the stresses of plates in three constructs were in the safe range, far lower than the allowable stresses (As the factor of safety ed line) . Under ting mode . At 1000stresses .For the fatigue life analysis results shown in The micro-nano tomography results were shown in 2 had a negative effect on the core structure.The influence of the three design parameters on the optimal objects during the optimization process was calculated. Through regularized responses on factors, it can be seen that the square term has a considerable effect on the result (over 45%). This study provided a design method for the sandwich composite metal locking screw, and optimized a new construct with controllable two-phase stiffness, near parallel interfragmentary motion, and high fixation construct safety to promote secondary fracture healing. With the development of sandwich structure 3D print technology , we can The sandwich screw is a cantilever beam with different working lengths under increasing load. The groove ensured the bone as a rigid body under a longer cantilever length, until the screw contacted with the near cortex. The larger is the width of the groove, the greater is the contact load. The above theory concurred with the sensitivity analysis shown in In axial compression, the initial stiffness of the far cortical locking construct was 52% lower than that of the locked plating construct . SecondaTo ensure the safety of the new constructs, it is necessary to choose allowable stresses and restrict the applied load to a lower value than the construct can fully support . ConsideThe limitations of this study are as follows: In the model verification experiment, because at present the current technology cannot realize the grafting and 3D printing of 0.65-mm Ti-6Al-4V on titanium alloy (Ti-13V-11Cr-3Al), we only used the SWL screws made of core material titanium alloy (Ti-13V-11Cr-3Al). However, the SWL model in this study is in an ideal condition that Ti-6A1-4V can fully bond with Ti-13V-11Cr-3Al. This combined structure relies on the manufacturing level and needs more fabricating cost. The current metal 3D printing technology is still difficult to achieve fine grafting and printing on complex 3D structures. Thus, the use of this new structure depends on the development of the manufacturing process. In addition, this study was based on an ideal fracture model on cylindrical bone models and did not represent the various complex situations of actual clinical fractures (such as more complex fracture lines and force lines).in vivo.In conclusion, in this study, the finite numerical simulation calculation was used to intelligently optimize the locking screws of the FCL construct under multiple working conditions. The SWL construct theoretically maintains its biomechanical safety and fatigue resistance while maintaining excellent mechanical properties for fracture internal fixation. The newly designed composite metal sandwich locking screw can theoretically achieve elastic fixation, progressive stiffness, uniform load distribution, and parallel interfragmentary motion of the fracture end in the far cortical locking construct, while maintaining better fatigue resistance and torsion resistance to the internal fixation construct. Additional studies are required to assess SWL screws performance in combined loading modes and to determine if SWL constructs effectively promote secondary bone healing


Fig/wasp pollination mutualisms are extreme examples of species-specific plant-insect symbioses, but incomplete specificity occurs, with potentially important evolutionary consequences. Why pollinators enter alternative hosts, and the fates of pollinators and the figs they enter, are unknown.Ceratosolen emarginatus, which concurrently interacts with its typical host Ficus auriculata and the locally sympatric alternative host F. hainanensis, recording frequencies of the wasp in figs of the alternative hosts. We measured ovipositor lengths of pollinators and style lengths in female and male figs in the two host species. Volatile organic compounds (VOCs) emitted by receptive figs of each species were identified using GC-MS. We tested the attraction of wasps to floral scents in choice experiments, and detected electrophysiologically active compounds by GC-EAD. We introduced C. emarginatus foundresses into figs of both species to reveal the consequences of entry into the alternative host.We studied the pollinating fig wasp, C. emarginatus entered a low proportion of figs of the alternative host, and produced offspring in a small proportion of them. Despite differences in the VOC profiles of the two fig species, they included shared semiochemicals. Although C. emarginatus females prefer receptive figs of F. auriculata, they are also attracted to those of F. hainanensis. C. emarginatus that entered male figs of F. hainanensis produced offspring, as their ovipositors were long enough to reach the bottom of the style; however, broods were larger and offspring smaller than in the typical host. Female figs of F. hainanensis failed to produce seeds when visited by C. emarginatus. These findings advance our current understanding of how these species-specific mutualisms usually remain stable and the conditions that allow their diversification. Obligate species-specific pollination mutualisms are important and unique parts of natural ecosystems that facilitate efficient reproductive isolation between plant species . HoweverFicus (Moraceae) and their fig wasp pollinators  that mediate host/pollinator encounter, and matching morphological traits that enforce specificity of the interaction . Such relinators , Yucca  an alternative host  Do the two fig species share semiochemicals that are electrophysiologically active in C. emarginatus? (v) Does relative ovipositor length of C. emarginatus among both typical and alternative hosts vary? (vi) What are the potential evolutionary consequences of entry by C. emarginatus into an alternative host, for both wasps and figs?The fig pollinating wasp association between annensis ; furtherFicus auriculata is distributed in moist valleys in rain forests, whereas F. hainanensis is distributed in limestone areas along rivers. We chose the natural habitats of the two species as study sites, the former in the rain forest in XTBG  in Menglun town, and the latter along the Mengxing River in Mengxing town. All study trees are non-cultivated and their identity was previously validated by genetic data presented in This study was conducted in Xishuangbanna, Yunnan, southwestern China. Ficus species, F. auriculata and F. hainanensis, are dioecious species . F. auriculata exhibits a heart-shaped to round leaves (15–55 × 10–27 cm), and its figs are produced on cauliforous branches on the main trunk and main branches. They are pear-shaped, 3–6 cm in diameter, and green-reddish when unmature (A-D phase). F. hainanensis is distinguishes from F. auriculata by its narrower obovate to elliptic leaves (12–25 × 6–23 cm). The figs are borne on long stoloniflorous branches expanding from the base of the trunk. The pear-shaped, 1–2 cm diameter figs are sparsely speckled with globose tuberculate over the reddish surface (A-E phase). They usually bear figs in December every year at our study site. Their fruiting period is synchronous within species. F. auriculata is typically pollinated by the species traditionally identified as Ceratosolen emarginatus Mayr , we introduced a single female wasp of C. emarginatus into each fig so that we could use the resulting offspring for experimentation without need for species identification. These offspring were temporary stored into a 120-mesh nylon bag (to prevent escape) at room temperature after emergence from the male-phase figs.The two study nanensis . Howevere 88.3%, . This isrphology : among oC. emarginatus and Ceratosolen sp. that had entered and produced offspring into the figs of F. auriculata and F. hainanensis by collecting post receptive figs, five days after the end of receptivity and male-floral phase, respectively. For the former, we collected at least 20 post-receptive figs from each of four male and four female trees. For the latter, we collected 20 male-phase figs per male tree at the same tree about two months later. Each post-receptive fig was cut open and effective foundresses were collected from the cavity and preserved in 75% alcohol for morphological species identification and counting. Every male-phase fig was put into a nylon bag (120 mesh) shortly before wasp emergence. Once wasps had emerged into the bag, ten wasps per bag were randomly selected and preserved in 75% alcohol for subsequent species identification. Combing with the aforementioned morphological criteria  containing a source of odour. Airflow was pumped by a mini-vacuum pump  through the bags into the arms of the Y-tube, after being purified by passing through an activated charcoal filter. The flow rate through each arm was maintained at 100 ml/min. A wasp was deposited in the third arm of the olfactometer for behavioral observation. Preliminary experiments showed that C. emarginatus was equally attracted by male and female figs of F. auriculata , so we used only male figs as a source of odours.To test whether F. auriculata and F. hainanensis were enclosed in nylon bags to protect them from oviposition by wasps until the figs became receptive. Male figs were collected just before pollinator emergence to obtain freshly emerged fig wasps, and only one wasp was selected from each fig. To test the response of C. emarginatus to receptive figs of typical and alternative host, three treatments were carried out: receptive figs of F. auriculata versus air, receptive figs of F. hainanensis versus air, and receptive figs of F. auriculata versus receptive figs of F. hainanensis. Because of differences in fig size between species, equal weights of figs of the two hosts were used . All bioassays were performed in a darkened room, between 10:00–12:00 h, within three hours after collection of fresh figs from trees. Each of the female fig wasps tested was positioned at the entrance to the stem of the olfactometer, and the arm it selected was recorded as well as the time to decision. If the wasp did not make a decision within 5 min, it was excluded from the total number counted and from statistical analysis. After testing five successive wasps, Y-tube arms were reversed to cancel out any orientation-bias effect between the two arms. Each wasp individual was tested only once, and, after ten successive wasps, the Y-tube was replaced with a new one to avoid any influence of residual materials remaining in the apparatus. Three treatments were repeated with total 31, 38 and 41 fig wasps that made a decision.To prepare the odour sources, pre-receptive stage figs of F. auriculata and F. hainanensis were enclosed in nylon bags on trees to prevent wasps from entering and ovipositing. When the figs had reached receptive phase , they were enclosed in polyethylene terephthalate bags for the collection of VOCs. Airflow, purified by passing through a filter of activated charcoal , was maintained through the bag by a mini-vacuum pump connected to the entrance by flow-meters with a flow rate of 300 ml/min, while a VOC trap containing 80 mg Porapak® Q adsorbent  was connected to the exit of the bag at a flow rate of 300 ml/min. To check for possible contaminant compounds sampled during collection, empty bags were used as blanks for extraction by means of the same dynamic headspace technique and equipment. VOC collection was performed for four hours from 10:00 to 14:00, the period of the day when fig wasps are most active. Three repeats were performed for each sex of each tree species. After VOC collection, the adsorbents were eluted three times with a total of 500 µl of dichloromethane and concentrated down to 100 µl with 99.99% N2. Then, two internal standards  were added to every sample prior to gas chromatography.VOCs were collected using the dynamic headspace technique . Small phttp://webbook.nist.gov) and the RI database  with an HP-5MS column . Helium was used as carrier gas at a flow rate of 1ml/min. The injector split vent was set at a ratio of 1:4 and the injector temperature was 250 °C. Oven temperature was set at 40 °C, and then programmed to rise to 150 °C at a rate of 3 °C/min, then at 10 °C/min to 260 °C, and finally temperature was maintained at 260 °C for 5 min. Compound identification was based on comparison of retention times (RT), matching of the mass spectra with the NIST 08 MS library, and Kovats retention indices (RI) taken from both the NIST Chemistry Web Book . VOCs were collected for injection in the GC-EAD as for the VOC analysis procedures except that we extended the collection duration to six hours in order to extract larger quantities of VOCs. The GC program was the same as that used for the analysis of VOCs presented above. A head with an antenna was placed on a micro-operating platform , and two glass electrodes filled with saline solution  were connected to the distal tip of the antenna and to the antennal scape. Antenna depolarization was recorded using the Electroantennography version 2.5 software package . Three antennae from C. emarginatus were tested for F. auriculata VOCs and three for F. hainanensis VOCs.To identify which VOCs of the receptive fig odours were detected by the wasps and thus constituted candidate semiochemicals, we performed electrophysiological tests. The responses of Ficus species and ovipositor length of the pollinator C. emarginatus. We enclosed the pre-receptive figs of F. auriculata and F. hainanensis in nylon bags (120 mesh) to prevent wasp entrance. When the figs became receptive to the pollinators, we collected 32 figs from four F. auriculata male trees and 30 figs from three female trees; we collected 30 figs from three F. hainanensis male trees and 31 figs from three female trees. Then 20 pistillate flowers per fig were randomly selected to measure style length. Ninety male figs of F. auriculata were collected and placed in separate bags before female wasp emergence. After emergence we selected one C. emarginatus from every fig and measured the length of the ovipositor, which was excised from the wasp abdomen and removed the ovipositor sheath. All measurements of style length of figs and ovipositor length of pollinators were conducted using a micrometer in a dissection microscope .As pollinating wasps oviposit by inserting their ovipositors into the female styles of pistillate flowers, we investigated matching relationship between style length in both host F. auriculata and F. hainanensis were enclosed in large nylon bags (120 mesh) to prevent wasp oviposition. When these figs reached receptive phase, one C. emarginatus emerging from F. auriculata was introduced into each fig. Wasps were introduced into at least 20 figs for each tree. We chose three trees of each sex for each host species. After introduction, figs were re-enclosed in large nylon bags until just before wasps emerged from the figs. The figs were then removed from the tree and enclosed in individual nylon bags. All emerging wasps were preserved in 75% alcohol for subsequent counting and measurements. According to morphological convergence produced by the selective pressures from the ostiole : the agonid wasps, C. emarginatus and Ceratosolen sp., are common insects and collection permitted by the leader of Department of Horticulture and Landscape, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Science.The following information was supplied relating to field study approvals (F. auriculata (C. emarginatus (one exception), but the total number of foundresses entering into a fig showed a significant variation . There was no difference in average of foundresses numbers between male (11.4 ± 7.8) and female fig (11.6 ± 6.5). Totally, the proportions of C. emarginatus entering figs of F. auriculata were 94.31% in male figs and 97.69% in female figs, respectively. We collected a total of 931 wasps  that had entered 120 receptive F. hainanensis figs. There was a small number of C. emarginatus . Among these, 24 out of 60 male figs and 11 out of 60 female figs contained C. emarginatus. The total number of C. emarginatus in a male fig was larger than in a female fig . Results for offspring production were qualitatively similar to those for fig visitation, but the differences between typical and alternative hosts were more extreme  were found while from 11 figs from 60 male F. hainanensis figs (occurring rate of 18.3%), there were of total 30 C. emarginatus individuals.We collected a total of 2,079 wasps  that had entered 174 receptive figs of riculata . Among t extreme . In F. aC. emarginatus individuals were strongly attracted by receptive figs of their typical host, F. auriculata, when confronted with a choice between it and air or the alternative host F. hainanensis. The wasps preferred F. hainanensis receptive figs over air . When given a choice between F. auriculata odour and air, C. emarginatus spent the shortest mean recorded time before entering a branch of the olfactometer . C. emarginatus preferred F. auriculata odour when given a choice between it and F. hainanensis, with the time taken independent of final arm choice . In the F. hainanensis versus air treatment, C. emarginatus took longer before entering an arm (63.99 ± 38.29 s) in comparison to the F. auriculata versus air treatment .Female over air . When thF. auriculata and F. hainanensis. VOCs emitted by receptive figs did not differ significantly between sexes within species . Nevertheless, the complement of VOCs produced by F. auriculata and F. hainanensis were distinguishable .We identified a total of 78 VOCs in scents emitted by receptive figs of C. emarginatus presented electroantennographic responses to nine compounds in the odours from F. auriculata and to six in the odours from F. hainanensis  were much longer than in figs of male trees in both Ficus species . Style length in figs of male trees of F. auriculata (0.97 ± 0.11 mm) was around 0.05 mm longer than that for figs of male trees of F. hainanensis (0.92 ± 0.05 mm) . Ovipositor of C. emarginatus was 1.19 ± 0.09 mm (n = 90) in length, which were very similar to the styles of male figs from the two Ficus hosts .Style lengths were bimodally distributed in both nd trees ; Fig. 6.C. emarginatus females produced significantly fewer offspring in F. auriculata figs than in F. hainanensis figs  and F. hainanensis  among figs and trees, respectively. All components of these same data types were combined into one for subsequent analysis. Further, female C. emarginatus reared from F. hainanensis were significantly smaller than those reared from their typical host F. auriculata , but similar to those Ceratosolen sp. associated with F. hainanensis . their ovipositor was shorter than those of both C. emarginatus and of Ceratosolen sp. (P = 0.034) raised on their respective typical hosts , but only two that were shown to elicit wasp antennal response as semiochemicals. The shared semiochemicals eliciting antennal response represented 19% of receptive male fig odours and 13% of receptive female fig odours in F. auriculata and 38% and 31% for male and female F. hainanensis figs respectively, indicating a large degree of chemical attractant overlap. Wasp attraction to receptive figs may be from long and short distance alongside contact attractants. In F. curtipes it has been shown that one VOC is mainly responsible for long distance attraction while another is more important for fig entry , compared with those of F. auriculata (0.88 ± 0.01 mm), which may cause a concomitant reduction in fitness due to reduced body size featuring shorter ovipositors which should impinge subsequent oviposition attempts  emerging from F. hainanensis  and literature; c compounds with their identity confirmed by comparison of MS and retention time with laboratory standards .a compounds with their identity confirmed by mass spectrometry analysis; b compounds with their identity confirmed by comparison of Kovats retention index with the NIST chemistry Web Book .Click here for additional data file.10.7717/peerj.13897/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.13897/supp-7Supplemental Information 7Click here for additional data file.


Thoracic Endovascular Aortic Repair (TEVAR) is the preferred treatment option for thoracic aortic pathologies and consists of inserting a self-expandable stent-graft into the pathological region to restore the lumen. Computational models play a significant role in procedural planning and must be reliable. For this reason, in this work, high-fidelity Finite Element (FE) simulations are developed to model thoracic stent-grafts. Experimental crimp/release tests are performed to calibrate stent-grafts material parameters. Stent pre-stress is included in the stent-graft model. A new methodology for replicating device insertion and deployment with explicit FE simulations is proposed. To validate this simulation, the stent-graft is experimentally released into a 3D rigid aortic phantom with physiological anatomy and inspected in a computed tomography (CT) scan at different time points during deployment with an ad-hoc set-up. A verification analysis of the adopted modeling features compared to the literature is performed. With the proposed methodology the error with respect to the CT is on average 0.92 ± 0.64%, while it is higher when literature models are adopted (on average 4.77 ± 1.83%). The presented FE tool is versatile and customizable for different commercial devices and applicable to patient-specific analyses. The endograft is composed of a fabric graft component attached to a metallic stent, which gives structural support to both the graft and the treated aorta.35Thoracic Endovascular Aortic Repair (TEVAR) is a minimally invasive technique to treat thoracic aortic pathologies. It has become the most adopted treatment option since the FDA approval of the first endograft in 2005.39 However, if used for clinical applications, numerical models must be reliable, thus the verification and validation (V&V) process is of foremost importance. To this aim, in 2018, the American Society of Mechanical Engineering (ASME) introduced its first V&V standard for specific applications to medical devices for establishing the credibility needed to support the use of a computational model.2 Verification and validation are two different concepts generally used together to demonstrate the credibility of an in-silico model. The verification process is referred to the process of determining if the computational model – together with the code used for its implementation – is sufficiently accurate to reproduce the underlying mathematical model. The validation is the process of determining if the mathematical model is accurate in representing the interested physical scenario.40Computational models are one of the most widely used tools to investigate the TEVAR procedure since they play a significant role either in supporting pre-operative planning and understanding the device performance. Patient-specific simulations can be performed to improve the device development and optimize the procedure.29 or 3D hexahedral elements33 are adopted; the graft is discretized using 2D triangular or quadrangular shell elements30 or membrane elements.33 Regarding the material properties, the graft fabric is modelled as a linear elastic material in most cases or as a linearized orthotropic linear elastic material.12 For the stent component, some studies simplify the superelastic Nitinol behavior with a linear elastic material model with Young’s modulus equal to the austenitic one.29 In addition, a few works introduce the pre-stress field in the unloaded stent component.34In the context of endograft numerical simulations, the recent literature involves several finite element (FE) structural studies regarding stent-graft modeling by adopting different strategies. Regarding the stent mesh, either 1D beam elementsin-silico analysis with real device configurations reconstructed from CT images performed on an endograft released in a mock aorta; (v) sensitivity study on different modelling choices to compare the proposed TEVAR model with those found in the literature.This study aims at developing high-fidelity FE simulations to virtually reproduce the TEVAR procedure. Experimental tests are used to add V&V evidence in the state-of-the-art on the TEVAR modeling. In particular, it includes (i) calibration of stent and graft materials based on experimental data; (ii) validation of the stent-graft model with experimental data; (iii) deployment simulation of the device into an idealized aorta; (iv) validation of the deployment simulation by comparing the For the purpose of the study, three models of the commercially available Valiant™ thoracic aortic stent graft with the Captivia™ delivery system   were created with SolidWorks  from measurements directly taken on the device with a calliper. Sizes 30 × 30 × 150 Closed-Web (device A), 46 × 46 × 150 Free-Flo (device B) and 34 × 34 × 200 Free-Flo (device C) were generated ; finite element simulations were performed on 20 CPUs of an Intel Xeon64 with 120 GB of RAM using the commercial explicit finite element solver LS-DYNA 971 Release 13.0 . The post-processing analysis was done with META Post Processor v22.0 .Different material models with sometimes discordant values are reported in the literature, the reason why a proper characterization and calibration of the material models were needed.8 Crimping and release experimental tests were carried out at 37 °C ± 2 °C (dry air) by using the Blockwise Crimper system  . Thus, to correctly model and calibrate the Nitinol material, it is necessary to refer to the stress-free configuration.22 In particular, the convergent meshes counted 240 and 160 elements for the 8-peaks and 5-peaks respectively, with an average element size of 1 mm. The stent was modelled adopting the superelastic Nitinol formulation. This model describes the superelastic response present in shape-memory alloys following the description proposed by Auricchio and Taylor,6 where the material has the ability to undergo large deformations with a full recovery during loading–unloading cycles. The crimping/release system was reproduced by imposing on 12 rigid planes placed around the stent strut displacement boundary conditions until the 5 mm stent diameter was reached .From the numerical side, after performing a mesh convergence analysis (reported in the Appendix), each stent ring was discretized with 2 nodes (linear) beam elements with Hughes Liu formulation and 2 × 2 gauss nodes in the circular cross-ection.et al..20 In particular, the Nitinol was calibrated starting from the 8-peaks and 5-peaks rings of device A  were carried out on graft samples cut from a Valiant Captivia stent-graft. The analyzed polyethylene terephthalate (PET) revealed a linear elastic behaviour in tension with a resultant Young’s modulus of 1080 MPa in the longitudinal directionThe stent-graft models were built by assembling the rings with the graft. The graft was discretized with 3-nodes membrane elements. 22,320, 35,136 and 23,272 elements were present in the graft part for devices A, B and C, respectively. The average element size was 1 mm: it was chosen to be consistent with the position of the sutures in the real device to create a merged node connection between the graft and stent elements . Penalty contacts between the planes and stent and graft were introduced with a friction value of 0.3 for the stent and 0.1 for the graft. Also, penalty self-contacts were introduced between the stent struts to prevent penetration. After a sensitivity analysis, a damping factor of 1 s25 was created and adopted to reproduce the TEVAR procedure. It was discretized with triangular rigid elements with an element size of 1.2 mm . The aorta had a diameter of 30 mm in the proximal landing zone, thus the Valiant Captivia 34 × 34 × 200 size stent-graft (device C) was chosen, as suggested by the Valiant Captivia instructions for use.38 The stent-graft initial configuration, as well as the stress/strain distribution, were imported from the pre-stress procedure. A new tracking method to reproduce the clinical procedure was proposed and the numerical simulation comprised 3 steps,23 as reported in Figure A CAD aortic model with physiological anatomyStent-graft crimping– the pre-stressed stent-graft was crimped in a catheter and penalty contacts with soft formulation between the stent-graft and crimping catheter were introduced with a friction coefficient of 0.1.(I) Stent-graft tracking – the stent-graft was displaced along the aortic centerline until the proximal landing zone was reached. Penalty contacts with soft formulation were imposed between the stent-graft and the catheter without friction.(II) Stent-graft gradual deployment – the stent-graft was deployed from a proximal toward a distal region by gradually unsheathing the catheter. The main body was firstly gradually released and then the proximal free flow ring, as happens in the real scenario with the tip capture mechanism.38 In this step, soft penalty contacts were introduced between the stent-graft and the aorta with a friction coefficient of 0.1.(III) −1 and 0.5 s−1 were imposed on the stent and the graft, respectively. The timestep was set to 0.001 ms .During the simulation damping factors of 1 s30 In addition, the maximum absolute distance between the simulation and segmentation for each stent strut was calculated to evaluate the stent strut alignment.The stent-graft configuration obtained from the simulation was compared with a real scenario to validate the numerical results. In particular, the same aorta CAD model was 3D-printed using Material Jetting technology  and a rigid transparent photopolymer (Stratasys VeroClear RGD810), featuring the mechanical and physical properties reported in the Appendix. Threaded caps were designed to close the outflow at super-aortic branches. Two threaded ½ tube connectors were designed to connect the aortic root and the abdominal aorta to the tubing system. Both caps and connectors were 3D-printed using VeroClear along with integrated sealings 3D-printed with a deformable photopolymer (Agilus30 Black). The vessel was closed filled with water at 37 °C to reproduce temperature physiological conditions. Device C was experimentally implanted in the model and the deployment was performed under CT scan . Four scans at different timing during the deployment were acquired: first stent ring releasing, half body releasing, main body fully deployment and free flow ring releasing. Using the open-source software VMTK (Orobix s.r.l.), the stent was segmented from each CT acquisition and compared with the simulation results. The experimental set-up is reported in Figure 32 were compared. With the literature approach, the stent-graft was simultaneously crimped and morphed by a catheter until the vessel centerline was reached. Then, the catheter was removed to let the device expand: the stent-graft was deployed at once and the free flow ring was not released as last, but simultaneously with all the stent struts. In particular, simulations with Nitinol modeled as linear elastic material  or with PET as linear elastic material (E = 1.84 MPa33 and E = 1.84 GPa24) were tested as well as the effect of the absence of the stent pre-stress.33 Also, to investigate the stent-graft releasing phase, a condition of modified “virtual catheter” method was studied where the stent-graft was deployed gradually.For the verification process of the stent-graft model, a sensitivity study on the impact of the modeling features was carried out. The list of the performed simulations is reported in Table The results of the different simulations were analysed in terms of simulation correctly running (positive or negative whether the simulation ended correctly or not) and, in the case of ended simulation, in terms of the opening area and deployed stent-graft length with respect to the CT configuration.The stent pre-stress procedure is reported in Figure Figure The OA of each stent strut Figure c was eva2 vs. CT OA of 579 mm2)  Figure a. In sim) Figure a. In simBy qualitatively analysing the deployed stent-graft length Figure b, in all40 This is particularly true for stent-graft simulations where different modelling and numerical strategies are adopted in the literature. In this context, investigating the mechanical behaviour of a commercial stent-graft by assigning the correct material properties to each component is necessary for the reliability of the numerical model itself.When performing numerical simulations, the reliability and truthfulness of numerical models are of primary importance and the verification and validation (V&V) process is crucial. In this sense, the synergy between numerical and experimental studies is fundamental.29 In addition, if modelled as superelastic, the Nitinol parameters are different among the different literature works. On the other hand, PET is described as a linear elastic material, but the Young’s modulus varies from 1.84 MPa33 to 1.84 GPa.24 Furthermore, in the work by Demanget et al.,12 a more elaborated linear elastic orthotropic material was proposed to differently model the Young’s modulus in the warp (225 MPa) and weft (1 GPa) directions. As regards the mechanical behaviour, the radial force is a fundamental indicator of the stent-graft effective fixation to the aortic wall41 and it is mainly investigated through ad-hoc crimping tests. For this reason, reproducing in silico a realistic behaviour obtained with crimping experimental tests42 is relevant for successfully replicating the TEVAR procedure. De Bock et al.10 detailed the mechanical behaviour of commercial abdominal stent-grafts, and the Nitinol and PET material parameters were calibrated on plate compression and crimping tests on the whole device. Similarly, Concannon et al.8 performed crimping/release experimental tests on a single Valiant Captivia stent strut without graft to calibrate the Nitinol parameters.In our study, the material parameters of a commercial stent-raft are deeply investigated, calibrated and validated based on experimental tests and compared with literature values. In fact, from the material modeling perspective, no consensus is present in the literature on the stent-graft material properties. Nitinol is modelled either as superelastic or it is simplified as linear elastic with a Young’s modulus equal to austenite.In our work, ad-hoc crimping experimental tests were performed both on single stent struts and on the stent-graft and were used for both material calibration and device model validation purposes. In addition, tests were carried out at 37 °C to guarantee the working condition of the stent-graft. Our final Nitinol calibrated material parameters of the single stent ring accounted also for the ring deformed shape since they were not directly derived from Nitinol wires. Also, an innovative PET fabric formulation was proposed to simulate the fabric behavior with no resistance to compression. The final PET material parameters include both the polymer woven stiffness and the presence of the sutures. In fact, the suture points were not directly modelled, but they were considered in the node-to-node connections between the stent and graft elements. The material parameters we identified allowed to simulate both the Nitinol and stent-graft model in the working ranges, 25–27 mm for the ring from device A and 41-42 mm for device B, since the obtained curves properly fit the experimental data (errors below 4% in the stent-grafts working ranges).et al.34 considered the stent pre-deformation by doubling the stent Young’s modulus. Derycke et al.13 included the stent pre-stress in the model for a custom-made thoracic stent-graft, but the Nitinol was simplified as linear elastic. Perin et al.30 included the stent pre-stress in a commercial stent-graft model for abdominal aneurysm adopting the superelastic Nitinol formulation. Recently, Hemmler et al.17 detailed a general stent-graft pre-deformation procedure for a Stainless Steel stent for the abdominal aorta and investigated the influence of the pre-stress on the vessel wall. The work presented by Concannon et al.8 studied the mechanical behaviour of a stent-graft including the stent pre-stress. However, they only calibrate Nitinol material parameters on one stent ring and simplified the complete stent-graft model by creating a homogenized model. To the best of the authors’ knowledge, our work is the first one that develops thoracic stent-graft numerical models accounting for the stent pre-stress in the stent-graft assembly phase and validating the numerical results on experimental data.Another key factor that highly affects the global behaviour of a stent-graft, is the stent pre-stress. Among the recent literature, few works dealing with stent-grafts introduced this feature in a FE model. Roy 38 Also, with this new method, the stent-graft follows the vessel curvature during the tracking phase, as happens in the real scenario when the device is inserted into the patient. The use of the FE method to reproduce the TEVAR technique is supported by many literature studies33 which performed validation on the final stent-graft configuration by comparing the simulation results with stent segmented from patient-specific CT images. However, the V&V process on the TEVAR procedure simulations is not always done to assess the credibility of the in silico results.37 To the best of the authors’ knowledge, the finite element TEVAR simulations were never compared at different timing during the deployment with an ad-hoc experimental setup. In our framework, the aorta was 3D printed with a rigid material21 to prevent numerical uncertainties related to the arterial wall mechanical properties. Furthermore, the vessel was closed and filled with water at 37 °C. The comparison between the numerical result and the segmented stent from CT images showed a good agreement both in terms of qualitative overlapping and quantitative area evaluation of the two final positions. The opening area error is small  and almost negligible in the proximal strut where incorrect sealing and malapposition may compromise the outcome of the TEVAR procedure. Moreover, the OA error resulting from the proposed tracking method is found to be much smaller than OA errors found in literature studies which range from 5 to 30%.30 The opening area is a fundamental indicator of the stent-graft anchoring to the aortic wall. If the stent struts are well attached to the vessel, the possibility of developing stent-graft related complications such as bird beak, endoleak or migration is reduced.26Concerning the deployment simulation, a new tracking methodology for replicating the TEVAR procedure was developed and validated with experimental results. The numerical simulation truthfully reproduced the deployment mechanism of a stent-graft also replicating the tip capture mechanism.33 With this approach, the simulation works correctly only if PET is modelled as fabric or, despite is not realistic, as linear elastic with E = 1.84 MPa.33 From our analysis, the “virtual catheter” method with fabric introduces errors in the stent struts OA (underestimated with respect to the CT). Also, the deployed stent-graft was longer with respect to the CT because the gradual deployment was not considered. In fact, with the gradual expansion, the stent-graft shortens a bit its length every time each stent strut is released. This is verified with the modified “virtual catheter” approach in which the deployed stent-graft length was captured because the device was deployed gradually.Since the goal of the study was to develop high fidelity FE stent-graft models, it was necessary to perform a verification analysis on the modeling choices. By exploiting the most common literature adopted options, the OA error with respect to the CT configuration was always higher than the proposed tracking model. Considering the Nitinol as a linear elastic material, a strong limitation is introduced in the stent strut behaviour and the stress and strain distributions developed during the procedure. When PET was simulated as linear elastic, the tracking simulation failed due to extremely high and bad deformations. The results of the simulation without stent pre-stress confirmed that the pre-stress plays a significant role in the stent strut opening area, especially in the free flow ring and proximal stent struts. In the literature, the TEVAR procedure is mainly modelled by following the “virtual catheter” method.The study is not free from limitations like the adoption of an idealized and rigid aortic model or the consideration of only one design of stent-graft. Also, the effect of the presence of the guidewire was neglected. However, the simplifications are reasonable since the focus of the study is on the development of high-fidelity stent-graft models and realistic modeling of the TEVAR procedure, which can be followed for any specific stent-graft design. Indeed, the obtained results can be generalized to any size of a stent-graft and patient-specific deformable anatomies reconstructed from clinical images can be easily adopted for pre-procedural planning.


Congenital abdominal aortic aneurysm (AAA) with coarctation has been considered an extremely rare condition. In this study, we present a 3-year-old boy, who was diagnosed by chance with congenital AAA at first operation. We replaced the AAA+coarctation with a 6-mm polytetrafluoroethylene (PTFE) graft. Histological examination of the aortic wall revealed no particular abnormalities. Collateral vessels were noted to develop over 14 years of followup. Good blood flow to both lower limbs and no intermittent claudication were observed. After growth, at the age 17, he underwent extra-anatomical bypass using a 12-mm PTFE graft. This is the first report of successful treatment of congenital AAA+coarctation with longterm followup. Pediatric abdominal aortic aneurysm (AAA) is uncommon; it is known to be usually caused by infection, aortitis, or iatrogenic trauma. Congenital AAA with coarctation is an extremely rare condition that is thought to carry a high mortality rate. In this study, we present a patient with congenital AAA with coarctation, whom we followed up for 14 years.The male patient was 3 years old during his first operation. He was admitted into our hospital because of a respiratory infection. A physical examination revealed a vascular murmur in his abdomen. Laboratory tests showed no abnormal data, and other general examinations revealed no abnormal findings. He had no history of trauma and no family history of AAA. However, after conducting an enhanced computed tomography (CT) on his abdomen, an irregularly shaped AAA was detected. Angiography of the abdominal aorta was then performed that showed an irregularly shaped AAA of 25 mm in size and atypical coarctation of the proximal segment of the AAA (During the operation, the AAA was exposed through a midline incision. The operative view showed the irregular shape of the AAA (We did not use drugs to prevent graft occlusion because the patient and his parents refused such drugs. The 6-mm PTFE graft was becoming relatively narrow due to the boy’s growth. He had no pain in his feet during exercise because a number of collateral vessels had developed, and both limbs have grown normally. However, when he stopped growing, he began to feel slight pain in his lower limbs during exercise. Replacement of the graft was thought to be difficult because both the proximal and distal sites of anastomosis were still too narrow ( This arterial malformation occurs by developmental arrest of the arterial system in the later embryogenetic stage. The molecular and genetic origins of congenital AAA are almost unknown because of the rarity of congenital AAA. Pediatric AAA is known to be rare; however, its common causes are connective tissue disorders, vasculitis, iatrogenic trauma, and infection. Congenital AAA is idiopathic and has an unknown etiology. One paper suggests that congenital AAA occurs from a developmental defect during embryogenesis that causes a narrow segment of the abdominal aorta, which leads to poststenotic turbulent blood flow and subsequent aneurysm formation. Our patient with congenital AAA with coarctation matches this hypothesis because there was a narrow segment of coarctation. Only 32 cases have been reported recently, including our case. A review showed that males were almost twice as likely to develop congenital AAA than females, half of the patients were neonates and infants, and only 4 of 26 patients were diagnosed at over 3 years of age. Our patient was diagnosed later than normal. One-third of the patients were asymptomatic, and the location of the AAA was infrarenal in 70% of the patients. The mortality rate of congenital AAA rupture and renal failure was as high as around 30%.According to one classification system, congenital AAA is defined as a localized truncal form of arterial defect. The 6-mm PTFE graft used in our case was at the lower allowable selection limit, but an 8-mm graft was too large to anastomose. In the long run, the 6-mm PTFE graft has shown longterm patency. However, a PTFE graft does not grow, and ischemic symptoms in the lower limbs may occur as the patient grows. At the initial surgery, we thought that the second surgery would be the replacement of the graft so we did not choose the extra-anatomical bypass. We have followed our patient regularly in an outpatient clinic for 14 years, and he has shown no signs of ischemia and no delay of growth on his lower limbs. Thus, we could wait until his growth stopped to perform the second surgery. To our knowledge, the previous longest followup of congenital AAA after surgical repair is 48 months in a case report. Our patient ultimately developed decreased lower ankle-brachial indexes in both limbs and felt slight pain in both limbs during exercise. At this time, we decided to perform the second operation to ensure increased blood supply to both lower limbs. Finally, the extra-anatomical bypass was successfully performed. To our knowledge, this is the first report of a second surgical repair for congenital AAA following a long period of followup.A standard approach to the treatment of congenital AAA is yet to be established. Seven of 26 patients underwent conservative treatment because of extensive aneurysms, small vessel diameter, and poor hemodynamic conditions. The mortality of those receiving conservative treatment was noted to be very high: four deaths were reported. Seventeen patients underwent surgical repair, with artificial grafts most frequently chosen for replacement. A Dacron graft was used in six cases, a PTFE graft in three cases, allografts in two cases, and aneurysmorrhaphy in two cases. One case was repaired with native iliac vessels. We had no experience of using a native vessel as a graft, and we were familiar with using a PTFE graft. Thus, we chose a PTFE graft to replace the abdominal aortic aneurysm. The worrisome complications of revascularization were graft stenosis and occlusion. A high risk of occlusion in artificial grafts with a diameter of less than 6 mm is reported.Congenital AAA is very rare, and there remains no universal treatment strategy for this condition. Longterm followup after surgery for this condition has not been previously reported. Our operative strategy of performing primary graft replacement and then performing an extra-anatomical bypass 14 years after the first operation may be one of the best treatments for congenital AAA.


Carbohydrates are one of the three macronutrients that provides energy in diets and are classified by their structures. Starch is a nonstructural carbohydrate and polysaccharide made of glucose monomers used for storage in plant cells. When starch makes up greater than 30% of the DM in diets there can be adverse effects on NDF digestibility due to decreases in ruminal pH. Sugars are water soluble carbohydrates that consist of monosaccharide and disaccharide units. Sugars ferment faster than starch because microorganisms in the rumen can ferment carbohydrates at different rates depending on their structure; however, this has not been shown to have negative effects on the ruminal pH. Sources of sugars such as molasses (sucrose) or whey (lactose) can be included in the diet as a partial replacement for starch in dairy cow diets. The purpose of replacing starch with sugars in a diet would be to add differing sources of carbohydrates in the diet to allow for continual fermentation of carbohydrates by the microorganisms in the rumen. It has been seen in studies and previous literature that the partial replacement of starch with sugars has the potential to maintain the ruminal environment and milk yield and composition in dairy cows without reducing NDF digestibility. The objective of this review is to evaluate the effects of partially replacing starch with sugars in dairy diets and its implication on ruminal fermentation, nutrient utilization, milk production, and feeding replacement strategy. Carbohydrates are one of the three macronutrients providing energy for continued bodily function and can constitute up to 70% of the lactating dairy cow diet . The impStarch is made of glucose monomers that can be fermented in the rumen by the microbial population. Starch fermentation in the rumen can provide precursors in the form of volatile fatty acids (VFA) for lactose, protein, and fat synthesis in the mammary gland . FurtherThe decline in NDF digestibility due to over feeding starch can be mitigated by the partial replacement of starch with sugars in the diet . Sugars Google Scholar and PubMed searches were implemented to find articles relevant for describing the structure of different carbohydrates and their inclusion and use in dairy cow diets. More specifically journal articles that implemented the partial replacement of starch in the diet for sugars including sucrose, lactose, molasses, and whey were searched to summarize the findings in these experiments.Carbohydrates consist of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Monosaccharides include xylose, fructose, galactose, and glucose. Disaccharides are combinations of two monosaccharides. Disaccharides include sucrose (fructose and glucose) and lactose . Oligosaccharides are chains of 2 to 20 monomeric units of sugars , such asStarch is made of glucose chains, and it is used for energy storage in plants . It is cWhen feeding cows, grains are usually processed to aid in their digestibility. Processing will make starch more available to the microorganisms and thus more available to the animal . DecreasHealth issues may arise with excess inclusion of starch in the diet from associated effects of decreased NDF, which is the fiber component of the cell and includes hemicellulose and cellulose components that are structural carbohydrates. More concentrate and less fiber in the diet yields an increase in VFA concentration which when paired with no change in the clearance of VFA from the rumen can decrease the ruminal pH . Milk faSugars are soluble carbohydrates as they are soluble in the cell’s aqueous environment . They arWater-soluble carbohydrates (WSC) provide a source of readily available energy to microorganisms in the rumen. Unlike structural carbohydrates which need more time to be fermented by bacteria, WSC are fermented at a faster rate . Starch 3–N). The authors also observed that increasing WSC concentration by 50%, increased microbial N concentration (158 vs. 144.6 mg/d) and the efficiency of microbial protein synthesis (12.7 vs. 9.9 g of N/kg of OM apparently digested) compared with the basal grass diet. The changes observed indicate a potential for improvement in microbial protein growth and efficiency with increasing WSC concentration.In an in vitro study by Supplemental dietary sugar has been shown to increase butyrate concentrations in the rumen . ButyratThe interest in incorporating sugars into dairy cow diets as a replacement for starch stems from the benefit in meeting energy requirements while maintaining ruminal fermentation and production. Since corn grain is typically the main source of starch, when replacing starch with sugars, corn, or corn starch will typically be the feedstuff replaced. Replacing corn can help improve farm profitability and increase supply for human consumption. Incorporating sugars in the diet as a replacement for starch has the potential to increase DMI, maintain ruminal pH, and improve microbial protein synthesis, and milk components . Sugars 3–N. Hence, sucrose can be included in the diet, to a certain extent, without compromising bacterial fermentation in the rumen.Sucrose is a fructose and glucose bonded by an α-1,4 glycosidic bond . Sucrose3–N concentration from 13.9 mg/dL in the diet with no sugar inclusion to 11.5 mg/dL in the diet with 7.5% of sugar inclusion. This could be indicative of more N being utilized for microbial protein synthesis. There was also a decrease in urinary excretion of urea N and total N. Overall, replacing starch with sugars or WSC could potentially lead to improvements in production and utilization of nitrogen.Diets partially replacing starch for sucrose using the same percentages as the ones tested by In another study by Similar to sucrose, lactose can be included in dairy cow diets. Lactose is a galactose and glucose connected by a β 1,4-glycosidic bond . Lactose3–N were observed, but there was an increase in butyrate proportion and a decrease in the proportion of acetate and branched chained fatty acids in the sugar diets. With the increase of ruminal butyrate, there was also an increase in the plasma concentration of β-hydroxy butyric acid (BHBA); however, this increase was not enough to place cows at risk for developing ketosis. Additionally, the DMI of cows tended to increase linearly, intake was increased by 1.6 kg/d when comparing the control to the high lactose diet. However, it was noted that the greatest effect when feeding lactose is its apparent ability to change ruminal fermentation not necessarily through an increase on DMI; hence, there have been other reports that do not note an increase in DMI  and whey (lactose) have the potential to replace starch in dairy cow diets without adversely affecting the ruminal environment or milk yield and composition. It has been observed that the addition of sugars in the diet to a total of 5% of the DM could have positive effects in ruminal fermentation and milk production. Future steps for feeding sugars in the diet will further consider diet basal levels of sugar when incorporating additional sugar from byproducts in the diet.