Effect of black wolfberry anthocyanin and maltitol on the gelation and microstructural properties of curdlan/gellan gum hybrid gels.

BACKGROUND: Laboratory scale experiments have shown that curdlan and gellan gum gelled together as curdlan/gellan gum (CG) hybrid gels showed better gel properties than the individual curdlan and gellan gum. In this study, CG and black wolfberry anthocyanin (BWA), CG and maltitol (ML) hybrid gels were constructed using CG hybrid gel as matrix. The effects of BWA or ML on the gel properties and microstructure of CG hybrid gels were investigated and a confectionery gel was developed. RESULTS: The presence of BWA increased the storage modulus (G') value of CG at 0.1 Hz, whereas ML had little effect on the G' value of CG. The addition of BWA (5 g L-1 ) and ML (0.3 mol L-1 ) increased the melting and gelling temperatures of CG hybrid gels to 42.4 °C and 34.1 °C and 44.2 °C and 33.2 °C, respectively. Meanwhile, the relaxation time T22 in CG-ML and CG-BWA hybrid gels was reduced to 91.96 and 410.27 ms, indicating the strong binding between BWA and CG, ML and CG. The hydrogen bond interaction between BWA or ML and CG was confirmed by the shift in the hydroxyl stretching vibration peak. Moreover, the microstructures of CG-ML and CG-BWA hybrid gels were denser than that of CG. In addition, confectionery gel containing CG-BWA-ML has good chewing properties. CONCLUSION: These results indicated that the incorporation of BWA or ML could improve the structure of CG hybrid gels and assign a sustainability potential for the development of confectionery gels based on CG complex. © 2024 Society of Chemical Industry.

PE6c greatly enhances prime editing in transgenic rice plants.

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a-d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

Optimized prime editing efficiently generates heritable mutations in maize.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime-editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Direct Liquid Phase Exfoliation of Graphite to Produce Few-Layer Graphene by Microfluidization.

Graphene has attracted a great number of attentions due to the excellent physical and chemical properties. For the convenience of investigations and applications, it is crucial to produce the grapheme with high-quality and high-yield by an easy-obtained method. In this research, a promising method is demonstrated to produce a high-concentration few-layer graphene (FLG) dispersion by direct microfluidization in water/surfactant systems. The effects of surfactant selection, chamber pressure and microfluidization cycles on the graphitic material exfoliation efficiency are systematically studied. The FLG concentration and the quality of the as-prepared FLG were determined by a series of characterizations. The graphene dispersions, with an average lateral size of 0.6 µm and a few-layer structure, were stabilized by surfactants at a high concentration of up to 1.7 mg/mL and exhibited a relatively high quality (ID/IG = 0.07-0.56, C/O ~ 19.36) within a processing time of a few hours. This method should facilitate the mass production of high-quality graphene by liquid-phase exfoliation and promote the industrial application of graphene.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

KEY MESSAGE: We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations.

Cas12a (Cpf1), a Class 2 Type V CRISPR/Cas nuclease, has several unique attributes for genome editing and may provide a valuable alternative to Cas9. However, a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application. In this report, we generated two variants, ttAsCas12 Ultra and ttLbCas12a Ultra harboring three (E174R, M537R, and F870L) or two (D156R and E795L) mutations, respectively, by combining the mutations from the temperature-tolerant variants ttAsCas12a (E174R) and ttLbCas12a (D156R), and those from the highly active variants AsCas12a Ultra (M537R and F870L) and LbCas12a Ultra (E795L). We compared editing efficiencies of the five resulting Cas12a variants (LbCas12a, ttLbCas12a, ttLbCas12a Ultra, AsCas12a Ultra, and ttAsCas12 Ultra) at six target sites of four genes in Arabidopsis (Arabidopsis thaliana). The variant ttLbCas12a Ultra, harboring the D156R and E795L mutations, exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 °C. In addition, optimization of ttLbCas12a Ultra, by varying nuclear localization signal sequences and codon usage, further greatly improved editing efficiency. Collectively, our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00144-w.

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

Cucumber mosaic virus movement protein severs actin filaments to increase the plasmodesmal size exclusion limit in tobacco.

Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)-stabilizing agent phalloidin but not by treatment with the F-actin-destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins.

Formation and characteristics of curcumin-loaded binary gels formed from large yellow croaker (Pseudosciaena crocea) roe protein isolate and gellan gum.

The aim of this study was to investigate the effect of gellan gum (GG) on the cold gelation of large yellow croaker roe protein isolate (pcRPI). The water-holding ability and storage modulus of the pcRPI-GG binary gels increased with the GG concentration, where the storage modulus of the pcRPI-0.2% GG gel was approximately 30.7 times that of the pure pcRPI gel. Compare to the other binary gels, pcRPI-0.2% GG gels exhibited a lower lacunarity and higher junction density, with a denser, more aggregated microstructure. Consequently, curcumin was embedded in pcRPI-0.2% GG gels, and simulated gastrointestinal digestion test results showed that GG addition effectively protected and slowed curcumin release in the gastrointestinal environment. These findings may contribute to elucidating the interaction of pcRPI with GG and demonstrate the potential of binary gels for the embedding and delivery of active substances.

MISSA is a highly efficient in vivo DNA assembly method for plant multiple-gene transformation.

We describe a highly efficient in vivo DNA assembly method, multiple-round in vivo site-specific assembly (MISSA), which facilitates plant multiple-gene transformation. MISSA is based on conjugational transfer, which is driven by donor strains, and two in vivo site-specific recombination events, which are mediated by inducible Cre recombinase and phage lambda site-specific recombination proteins in recipient strains, to enable in vivo transfer and in vivo assembly of multiple transgenic DNA. The assembly reactions can be performed circularly and iteratively through alternate use of the two specially designed donor vectors. As proof-of-principle experiments, we constructed a few plant multigene binary vectors. One of these vectors was generated by 15 rounds of MISSA reactions and was confirmed in transgenic Arabidopsis (Arabidopsis thaliana). As MISSA simplifies the tedious and time-consuming in vitro manipulations to a simple mixing of bacterial strains, it will greatly save time, effort, and expense associated with the assembly of multiple transgenic or synthetic DNA. The principle that underlies MISSA is applicable to engineering polygenic traits, biosynthetic pathways, or protein complexes in all organisms, such as Escherichia coli, yeast, plants, and animals. MISSA also has potential applications in synthetic biology, whether for basic theory or for applied biotechnology, aiming at the assembly of genetic pathways for the production of biofuels, pharmaceuticals, and industrial compounds from natural or synthetic DNA.

Overexpression of AtDOF4.7, an Arabidopsis DOF family transcription factor, induces floral organ abscission deficiency in Arabidopsis.

After flower pollination, a programmed process called abscission occurs in which unwanted floral organs are actively shed from the main plant body. We found that a member of the DOF (for DNA binding with one finger) transcription factor family, Arabidopsis (Arabidopsis thaliana) DOF4.7, was expressed robustly in the abscission zone. The Arabidopsis 35S::AtDOF4.7 lines with constitutive expression of AtDOF4.7 exhibited an ethylene-independent floral organ abscission deficiency. In these lines, anatomical analyses showed that the formation of the abscission zone was normal. However, dissolution of the middle lamella failed to separate between the cell walls. AtDOF4.7 was identified as a nucleus-localized transcription factor. This protein had both in vitro and in vivo binding activity to typical DOF cis-elements in the promoter of an abscission-related polygalacturonase (PG) gene, PGAZAT. Overexpression of AtDOF4.7 resulted in down-regulation of PGAZAT. AtDOF4.7 interacted with another abscission-related transcription factor, Arabidopsis ZINC FINGER PROTEIN2. Taken together, our results suggest that AtDOF4.7 participates in the control of abscission as part of the transcription complex that directly regulates the expression of cell wall hydrolysis enzymes.

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

Overexpression of the Arabidopsis α-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus.

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.

[Characteristics and Source Apportionment of VOCs and O3 in Shijiazhuang].

To study the composition characteristics and sources of volatile organic compounds (VOCs) in Shijiazhuang City, three national control points were selected to conduct VOCs sampling and analysis from March 2017 to January 2018. The correlation of VOCs through combination with meteorological and ground-level O3 data, and the sources of VOCs were analyzed by positive matrix factorization (PMF). To quantify the pollution period of O3 in summer, its temporal sequence characteristics were studied by wavelet analysis. During the sampling period, the average concentration of ambient total VOCs (TVOCs) was (137.23±64.62) µg·m-3. Haloalkanes were the most dominant VOC compounds, accounting for 31.77% of total VOCs mass, followed by aromatic (30.97%) and oxygenated VOCs (OVOCs, 23.76%). The seasonal variation in VOC concentration followed the trend in winter (187.7 µg·m-3) > autumn (146.8 µg·m-3) > spring (133.24 µg·m-3) > summer (107.1 µg·m-3); the concentration of VOCs shows a trend of increasing gradient from west to east. The O3 concentration correlated negatively with VOCs and NO2, and positively with temperature, sunshine duration, wind speed, and visibility. Changes in meteorological elements were concerned before the occurrence of ozone pollution in summer, especially in 4-5 days in June and 7-8 days during July to August after the occurrence of increasing temperature. Finally six potential sources of VOCs were quantified by the PMF model, including from gasoline emissions (24.78%), diesel vehicle emissions (24.69%), solvent usage (18.64%), the chemical industry (11.87%), regional background (10.84%), and the pharmaceutical industry (9.17%). Ozone formation potential (OFP) contribution of emission sources of gasoline and diesel vehicles (54.98%) was over half of the total contribution. Meanwhile, these findings illustrated that control of vehicle emissions and industrial sources would be an important way to reduce VOCs concentrations and improve air quality in Shijiazhuang.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement.

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.

Ethylene response factor TERF1 enhances glucose sensitivity in tobacco through activating the expression of sugar-related genes.

Ethylene response factor (ERF) proteins are important plant-specific transcription factors. Increasing evidence shows that ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interaction with different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activates a cluster gene expression through interacting with GCC box and dehydration responsive element (DRE), resulting in enhanced sensitivity to abscisic acid (ABA) and tolerance to drought, and dark green leaves of mature plants, indicating that TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpression of TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides information indicating that there are many cis-acting elements, including sugar responsive elements (SURE) and W box, suggesting that TERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reaction amplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Further investigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination, root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment. Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE and activates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response. Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.

Peroneus Longus Tendon Autograft is a Safe and Effective Alternative for Anterior Cruciate Ligament Reconstruction.

The ipsilateral peroneus longus tendon (PLT) was utilized as an autograft for anterior cruciate ligament (ACL) reconstruction of patients with acute ACL rupture and grade III medial collateral ligament (MCL) injury. We investigated the efficacy and safety of this alternative autograft compared with autologous hamstring tendon (HT). Biomechanical testing of the graft options was performed and compared with the native ACL. Thirty-eight patients with acute ACL ruptures and grade III MCL injuries were treated with ACL reconstruction with a doubled autologous PLT or quadrupled autologous HT. Knee stability and function was evaluated clinically with the Lachman test and KT-2000 arthometer as well as subjectively with functional scores. Effects on the donor ankle were evaluated by biomechanical testing. The ultimate tensile strengths of doubled PLT and quadrupled HT were significantly higher than that of the native ACL and the ultimate tensile strength of doubled PLT was comparable with that of quadrupled HT. There were no significant differences in clinical or functional scores between the two groups. There were no significant differences in pre- and postoperative biomechanical testing of the donor ankle. PLT is a suitable alternative autograft for an ACL reconstruction in patients with a concomitant grade III MCL injury without a significant biomechanical disadvantage to the ankle donor site.