Directly measured strain patterns of the anterior talofibular and calcaneofibular ligaments after isolated ATFL repair in a combined ATFL and CFL injury: A cadaver study.

BACKGROUND: Even though 20% of chronic lateral ankle instability results from a combined anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL) injury, only the ATFL is sutured using arthroscopic ligament repair techniques. Although some biomechanical and clinical studies have proved that isolated ATFL repair yields excellent results, previous biomechanical studies were performed using systems that only allow indirect estimations. The purpose of this study was to clarify strain patterns by directly measuring repaired ATFL and CFL strain patterns on cadaveric models that underwent isolated ATFL repair of a combined ATFL and CFL injury. METHODS: The miniaturization ligament performance probe (MLPP) system was used for directly measuring the strain patterns to insert the strain gauges into the mid-substance of normal and repaired ATFL and CFL fibers in five cadaveric specimens to allow measurement of strain patterns in the axial and three-dimensional motion of the ankle. RESULTS: The normal and repaired ATFL showed similar strain patterns in axial and three-dimensional motions. During the axial range of motion of the ankle, the repaired CFL showed a strain pattern almost similar to that of normal CFL, but the strain increased as the plantar flexion or dorsiflexion angle increased to the maximum value of 100 at 30° plantarflexion or strain values of 17-55/100 at 15°dorsiflexion. During three-dimensional motion, the repaired CFL was under the maximum value of 100 during dorsiflexion-inversion and exhibited less strain (7-38/100) during plantar flexion-eversion. CONCLUSION: The repaired CFL did not show a strain pattern that was completely consistent with a normal strain pattern; however, it did have some degree of tension similar to a normal strain pattern, even though it was not directly repaired.

Genotype-phenotype correlation of small-intestinal polyps on small-bowel capsule endoscopy in familial adenomatous polyposis.

BACKGROUND AND AIMS: In familial adenomatous polyposis (FAP), neoplastic lesions outside the colon have become increasingly important. The genotype-phenotype correlation has been established for duodenal polyps, and regular screening is recommended. However, this correlation remains unclear for small-intestinal lesions, except for reports on the relationship between their occurrence and Spigelman stage. Here, we used small-bowel capsule endoscopy (SBCE) to investigate the genotype-phenotype correlation of small-intestinal polyps in FAP. METHODS: The genotype-phenotype correlation of small-intestinal polyps was investigated in patients with FAP who underwent SBCE, Esophagogastroduodenoscopy (EGD), and adenomatous polyposis coli (APC) gene analysis. Of 64 patients with FAP who underwent SBCE, 41 were included in the final analysis, 4 did not undergo a complete small intestine examination, and 19 did not undergo genetic analysis. RESULTS: The prevalence (median number) of small-intestinal polyps by Spigelman stage was 26% (1.5), 0% (0), 44% (5), 60% (4), and 73% (25.5) for stages 0 to IV, respectively. Significantly more small-intestinal polyps were found in Spigelman stage III and IV groups than in the stage 0 group (P < .05). The APC variant was negative for 6 patients (15%), and the sites associated with more than 5 small-intestinal polyps were codons 278, 1062, 1114, 1281, 1307, 1314, and 1504. CONCLUSIONS: In FAP patients, SBCE surveillance is potentially recommended for patients with pathogenic variants in the APC gene at codons 278 and 1062 to 1504 or with Spigelman stage III or higher.

Strain patterns in normal anterior talofibular and calcaneofibular ligaments and after anatomical reconstruction using gracilis tendon grafts: A cadaver study.

BACKGROUND: Inversion ankle sprains, or lateral ankle sprains, often result in symptomatic lateral ankle instability, and some patients need lateral ankle ligament reconstruction to reduce pain, improve function, and prevent subsequent injuries. Although anatomically reconstructed ligaments should behave in a biomechanically normal manner, previous studies have not measured the strain patterns of the anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL) after anatomical reconstruction. This study aimed to measure the strain patterns of normal and reconstructed ATFL and CFLs using the miniaturization ligament performance probe (MLPP) system. METHODS: The MLPP was sutured into the ligamentous bands of the ATFLs and CTLs of three freshly frozen cadaveric lower-extremity specimens. Each ankle was manually moved from 15° dorsiflexion to 30° plantar flexion, and a 1.2-N m force was applied to the ankle and subtalar joint complex. RESULTS: The normal and reconstructed ATFLs exhibited maximal strain (100) during supination in three-dimensional motion. Although the normal ATFLs were not strained during pronation, the reconstructed ATFLs demonstrated relative strain values of 16-36. During the axial motion, the normal ATFLs started to gradually tense at 0° plantar flexion, with the strain increasing as the plantar flexion angle increased, to a maximal value (100) at 30° plantar flexion; the reconstructed ATFLs showed similar strain patterns. Further, the normal CFLs exhibited maximal strain (100) during plantar flexion-abduction and relative strain values of 30-52 during dorsiflexion in three-dimensional motion. The reconstructed CFLs exhibited the most strain during dorsiflexion-adduction and demonstrated relative strain values of 29-62 during plantar flexion-abduction. During the axial motion, the normal CFLs started to gradually tense at 20° plantar flexion and 5° dorsiflexion. CONCLUSION: Our results showed that the strain patterns of reconstructed ATFLs and CFLs are not similar to those of normal ATFLs and CFLs.

Three-dimensional analysis of anterior talofibular ligament strain patterns during cadaveric ankle motion using a miniaturized ligament performance probe.

BACKGROUND: Measuring the strain patterns of ligaments at various joint positions informs our understanding of their function. However, few studies have examined the biomechanical properties of ankle ligaments; further, the tensile properties of each ligament, during motion, have not been described. This limitation exists because current biomechanical sensors are too big to insert within the ankle. The present study aimed to validate a novel miniaturized ligament performance probe (MLPP) system for measuring the strain patterns of the anterior talofibular ligament (ATFL) during ankle motion. METHODS: Six fresh-frozen, through-the-knee, lower extremity, cadaveric specimens were used to conduct this study. An MLPP system, comprising a commercially available strain gauge (force probe), amplifier unit, display unit, and logger, was sutured into the midsubstance of the ATFL fibers. To measure tensile forces, a round, metal disk (a "clock", 150 mm in diameter) was affixed to the plantar aspect of each foot. With a 1.2-Nm load applied to the ankle and subtalar joint complex, the ankle was manually moved from 15° dorsiflexion to 30° plantar flexion. The clock was rotated in 30° increments to measure the ATFL strain detected at each endpoint by the miniature force probe. Individual strain data were aligned with the neutral (0) position value; the maximum value was 100. RESULTS: Throughout the motion required to shift from 15° dorsiflexion to 30° plantar flexion, the ATFL tensed near 20° (plantar flexion), and the strain increased as the plantar flexion angle increased. The ATFL was maximally tensioned at the 2 and 3 o'clock (inversion) positions (96.0 ± 5.8 and 96.3 ± 5.7) and declined sharply towards the 7 o'clock position (12.4 ± 16.8). Within the elastic range of the ATFL (the range within which it can return to its original shape and length), the tensile force was proportional to the strain, in all specimens. CONCLUSION: The MLPP system is capable of measuring ATFL strain patterns; thus, this system may be used to effectively determine the relationship between limb position and ATFL ankle ligament strain patterns.

3D-CT stress test for the assessment of CFL insufficiency.

BACKGROUND: Following ankle sprains, some patients complain of their ankles "giving way," characterized by functional instability with no positive findings in traditional stress tests. The calcaneofibular ligament (CFL) may contribute to the stabilization of the subtalar and talocrural joints, and some functional instability may be due to CFL insufficiency. We aimed to clarify and quantitatively assess CFL insufficiency with three-dimensional stress computer tomography (CT) using the Pronation-External Rotation Stress Test (PERST). METHODS: Ten patients who tested positive under PERST and underwent an isolated CFL reconstruction were included. Using a custom-made loading jig, we used the Supination-Internal Rotation Stress Test (SIRST) and PERST to assess the function of anterior talofibular ligament (ATFL) and CFL, respectively. 3D-CT in neutral position was used as a baseline, and we quantified the distance between the origin and insertion of the CFL and ATFL at 2 years pre- and postoperatively. RESULTS: Postoperative scores improved in all patients with no giving way symptoms. The preoperative length of the CFL increased by 14.0% from baseline under PERST, while the postoperative length only increased by 2.0% and was significantly restricted (P < .01). The pre- and postoperative length of ATFL was increased by 7.5% and 9.0% from baseline under SIRST, respectively, with no significant difference (P = .41). The clinical function improved with significantly less change in distance between the origin and insertion under PERST and showed no difference under SIRST. CONCLUSION: The 3D-CT stress test may be useful for quantifying pre- and postoperative CFL function. CFL insufficiency is one of the main causes of subtalar joint instability; therefore, measuring the distance between the origin and insertion of the CFL could provide the means to quantify the instability of the subtalar joint.

Strain pattern of each ligamentous band of the superficial deltoid ligament: a cadaver study.

BACKGROUND: There are few reports on the detailed biomechanics of the deltoid ligament, and no studies have measured the biomechanics of each ligamentous band because of the difficulty in inserting sensors into the narrow ligaments. This study aimed to measure the strain pattern of the deltoid ligament bands directly using a Miniaturization Ligament Performance Probe (MLPP) system. METHODS: The MLPP was sutured into the ligamentous bands of the deltoid ligament in 6 fresh-frozen lower extremity cadaveric specimens. The strain was measured using a round metal disk (clock) fixed on the plantar aspect of the foot. The ankle was manually moved from 15° dorsiflexion to 30° plantar flexion, and a 1.2-N-m force was applied to the ankle and subtalar joint complex. Then the clock was rotated every 30° to measure the strain of each ligamentous band at each endpoint. RESULTS: The tibionavicular ligament (TNL) began to tense at 10° plantar flexion, and the tension becomes stronger as the angle increased; the TNL worked most effectively in plantar flex-abduction. The tibiospring ligament (TSL) began to tense gradually at 15° plantar flexion, and the tension became stronger as the angle increased. The TSL worked most effectively in abduction. The tibiocalcaneal ligament (TCL) began to tense gradually at 0° dorsiflexion, and the tension became stronger as the angle increased. The TCL worked most effectively in pronation (dorsiflexion-abduction). The superficial posterior tibiotalar ligament (SPTTL) began to tense gradually at 0° dorsiflexion, and the tension became stronger as the angle increased, with the SPTTL working most effectively in dorsiflexion. CONCLUSION: Our results show the biomechanical function of the superficial deltoid ligament and may contribute to determining which ligament is damaged during assessment in the clinical setting.

Visibility of early gastric cancer in texture and color enhancement imaging.

Objective: There are little data regarding the efficacy of texture and color enhancement imaging (TXI) for early gastric cancer (EGC) diagnosis. This study aimed to compare the color difference and visibility of EGC between white light imaging (WLI) and TXI. Methods: This study included 20 EGCs of 18 patients undergoing endoscopic submucosal dissection. Still images of EGC in WLI, TXI mode 1 (with color enhancement), and TXI mode 2 (without color enhancement), which were consistent in distance, angle, and air insufflation, were constructed by computer simulation. The center of the lesion, eight equal peripheral points 5 mm outside the lesion, and eight inner points two-thirds of the distance from peripheral points to the EGC lesion center were annotated. Mean color differences (ΔE) of the area between peripheral and inner points per lesion in WLI, TXI mode 1, and TXI mode 2 were analyzed. In addition, four endoscopists independently scored the visibility of EGC images of TXI mode 1 and 2 compared with WLI. Results: Clinicopathological characteristics were as follows: 0-IIa/0-IIb/0-IIc/0-IIa+IIc = 6/1/11/2, reddish/pale = 10/10, differentiated/undifferentiated = 18/2, median tumor size = 13.5 mm. Mean ΔE ± SD = WLI/TXI mode1/TXI mode2 = 10.3 ± 4.7, 15.5 ± 7.8, and 12.7 ± 6.1, respectively. Mean ΔE was significantly higher in TXI mode 1 than in WLI. Visibility (improved/no change/decreased) was 7/13/0 and 4/16/0 in TXI mode 1 and 2, respectively. The visibility was significantly more commonly improved in the macroscopic type 0-IIc or 0-IIb than in 0-IIa or IIa+IIc in TXI mode 1. Conclusions: TXI could improve the visibility of EGC compared with WLI.

Suture anchor stabilization of symptomatic accessory navicular in adolescents: Clinical and radiographic outcomes.

BACKGROUND: Screw fixation used in modified Kidner procedures to treat persistent symptomatic accessory navicular in adult cases is often challenging in adolescent cases with a small accessory fragment. The present study aimed to document the clinical effect of a suture anchor stabilization technique applicable to such cases where osteosynthesis is considered an ideal outcome. METHODS: Consecutive clinical cases who received this surgical treatment from 2009 to 2016 were retrospectively reviewed. The focus of interest included radiographic union of the accessory bone, changes in symptoms evaluated using a validated clinical outcome scale introduced by the Japanese Society for Surgery of the Foot, and changes in the medial arch bony alignment measured in lateral weight-bearing plain radiographs. RESULTS: Twenty-two feet in 15 individuals (11 females and 4 males, age at surgery 10-16 years) were identified. In 14 feet (64%), radiographic bone union was confirmed within 8 weeks postoperatively. At the final follow-up ranging 12-51 months postoperation, the clinical scores have significantly improved (p < 0.001) to 96 ± 5.71 (mean ± standard deviation, range 87-100), from 54 preoperatively. Radiographic measurements revealed significant postoperative increase of the sagittal talar tilt angle (p < 0.001, increment 4 ± 3°, range 0-11) and the talo-first metatarsal angle (p < 0.001, increment 5 ± 4°, range 0-12). No significant changes were identified in the calcaneal pitch angle, first metatarsal tilt angle, calcaneo-navicular angle, and the navicular height. CONCLUSION: Despite the modest bone union rate, the clinical outcomes suggest distinct symptom-relieving effect, at least in the short- to midterm, while the radiographic measurements suggest positive biomechanical effects. The present suture-anchor stabilization concept appears to be a promising treatment option for persistent symptomatic accessory navicular in adolescent cases.

Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3'-sulfoquinovosyl-1'-monoacylglycerol, targeting angiogenesis.

We previously reported that 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) was effective in suppressing the growth of solid tumors due to hemorrhagic necrosis in vivo. In the present study, we investigated the antiangiogenic effect of SQMG. In vivo assessment of antitumor assays showed that some tumor cell lines, but not others, were sensitive to SQMG. Microscopic study suggested that in SQMG-sensitive tumors, but not SQMG-resistant tumors, angiogenesis was reduced. We next investigated gene expression relating to angiogenesis in tumor tissues by quantitative real-time polymerase chain reaction. Consequently, although vascular endothelial growth factor gene expression was not detected with significant differences among the cases, significant downregulation of Tie2 gene expression was observed in all SQMG-sensitive tumors as compared with controls, but not in SQMG-resistant tumors. These data suggested that the antitumor effects of SQMG could be attributed to antiangiogenic effects, possibly via the downregulation of Tie2 gene expression in SQMG-sensitive tumors.

Alpha-sulfoquinovosylmonoacylglycerol is a novel potent radiosensitizer targeting tumor angiogenesis.

Angiogenesis is a promising target for the treatment of cancer, and varying types of antiangiogenic agents have been developed. However, limitations and problems associated with antiangiogenic therapy have recently arisen. Although radiotherapy can be combined with antiangiogenic compounds to overcome these difficulties, almost all previously described angiogenesis inhibitors could still cause side effects at effective doses, and only additive effects are seen in current combination therapy. In this study, we identified a member of the sulfoquinovosylacylglycerols, alpha-sulfoquinovosylmonoacylglycerol (alpha-SQMG), originally derived from sea urchins, as a potent radiosensitizer. The agent synergistically inhibits angiogenesis at low doses when combined with ionizing radiation. Combined treatment with alpha-SQMG and radiation seems to promote the adoption of a senescence-like phenotype by vascular endothelial cells. Finally, the agent remarkably enhances the radioresponse of human tumors transplanted into nude mice, accompanied by a significant reduction in the vascularity of the tumors. Collectively, alpha-SQMG may be a novel potent radiosensitizer targeting angiogenesis.

Identification of antibiotic clarithromycin binding peptide displayed by T7 phage particles.

Peptide libraries displayed by T7 phage, which contain random cDNA fragments insets, were screened for their ability to bind to a biotinylated derivative of clarithromycin. Phage particles bound to an immobilized derivative of the antibiotic were isolated and the inserted cDNA was amplified and sequenced. A common selected peptide sequence, composed of 19 amino acids, was obtained and a synthetic peptide with this sequence was produced. Surface plasmon resonance experiments showed that the synthetic peptide immobilized on a sensor chip bound to clarithromycin and the dissociation constant was determined to be 2.1 x 10(-3) M. The dissociation constants of other macrolide antibiotics, erythromycin, roxithromycin, azithromycin and josamycin were also determined to be 5.4 x 10(-3) M, 6.2 x 10(-5) M, 1.1 M and 3.4 x 10(-2) M, respectively. These results indicated that T7 phage display method might be useful to determine relatively weak interactions between small molecule drugs and the selected peptides which could represent a possible binding site conserved in binding proteins.

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid.

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.

Mammalian mitotic centromere-associated kinesin (MCAK): a new molecular target of sulfoquinovosylacylglycerols novel antitumor and immunosuppressive agents.

Sulfoquinovosylacylglycerols (SQAGs), in particular compounds with C18 fatty acid(s) on the glycerol moiety, may be clinically promising antitumor and/or immunosuppressive agents. They were found originally as inhibitors of mammalian DNA polymerases. However, SQAGs can arrest cultured mammalian cells not only at S phase but also at M phase, suggesting they have several molecular targets. A screen for candidate target molecules using a T7 phage display method identified an amino acid sequence. An homology search showed this to be a mammalian mitotic centromere-associated kinesin (MCAK), rather than a DNA polymerase. Analyses showed SQAGs bound to recombinant MCAK with a K(D)=3.1x10(-4) to 6.2x10-5 M. An in vivo microtubule depolymerization assay, using EGFP-full length MCAK fusion constructs, indicated inhibition of the microtubule depolymerization activity of MCAK. From these results, we conclude that clinically promising SQAGs have at least two different molecular targets, DNA polymerases and MCAK. It should be stressed that inhibitors of MCAK have never been reported previously so that there is a major potential for clinical utility.

Sulfoquinovosylmonoacylglycerol inhibitory mode analysis of rat DNA polymerase beta.

We have previously reported that sulfoquinovosylmonoacylglycerol (SQMG) is a potent inhibitor of mammalian DNA polymerases. DNA polymerase beta (pol beta) is one of the most important enzymes protecting the cell against DNA damage by base excision repair. In this study, we characterized the inhibitory action of SQMG against rat pol beta. SQMG competed with both the substrate and the template-primer for binding to pol beta. A gel mobility shift assay and a polymerase activity assay showed that SQMG competed with DNA for a binding site on the N-terminal 8-kDa domain of pol beta, subsequently inhibiting its catalytic activity. Fragments of SQMG such as sulfoquinovosylglycerol (SQG) and fatty acid (myristoleic acid, MA) weakly inhibited pol beta activity and the inhibitory effect of a mixture of SQG and MA was stronger than that of SQG or MA. To characterize this inhibition more precisely, we attempted to identify the interaction interface between SQMG and the 8-kDa domain by NMR chemical shift mapping. Firstly, we determined the binding site on a fragment of SQMG, the SQG moiety. We observed chemical shift changes primarily at two sites, the residues comprising the C-terminus of helix-1 and the N-terminus of helix-2, and residues in helix-4. Finally, based on our present results and our previously reported study of the interaction interface of fatty acids, we constructed two three-dimensional models of a complex between the 8-kDa domain and SQMG and evaluated them by the mutational analysis. The models show a SQMG interaction interface that is consistent with the data.

Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display.

SQAG9, a new class of immunosuppressive sulfoquinovosylacylglycerol, and its biotinylated derivatives have been synthesized. A T7 Phage library, composed of random cDNA fragments from Drosophila melanogaster, displayed a possible binding peptide of 14 amino acids. The immobilized synthetic peptide on a sensor chip showed a dissociation constant of K(D)=1.5 x 10(-6) against SQAG9 in a surface plasmon resonance experiment.

Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors.

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase alpha and beta and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure-function relationship of the synthetic sulfo-glycolipids to DNA polymerase alpha and beta and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase alpha with IC(50) values of 3-5 microM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.

Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmonoacylglycerols.

We recently reported that 3'-sulfonoquinovosyl-1'-monoacylglycerol (designated A-5) extracted from sea urchin intestine was effective in suppressing the growth of solid tumors. Although the major fatty acid component of A-5 was a saturated C(16) acid, there were five other fatty acids, 14:0, 18:0, 14:1, 16:1, and 18:1, which constitute minor components of A-5. Therefore, it remains unclear as to which of these six fatty acid components of A-5 has the anti-tumor effect. In this study, we synthesized sulfolipids each containing only one of these six fatty acids and tested their cytotoxicity against tumor cells and in vivo anti-tumor effects on nude-mice bearing solid tumors of human lung adenocarcinoma cell line A-549. The IC(50) values of all products against tumor cells were more than 10(-5) M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti-tumor assay showed that sulfoquinovosylmonoacylglycerols (SQMG) composed of 14:1 and 18:1 (designated SQMG(14:1) and SQMG(18:1), respectively) were significantly effective in suppressing the growth of solid tumors. Our data suggested that these two SQMGs had a substantial anti-tumor effect in vivo, and they are of interest as candidate drugs for anti-cancer treatment.