A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis

シツリョウ ブンセキ ソウチ オ モチイタ アミロイドーシス ノ シンダンホウ ノ カイハツ オヨビ ビョウタイ カイセキ

博士(医学)熊本大

Shorter recovery can be achieved from using walking boot after operative treatment of an ankle fracture

Background/Objective: Ankle fractures, even if treated surgically, usually take a long time to heal. For all patients with ankle fracture, immobilisation is a critical part of treatment. Short-leg walking boots (WBs) have been reported to be an effective alternative to plaster casts (PCs) that could shorten this postoperative recuperative period. The aim of this study was to compare the functional recovery of a conventional PC with that of a WB after surgery for ankle fractures. Methods: Forty-seven patients (mean age, 53.9±12 years) who had undergone surgical operation for an unstable ankle fracture from January 2008 to October 2014 were reviewed retrospectively. Either a PC or a WB was prescribed postoperatively, with 25 patients and 22 patients, respectively. The time that it took the patient to stand unipedal on the affected side after allowing full-weight bear and to walk without crutches were used for assessment of functional recovery. The prevalence of postoperative loss of reduction and nonunion was also reviewed. Results: Both the time of being able to stand unipedal on the injured side and to walk without crutches were significantly shorter in patients using WBs (WB, 2.6 weeks; PC, 4.5 weeks, p=0.01; WB, 1.4 weeks; PC, 3.1 weeks, p=0.03). There were no patients with loss of reduction or nonunion. Conclusion: Patients who used WBs showed a significantly faster recovery. WBs have an adjustable heel lift that allows users to change the ankle position slightly plantarflexed that helps walking in a postoperative swollen ankle. WBs are easy to slip on, and it is easy to adjust the ankle position in conformity with swelling so that the least painful position could be maintained during walking. WBs have good fixity to allow immediate weight-bearing postoperatively, and there were no cases with loss of reduction postoperatively. The Rocker bottom design minimises the sagittal plane motion in the specific joint of the foot, which also facilitates the course of recuperation. An ankle fracture fixed appropriately endures loading when a WB is used. The WB treatment results in faster functional recovery, allowing the patients to return to normal activity at a faster rate

Characterization of heterozygous ATTR Tyr114Cys amyloidosis-specific induced pluripotent stem cells

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC–MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis

Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma

Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma