9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of ßδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

Delayed reverse sural flap for staged reconstruction of the foot and lower leg.

BACKGROUND: Soft-tissue defects of the foot and lower leg caused by traumatic injury, tumor ablation, or infection associated with osteomyelitis often require coverage by flaps. One excellent option for reconstruction of these defects is the distally based neurofasciocutaneous sural flap. It allows rapid and reliable coverage of defects from the distal third of the lower leg to the forefoot without significant functional donor-site morbidity. However, the maximal size of the flap is limited by the delicate perfusion of the arterial network associated with the superficial sensory nerve. Delay procedures may increase the reliability of large sural flaps. METHODS: The authors successfully used delayed sural flaps based on a two-step procedure for the treatment of 11 patients (three women and eight men, age 50.1 +/- 20.0 years) with osteomyelitis (n = 3), melanoma (n = 3), sarcoma (n = 1), squamous cell carcinoma (n = 1), posttraumatic defects (n = 2), and recurrent gouty ulcer (n = 1). The delay period ranged from 7 to 15 days (9.7 +/- 3.1), the length of the flap was from 9 to 19 (14.8 +/- 3.0) cm, and the width of the flap from 7 to 12 (9.2 +/- 1.3) cm. Temporary wound coverage was achieved by vacuum-assisted closure during the delay period. RESULTS: All defects were covered successfully without major complications. CONCLUSIONS: The delay procedure positively affects the viability of large sural neurofasciocutaneous flaps. The authors recommend this modification for patients with large defects at the distal third of the lower leg or foot, requiring a two-step surgical approach due to the underlying disease.

Anti-TGFbeta1 antibody for modulation of expression of endogenous transforming growth factor beta 1 to prevent fibrosis after plastic surgery in rats.

The cytokine transforming growth factor beta 1 (TGFbeta1) substantially influences synthesis of extracellular matrix, fibrosis, and neoangiogenesis during wound healing in a dose dependent manner. We carried out experiments in rats to measure the degree of reduction of disorders of wound healing and fibrosis produced by inhibition of endogenous TGFbeta1 by polyclonal antibodies (poAB). A free myocutaneous gracilis flap was transplanted from the groin to the neck region in 30 Wistar rats (300-450 g body weight). In 15 animals intraoperatively and daily from days 3 to 7 postoperatively, 1 microg anti-TGFbeta1 poAB in 500 microl of phosphate buffered saline (PBS) were injected into the neck region. Fifteen animals served as controls. On postoperative days 3, 4, 5, 7, 14, and 28 the expression of endogenous TGFbeta1 in cytoplasm was analysed by immunohistochemistry (ABC-POX; AEC), in situ hybridisation of TGFbeta1-mRNA, and Sirus Red staining of collagen matrix; it was quantified using labelling indices. Neutralisation of the TGFbeta1 activity by specific poAB resulted in inhibition of the cytoplasmatic expression compared with untreated animals. In the transition area between grafted tissue and graft bed, a significant reduction of TGFbeta1 expression (mean (S.D.) 34.7 (6.5)) was found from day 5 in the group treated with anti-TGFbeta1 poAB compared with the control group (mean (S.D.) 48.1 (6.6)) (P <0.03). Up to day 14 the endogenous expression of TGFbeta1 (mean (S.D.) 30.0 (2.8)) was reduced after the application of TGFbeta1 poAB compared with the control group (mean (S.D.) 44.0 (12.3)). Sirus Red staining indicated a more complex packed structure and generally more prominent collagen types I-IV fibres in untreated animals than in animals that were given anti-TGFbeta1 poAB. Expression of TGFbeta-mRNA by in situ hybridisation was reduced in fibroblasts in animals that were given anti-TGFbeta1 poAB. The results indicate that anti-TGFbeta1 might improve the healing of free flaps in the graft beds of patients who are prone to excessive fibrosis.

Dosimetry-guided radioactive iodine treatment in patients with metastatic differentiated thyroid cancer: largest safe dose using a risk-adapted approach.

UNLABELLED: This study is a retrospective analysis of 124 differentiated thyroid cancer patients who underwent dosimetric evaluation using MIRD methodology over a period of 15 y. The objectives of the study were to demonstrate the clinical use of dosimetry-guided radioactive iodine ([RAI] (131)I) treatment and the safe and effective application of a 3-Gy bone marrow (BM) dose in patients with differentiated thyroid cancer. METHODS: Tumor and BM dose estimates were obtained. The administered activity that would deliver a maximum safe dose to the organ at risk (red BM or lungs) was determined as well as the resulting doses to the metastases. The clinical benefit of an individual RAI treatment was predicted on the basis of the dose estimates and the expected therapeutic response. Each patient's response to treatment was assessed clinically and by monitoring the hematologic profile. RESULTS: One hundred twenty-four patients underwent 187 dosimetric evaluations. One hundred four RAI treatments were performed. A complete response at metastatic deposits was attained with absorbed doses of >100 Gy. No permanent BM suppression was observed in patients who received absorbed doses of <3 Gy to BM. The maximum administered dose was 38.5 GBq (1,040 mCi) with the BM dose limitation. CONCLUSION: Dosimetry-guided RAI treatment allows administration of the maximum possible RAI dose to achieve the maximum therapeutic benefit. Estimation of tumor dose rates helps to determine the curative versus the palliative intent of the therapy.