Proteins unfold constantly in cells, especially under stress conditions. Degradation of denatured polypeptides by Lon and related ATP-dependent AAA + proteases helps prevent toxic aggregates formation and other deleterious consequences, but how these destructive enzymatic machines distinguish between damaged and properly folded proteins is poorly understood. Here, we show that Escherichia coli Lon recognizes specific sequences-rich in aromatic residues-that are accessible in unfolded polypeptides but hidden in most native structures. Denatured polypeptides lacking such sequences are poor substrates. Lon also unfolds and degrades stably folded proteins with accessible recognition tags. Thus, protein architecture and the positioning of appropriate targeting sequences allow Lon degradation to be dependent or independent of the folding status of a protein. Our results suggest that Lon can recognize multiple signals in unfolded polypeptides synergistically, resulting in nanomolar binding and a mechanism for discriminating irreversibly damaged proteins from transiently unfolded elements of structure.

The fibula free flap became popular in orthopedic oncology for limb-sparing long bone tumor resection. It is particularly suitable for intercalary or resection arthrodesis options. In the present series, a surgical reconstruction algorithm was used, enabling each patient to receive a personalized technique. During the years 1998 to 2002, 30 patients underwent limb-sparing surgery for long bone sarcoma. There were 18 males and 12 females. Their mean age was 23 years (range, 9 to 70 years). The diagnoses were Ewing's sarcoma (11 patients), osteogenic sarcoma (eight patients), chondrosarcoma (five patients), giant cell tumor of bone (three patients), high-grade soft-tissue sarcoma (two patients), and leiomyosarcoma of bone (one patient). The majority of tumors where located in the lower extremity (23 patients), mostly in the femur (15 patients with four tumors in the proximal femoral shaft, five tumors in the distal femoral shaft, five tumors in the whole femoral shaft, and one tumor in the proximal femoral head). In seven patients, the upper extremity was involved; in six patients, the radius was involved; and in one patient, the humerus was involved. The free fibula flap was used in three types of approaches: vascularized fibula as an osseous flap only (18 patients), a combination of a vascularized fibula flap in conjunction with an allograft (Capanna's technique; 10 patients), and a free double-barreled fibula (two patients). All flaps survived. Postoperatively, all patients were monitored clinically, radiologically, and by radioisotope bone scan studies. Callus formation and union were shown 2.6 to 8 months postoperatively. Patients who underwent lower extremity reconstruction were nonweightbearing for 3 to 9 months, with a transition period in which they used a brace and gradually increased weightbearing until full weightbearing was achieved. Eight patients had 11 recipient-site complications. Two patients (6.7 percent) had hematomas, and three patients (10 percent) had infection and dehiscence of the surgical wound with bone exposure in one patient; all complications resolved with conservative treatment only. Failure of the hardware fixation system occurred in two patients, mandating surgical correction. No fibula donor-site complications were recorded. In intercalary resections, the use of the vascularized fibula flap as an isolated osseous flap might be insufficient. Different body sites have different stress loads to carry, depending on the age of the patient and on his individual physical status. To achieve initial strength in the early period, the authors combined the free fibula flap with an allograft (Capanna's method) or augmented it as a double-barreled fibula. They propose a surgical algorithm to assist the surgeon with the preferred method for reconstruction of various long bone defects in different body locations at childhood or adulthood. Long bone reconstruction using a vascularized fibula flap, alone or in combination with an allograft, autogenous bone graft, or double-barreled fibula for limb-s...

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.

Objective: To present our method for anterior skull base reconstruction after oncological resections. Methods: One hundred nine patients who had undergone 120 anterior skull base resections of tumors (52 malignant [43%], 68 benign [57%]) via the subcranial approach were studied. Limited dural defects were closed primarily or reconstructed using a temporalis fascia. Large anterior skull base defects were reconstructed by a double-layer fascia lata graft. A split calvarial bone graft, posterior frontal sinus wall, or three-dimensional titanium mesh were used when the tumor involved the frontal, nasal, or orbital bones. A temporalis muscle flap was used to cover the orbital socket for cases of eye globe exenteration, and a rectus abdominis free flap was used for subcranial-orbitomaxillary resection. Pericranial flap wrapping of the frontonaso-orbital segment was performed to prevent osteoradionecrosis if perioperative radiotherapy was planned. Results: The incidence of cerebrospinal fluid (CSF) leak, intracranial infection, and tension pneumocephalus was 5%. Histopathological and immunohistochemical analysis of fascia lata grafts in reoperated patients (n ¼ 7) revealed integration of vascularized fibrous tissue to the graft and local proliferation of a newly formed vascular layer embedding the fascial sheath. Conclusion: A double-layer fascial graft alone was adequate for preventing CSF leak, meningitis, tension pneumocephalus, and brain herniation. We describe a simple and effective method of anterior skull base reconstruction after resections of both malignant and benign tumors.