A loss of FUS/TLS function leads to impaired cellular proliferation.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is a multifunctional RNA/DNA-binding protein that is pathologically associated with cancer and neurodegeneration. To gain insight into the vital functions of FUS and how a loss of FUS function impacts cellular homeostasis, FUS expression was reduced in different cellular models through RNA interference. Our results show that a loss of FUS expression severely impairs cellular proliferation and leads to an increase in phosphorylated histone H3, a marker of mitotic arrest. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with multiple processes, some of which influence cell proliferation including cell cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. FUS knockdown also correlated with increased expression of the closely related protein EWS (Ewing's sarcoma). We demonstrate that the maladaptive phenotype resulting from FUS knockdown is reversible and can be rescued by re-expression of FUS or partially rescued by the small-molecule rolipram. These results provide insight into the pathways and processes that are regulated by FUS, as well as the cellular consequences for a loss of FUS function.

Angular nerve: new insights on innervation of the corrugator supercilii and procerus muscles.

BACKGROUND: Elimination of glabellar frown lines by direct denervation of the corrugator and procerus muscles has proven elusive. Furrows often persist after the frontal branch of the facial nerve has been divided. In order to find an explanation for these observations and to provide an anatomic basis for future surgical strategies, the pattern of innervation of these muscles was studied. METHODS: Two separate investigations were performed: (1) cadaveric anatomical dissection in 18 fresh cadaver hemi-faces utilizing loupe magnification, operating microscope, and template tracings; and (2) electrophysiologic testing performed in 12 healthy volunteers utilizing a Viking Ile nerve stimulator (Nicolet, Madison, WI). RESULTS: The corrugator was found to have a dual nerve supply with contributions from branches of the frontal, zygomatic and buccal branches of the facial nerve. The frontal branch passes deep to the orbicularis at the level of the eyebrow; and the buccal branch, after receiving a contribution from the zygomatic, forms the angular nerve, which then passes superiorly in front of the medial canthus to supply both corrugator and procerus. The procerus muscle was found to receive contributions from the angular nerve exclusively. CONCLUSIONS: Procedures designed to denervate the corrugator should be directed at both components of its redundant nerve supply, the frontal branch as well as the angular nerve. Division of the angular nerve will also denervate the procerus muscle. Both frontal nerve branches and angular nerve components are accessible through an upper blepharoplasty incision.

Repair of a large septal perforation with a radial forearm free flap: brief report of a case.

We treated a 38-year-old man who had a large septal perforation that had been caused by chronic nasal inhalation of cocaine. We were able to repair the perforation with a left radial forearm free flap. Long-term followup indicates a successful closure of the defect and a natural thinning of the flap. The patient remains symptom-free more than 2 years following surgery.

Early treatment of severe mandibular hypoplasia with distraction mesenchymogenesis and bilateral free fibula flaps.

The technique of distraction has revolutionized the treatment of mandibular hypoplasia; however, presently large mandibular defects still require bone grafts. Microvascular grafting is commonly used in adults. Conversely, in pediatric reconstruction, nonvascularized rib grafts remain standard. Unfortunately, resorption of nonvascularized bone remains a major issue, particularly when soft tissue is hypoplastic. This case study represents a combination of techniques in the treatment of severe mandibular deficiency, and introduces the concept of distraction mesenchymogenesis. The patient was a 2 1/2-year-old boy with severe bilateral Pruzansky class III mandibular hypoplasia. He had a permanent open mouth posture, an overjet of 23 mm, and was unable to move the lower mandibular segment. His oropharyngeal airway diameter was 2.2 mm and he was tracheostomy dependent. The patient was treated with distraction of the lower jaw mesenchyme followed by bilateral functional free fibular microvascular flaps containing reinnervated muscle. This created a well-vascularized body, ramus, and condyle bilaterally within an adequate soft-tissue envelope. Postoperatively, the overjet was reduced to 5 mm. The patient can now actively move his mandible. Airway diameter increased to 10 mm, and the patient is able to tolerate intermittent tracheostomy plugging. This innovative combination of techniques allows early intervention, limits graft resorption, and improves airway control.

The vascularized pig fibula bone flap model: effect of segmental osteotomies and internal fixation on blood flow.

The free fibular flap is the flap of choice for reconstruction of complex mandibular defects, although two or more osteotomies may be required to recreate the normal mandibular contour. The effect of these surgical manipulations on the fibula has not been adequately investigated. This study was designed to study the effect of multiple segmental osteotomies and internal fixation techniques on blood flow in the vascularized pig fibula bone flap model. The hindlimbs of 15 Yorkshire pigs were randomized into 1 of 5 groups (n = 6 fibulae per group) consisting of: (1) a nonoperated, in situ fibula; (2) an elevated fibula flap; (3) an elevated fibula flap with two segmental osteotomies; (4) an elevated fibula with two segmental closing osteotomies rigidly fixed with 2-mm miniplates; (5) an elevated fibula with two segmental closing osteotomies rigidly fixed with 2-mm lag screws. Total and gradient blood flow was measured in the bone and soft-tissue components of these flaps using the 15-microm radioactive microsphere technique. The creation of two segmental osteotomies in the vascularized pig fibula bone flap model resulted in a significant decrease (p<0.05) in the gradient blood flow in the segment of bone distal to the second osteotomy. Application of miniplates or lag screws across closing osteotomies resulted in a significant decrease (p<0.05) in total and gradient blood flow to the bone component of the fibulae, as compared with the elevated and osteotomized fibulae groups. An increase in blood flow suggesting a hyperemic response was noted in the bone and soft tissue in the elevated and osteotomized flap groups as compared with the in situ, nonoperated controls. This study established the validity of the pig fibula as a suitable model for investigating the pathophysiology of blood flow changes in the face of standard surgical maneuvers necessary for the restoration of mandibular form and function. The results demonstrated that the creation of multiple segmental osteotomies and the application of internal fixation significantly decreases (p<0.05) blood flow to the distal portion of the flap. The effects of segmental osteotomies and internal fixation on healing and growth of the pig fibula bone flap model are investigated in a separate study.

Flap selection in cranial base reconstruction.

We have compared our local, pedicled, and free-flap reconstructions for 90 skull base defects performed over the past 10 years. The pericranial flap was found to provide a reliable dural seal. Free-flap reconstructions exhibited a significantly higher incidence of uncomplicated primary wound healing (95 versus 62.5 percent) and a much lower incidence of flap loss (0 percent), cerebrospinal fluid leak (5 percent), meningitis, and abscess (0 percent) when compared with defects reconstructed with pedicled myocutaneous flaps. We conclude that microvascular free-tissue transfer is the safest, most economical procedure when faced with moderate to large composite defects of the cranial base.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Lower extremity free flaps: a review.

OBJECTIVE: To identify factors related to free-flap coverage of lower extremity fractures that are linked to a negative outcome. DESIGN: A chart review. SETTING: A large microsurgical referral centre. PATIENTS: From 1981 to 1989, the records of all patients who underwent free-tissue transfer to the lower extremity with more than 1 year of follow-up were selected. From this was drawn a subgroup of 49 patients (mean age, 36 years) who had tibial fractures (55% were motor vehicle injuries) and in almost all cases established soft-tissue or bony defects. They formed the study group. INTERVENTION: Free-flap transfer. OUTCOME MEASURES: Factors that might be associated with free-flap failure: mechanism of injury, grade of tibial fracture, history of smoking, diabetes, peripheral vascular disease, ischemic heart disease, vascular compromise in the leg preoperatively, recipient artery used, type of anastomosis, and hypertension or hypotension intraoperatively. RESULTS: Type IIIB tibial fractures were the most frequent (67%) and carried a significantly (p = 0.02) higher risk of free-flap failure than other types of fracture. Patients underwent a mean of four procedures before referral for free-tissue transfer. The mean time from injury to flap coverage was 1006 days. Stable, long-term coverage of the free flaps was achieved in 78% of patients. Wound breakdown was most often caused by recurrent osteomyelitis (65%). Seventy-four percent of the fractures healed. The amputation rate was 10%. Four patients required repeat free-flap transfer for limb salvage. CONCLUSIONS: Only the grade of tibial fracture could be significantly related to postoperative free-flap failure.

Anatomic and hemodynamic study of the vascularized pig fibula bone flap model.

Despite much interest in studying the pathophysiology of experimental skin and muscle flaps to better understand the pathobiology of flap failure, relatively little has been published in the investigation of vascularized bone flaps. The aim of this study was to develop a suitable vascularized bone flap model in the pig in the hope that this model may prove useful in studying the pathophysiology of vascularized bone tissue transfer. Yorkshire pigs (17-26 kg) were used for all experiments. Anatomic studies revealed that the fibula in the hindlimb was the most suitable bone for investigation as a flap model. Anatomic dissections, radiologic investigations (plain x-rays, angiograms), and morphometric analyses of the fibulae in both hindlimbs of five animals were carried out. In a separate group of pigs (n = 6), the fibula was elevated as a vascularized flap and then blood flow was measured using the 15-microns radioactive microsphere technique. The fibula in the pig is supplied by a branch of the cranial tibial artery, running along an intermuscular septum between the posterior and anterior compartments of the hindlimb accompanied by one or two vena commitans. The bone flap is raised with a cuff of flexor hallucis longus with a length of 9.2 +/- 0.2 cm (mean +/- SEM). Blood flow measurement confirmed that the entire fibula was well vascularized when elevated on its pedicle. Gradient blood flow showed a bimodal distribution, with regions of highest blood flow noted at the proximal and distal ends of the bone flap, in areas where there were greater percentages of cancellous bone. The results of these experiments suggest that the pig fibula may be a suitable model for the study of vascularized bone flap pathophysiology.

The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene.

The D. melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes. Here we show that the predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins. There is also extensive functional overlap between mei-41 and ATM. Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability. We also demonstrate that mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells. Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene.

Primary mandibular reconstruction with the titanium hollow screw reconstruction plate: evaluation of 51 cases.

Fifty-one patients undergoing surgical reconstruction of mandibular defects with the titanium hollow screw reconstruction plate over a 4-year period were analyzed. Plate failure was defined as flap necrosis, plate extrusion, or plate fracture necessitating a further surgical procedure and occurred in 12 patients (24 percent). The incidence was highest for patients who had more than three mandibular regions resected. It was not affected by the primary site of the tumor or whether the patient had received radiation therapy. Although the failure rate was the same whether the patient attained full oral function or was fed by gastrostomy, plate fracture was limited to the former group. The overall cause-specific survival rate for this group of patients was 68 and 56 percent at 1 and 2 years, respectively. In addition, 81 percent of patients attained a full oral diet.

Histologic study of pulp capping using chlorhexidine in dogs.

This histological study was conducted to compare the pulp reactions to chlorhexidine and calcium hydroxide. Twenty permanent premolar teeth of beagle dogs were mechanically exposed under aseptic conditions. The pulp was then capped with one of the following: chlorhexidine 0.2% on the left side and calcium hydroxide (dycal) on the right side of the same dog. Maxillary second premolars were extracted from each dog three weeks after the pulp capping procedure. Maxillary third premolars and mandibular first premolars were extracted at 6 weeks and 9 weeks respectively. The mandibular second premolars were extracted three months after the pulp capping procedure. The specimens were fixed in 10% formalin and decalcified in 5% formic acid. Serial sections, 7 microns thick, were prepared and stained with hematoxylin and eosin. Teeth treated with chlorhexidine or with calcium hydroxide were well tolerated by the dental pulp at 3 and 6-week intervals. Pulps treated showed satisfactory pulp reaction and the odontoblastic layer appeared to be normal and in its configuration. Inflammatory response was not present at the 6-week time interval. At nine weeks, the inflammatory response was severe in those teeth treated with calcium hydroxide. There were no remarkable histological differences among the treatment groups at 12 and 15 weeks. These specimens showed complete loss of pulpal architecture. Regressive changes were seen and microscopic specimens revealed presence of spaces and intertwining bundles of films in the pulp.

The free flap and plate in oromandibular reconstruction: long-term review and indications.

The purpose of this study was to define the role of reconstruction plates as bone replacement in oromandibular reconstruction. From 1987 through 1991, 71 consecutive oral cancer patients underwent composite resection and reconstruction and were entered into one of two studies. In the first study of 31 patients, 15 underwent oromandibular reconstruction using a radial forearm osteocutaneous flap, while the remainder (16) received a radial forearm fasciocutaneous flap together with a mandibular reconstruction plate. The second study involved 40 subsequent patients, all receiving the latter form of reconstruction. Twenty-one of the plates were stainless steel, and the remaining 19 were of the titanium hollow screw (THORP) type. We followed the patients prospectively. We defined success as a reconstruction that we did not have to remove. Additionally, since the patients had limited life expectancy, we developed the idea of days of life lost and incorporated it into our definition of a successful outcome. Vascularized autogenous bone proved to be more successful than metallic plates used alone in terms both of reconstruction survival and of minimizing days of life lost. The overall success rate of mandibular plate reconstruction was 78.9 percent, but analysis by defect type revealed a failure rate of 35 percent when the defects were anterior and only 5 percent when they were lateral. THORP plates demonstrated a trend towards more durability. We would now recommend plate reconstruction only in lateral defects in patients with a poor prognosis.

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster.

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.

Reconstruction of the cranial base defect.

Cranial base resection and reconstruction is the newest frontier in head and neck cancer surgery. It is following in the footsteps of all previous surgical challenges. Impossibility is the gauntlet. Development of improved imaging allows us to define the extent of the tumor, and the collaboration of diverse disciplines allows us to work out approaches. Reconstruction always begins quite crudely, at first being simply an exercise in hole filling. This is followed by refinement and ultimately the ability to replace what has been lost. In many aspects of head and neck surgery we are already quite sophisticated. Mandibular reconstruction has gone through all these stages and we are now in a position to reconstruct mandibles with bone of sufficient quantity and quality to take dental implants, essentially replacing what was lost. The future of cranial base surgery holds exciting challenges to our ingenuity to refine and develop what has been started.

Mandibular reconstruction with osseointegrated implants into the free vascularized radius.

Complete oral rehabilitation after oromandibular resection is a goal that is frequently difficult to attain. Poor speech, inability to eat a solid diet, and cosmetic deformity are all potential problems. The introduction of osseointegrated implants and free vascularized bone grafting are two techniques that have permitted improved results of oromandibular reconstruction. When used together they provide the best possibility for providing oral continence, a stable denture, and minimal cosmetic deformity. The free vascularized iliac crest has been used widely to reconstruct mandibular defects with the placement of enosseus implants. However, the radius provides a good alternative to reconstruct the mandible. We report four cases of mandibular reconstruction with the use of the free vascularized radius with subsequent placement of osseointegrated implants. The results of these cases suggest that enosseus implants can be used successfully in the radius. The radius is ideal to reconstruct small to moderate-sized defects of the lateral mandible with loss of oral mucosa.

Donor site deficit of the osteocutaneous radial forearm flap.

A functional evaluation was made of osteocutaneous radial forearm flap donor upper extremities at a mean 19.4 months postoperatively. Donor deficits were evaluated as follows: (1) skin: subjective appearance and durability; (2) skeleton: range of motion of adjacent joints; (3) vessels: cold intolerance, digital temperature, digital--brachial index; (4) muscle: key pinch; (5) nerve: two-point discrimination of dorsal first web space. Ten consecutive patients with a mean donor site area of 51.5 cm2 and mean length of harvested radius of 10.9 cm were studied. Incomplete skin graft take at the donor site (7 of 10 patients) and appearance of fair or poor (6 of 10 patients) were frequent cutaneous complications. Wrist range of motion was decreased with pronation (90%), flexion (90%), and extension (89%). Mean digital temperature, comparing extremities that did (operated) and did not (nonoperated) undergo surgery, or comparing the radial artery-supplied fingers to the ulnar artery-supplied fingers within the same operated hand showed no effect from the lost radial artery. Mean digital-brachial index was 1.24 for the nonoperated and 1.15 for the operated extremity. Key pinch on the operated side was a mean of 74% of the nonoperated side. Although raising the flap creates multiple tissue deficits, each with the potential to alter upper extremity function, detailed functional evaluation failed to demonstrate significant alteration in upper extremity function after flap harvest.

Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.

Use of reconstruction plates in conjunction with soft-tissue free flaps for oromandibular reconstruction.

The plate-flap option has a place in oromandibular reconstruction: It allows an expedient method to obtain an excellent cosmetic and functional result with minimal donor site disability. It is best for lateral and posterior low-volume defects in the debilitated, the elderly, and in those patients with a poor prognosis. This technique may be used as a preliminary to vascularized bone grafts in patients receiving postoperative radiotherapy. This method is no panacea. These are very specific indications for its use. Failure usually requires rescue with a vascularized bone graft. With proper selection of patients, however, this will rarely be necessary. A major weakness in the plating systems available at present is their tendency to fracture if they remain in place too long. The gauntlet is, therefore, thrown down to those in a position to develop more reliable hardware.