Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system.

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.

Congenital cholesteatoma in a case of congenital aural atresia.

OBJECTIVE: A congenital cholesteatoma arising in the setting of congenital aural atresia is described. This represents the first case reported in the literature. STUDY DESIGN: Case report. SETTING: Tertiary otologic referral center, University of Virginia Health System, Charlottesville, Virginia. PATIENT: Eight-year-old patient with unilateral congenital aural atresia. INTERVENTIONS: Preoperative computer tomographic scanning and surgical atresia repair with removal of cholesteatoma. MAIN OUTCOME MEASURES: Recurrence of cholesteatoma; epithelialized ear canal; hearing improvement. RESULTS: A congenital cholesteatoma located medial to the ossicular mass in a child with congenital aural atresia was removed, with no evidence of recurrent disease at a revision operation 1 year later. Hearing was improved to a speech reception threshold of 25 dB. CONCLUSION: Congenital cholesteatoma can arise medial to the ossicular mass in congenital aural atresia. This finding lends support to the epidermoid rest theory for the cause of congenital cholesteatoma.

Stapedectomy: demographics in 2006.

OBJECTIVE: To evaluate whether the performance of stapes surgery by residents during residency has decreased during the last 40 years, and if so, whether such a decrease has impacted the number of general otolaryngologists performing stapedectomy in the private practice setting. STUDY DESIGN: Survey questionnaire of members of the American Academy of Otolaryngology-Head and Neck Surgery. METHODS: A short questionnaire was mailed to 1,700 members of the American Academy of Otolaryngology-Head and Neck Surgery. Data collected included age, year of residency graduation, fellowship (if any), practice setting, geographic location, number of stapedectomies performed in residency, and number of stapedectomies performed currently. RESULTS: Seven hundred fifty-six (44%) of 1,700 surveys were returned; 558 (74%) of 756 respondents did not complete a fellowship and were included in the general otolaryngologist group. Overall, 41% of general otolaryngologists are performing stapes surgery. There was a significant decline in the number of resident stapedectomies performed between 1960 and 1986; since 1986, this number has plateaued to an average of 8 to 9 (self-reported) cases per resident. More recent graduation from residency trended toward decreased inclination to do stapedectomy, although the result was not statistically significant. Increasing number of stapedectomy cases performed in residency correlated with an increased likelihood of performing the operation in practice. CONCLUSION: Despite a decreasing case volume of stapes surgery in residency, stapedectomy remains an operation performed by general otolaryngologists.

Intraparotid facial nerve schwannoma: diagnosis and management.

OBJECTIVE: Facial nerve schwannomas are rare neoplasms that present a challenge in diagnosis and management. We sought to gain insight into the clinical presentation and its implications in management. STUDY DESIGN AND SETTING: A group of patients diagnosed with schwannoma was reviewed, with focus on tumor arising in the intraparotid region of the facial nerve. The patient population consisted of 3722 patients evaluated retrospectively over 38 years at the Shadyside Facial Paralysis Center, a tertiary referral center. From this population, 29 patients (18 women and 11 men) with the diagnosis of schwannoma were selected for review. RESULTS: Twenty-nine patients had facial nerve schwannoma. The average age at diagnosis was 44 years (range, 7 to 78 years). Eight patients had intraparotid involvement, with 5 having a palpable mass in the parotid. The duration of symptoms averaged 8.4 years overall, with intraparotid involvement averaging 10.9 years. The 8 intraparotid schwannomas had a mean House-Brackmann grade of 1.5/6 at initial presentation. Postoperative facial nerve function was only 4.4/6 for resected intraparotid schwannomas. CONCLUSIONS: Intraparotid facial nerve schwannomas are slow growing tumors, which are usually asymptomatic but can be painful. Schwannoma should be suspected if the facial nerve cannot be found intraoperatively or if the tumor is intimately associated with the facial nerve. In cases where schwannoma is suspected, biopsy is recommended, while complete resection is postponed to obtain imaging studies to evaluate the extent of disease and to discuss possible outcomes with the patient. SIGNIFICANCE: This article provides guidance for the appropriate management of intraparotid facial nerve schwannoma.

Management of retrobulbar hematoma.

BACKGROUND: Retrobulbar hematoma (RH) is a complication that can result from both otolaryngic and ophthalmologic procedures. RH can occur during endoscopic sinus surgery and improper treatment can result in several morbidities, including visual loss. Despite serious consequences, management for RH is not well evaluated. However, lateral canthotomy with cantholysis is generally recommended. The objective of this study is to review the management for RH. METHODS: A retrospective study was performed at our tertiary hospital from 1979 to 2006 for patients with the ICD-9 code for orbital hematoma. The demographic information, comorbidities, presentation, management, follow-up period, and outcomes were evaluated. Data were analyzed. RESULTS: Twenty-two patients were identified with 13 male patients and an average age of 43 years (range, 11-80 years). The RH was broken into three categories: iatrogenic, six cases; trauma, eight cases; and spontaneous, eight cases. The most common symptom was diplopia followed by orbital pain. The average pretreatment and posttreatment tonometric pressures were 25.3 mm Hg (range, 11-60 mm Hg) and 14.5 mm Hg (range, 10-22 mm Hg), respectively. The average proptosis was 4.3 (range: 0-8) mm. Treatments were observation (13 cases), medical treatment alone (4 cases), and surgical treatment with and without medical treatment (5 cases). Sixty-eight percent of the patient's visual acuity improved with these treatments. Twenty-seven percent had no visual changes from the RH. The average follow-up was 5 years. CONCLUSION: Traditionally, lateral canthotomy with cantholysis is recommended for the treatment for RH. However, in certain patients and settings, there may be an acceptable alternative option for the management of RH.

Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.

Anatomic risk factors for sinus disease: fact or fiction?

BACKGROUND: Sinonasal anatomic variants have been postulated as a risk factor for sinus disease. Therefore, a study was conducted to examine the correlation of sinus disease to septal deviation, concha bullosa, and infraorbital ethmoid cells. METHODS: Two hundred fifty consecutive sinus and orbital computed tomography scans were examined at the University of Virginia over a 2-year period. Coronal, sagittal, and axial views were examined for the presence and size of concha bullosa and infraorbital ethmoid cells. Septal deviations were measured by examining the width of the nasal cavity at the level of the maxillary sinus ostium. The severity of mucosal thickening in the maxillary, ethmoid, and frontal sinuses was recorded. The correlation between mucosal disease of the sinuses to the anatomic variants was then compared. RESULTS: Computed tomography images were reviewed in 250 consecutive studies (500 sides). Of the 500 sides, 67.2% of sides had some level of mucosal thickening. Concha bullosa and infraorbital ethmoid cells were both present in 27% of the sides. Concha bullosa was associated with maxillary sinus disease (p < 0.01). Infraorbital ethmoid cells were associated with both ethmoid (p < 0.05) and maxillary (p < 0.01) mucosal disease. Frontal sinus disease had no significant correlation with these anatomic variants (p > 0.05). For sinuses with infraorbital ethmoid cells or concha bullosa, there were a higher number of diseased sinuses with larger anatomic variants (p < 0.01). Narrow nasal cavities were associated with maxillary sinus disease (p < 0.01). CONCLUSION: Septal deviations, concha bullosa, and infraorbital ethmoid cells, which contribute to the narrowing of the osteomeatal complex, are associated with mucosal disease.