MyoCount: a software tool for the automated quantification of myotube surface area and nuclear fusion index.

It is often desirable to characterise the morphology of myogenic cultures. To achieve this, the surface area of myotubes is often quantified, along with the nuclear fusion index (NFI). Existing methods of such quantification are time-consuming and subject to error-prone human input. We have developed MyoCount, an open-source program that runs via the freely available MATLAB Runtime and quantifies myotube surface area and NFI. MyoCount allows the user to adjust its parameters to account for differences in image quality, magnification and the colour channels used in generating the image. MyoCount measures of myotube surface area and NFI were compared to the mean of measures performed by two blinded investigators using ImageJ software (surface area R 2 = 0.89, NFI R 2 =0.87). For NFI, the mean coefficient of variation (CV) between two investigators (17.6 ± 2.3%) was significantly higher than that between the investigator mean and MyoCount (13.5 ± 1.4%). For measurements of myotube area, the CV did not differ between both analysis methods. Given these results and the advantages of applying the same image analysis method uniformly across all images in an experiment, we suggest that MyoCount will be a useful research tool and we publish its source code and instructions for its use alongside this article.

Obese subcutaneous adipose tissue impairs human myogenesis, particularly in old skeletal muscle, via resistin-mediated activation of NFκB.

Adiposity and adipokines are implicated in the loss of skeletal muscle mass with age and in several chronic disease states. The aim of this study was to determine the effects of human obese and lean subcutaneous adipose tissue secretome on myogenesis and metabolism in skeletal muscle cells derived from both young (18-30 yr) and elderly (>65 yr) individuals. Obese subcutaneous adipose tissue secretome impaired the myogenesis of old myoblasts but not young myoblasts. Resistin was prolifically secreted by obese subcutaneous adipose tissue and impaired myotube thickness and nuclear fusion by activation of the classical NFκB pathway. Depletion of resistin from obese adipose tissue secretome restored myogenesis. Inhibition of the classical NFκB pathway protected myoblasts from the detrimental effect of resistin on myogenesis. Resistin also promoted intramyocellular lipid accumulation in myotubes and altered myotube metabolism by enhancing fatty acid oxidation and increasing myotube respiration and ATP production. In conclusion, resistin derived from human obese subcutaneous adipose tissue impairs myogenesis of human skeletal muscle, particularly older muscle, and alters muscle metabolism in developing myotubes. These findings may have important implications for the maintenance of muscle mass in older people with chronic inflammatory conditions, or older people who are obese or overweight.

CodonLogo: a sequence logo-based viewer for codon patterns.

MOTIVATION: Conserved patterns across a multiple sequence alignment can be visualized by generating sequence logos. Sequence logos show each column in the alignment as stacks of symbol(s) where the height of a stack is proportional to its informational content, whereas the height of each symbol within the stack is proportional to its frequency in the column. Sequence logos use symbols of either nucleotide or amino acid alphabets. However, certain regulatory signals in messenger RNA (mRNA) act as combinations of codons. Yet no tool is available for visualization of conserved codon patterns. RESULTS: We present the first application which allows visualization of conserved regions in a multiple sequence alignment in the context of codons. CodonLogo is based on WebLogo3 and uses the same heuristics but treats codons as inseparable units of a 64-letter alphabet. CodonLogo can discriminate patterns of codon conservation from patterns of nucleotide conservation that appear indistinguishable in standard sequence logos. AVAILABILITY: The CodonLogo source code and its implementation (in a local version of the Galaxy Browser) are available at http://recode.ucc.ie/CodonLogo and through the Galaxy Tool Shed at http://toolshed.g2.bx.psu.edu/.

Vaccination of llamas, Llama glama, with an experimental killed encephalomyocarditis virus vaccine.

Encephalomyocarditis virus (EMCV) is a pandemic virus that has caused mortality in numerous captive wildlife species worldwide. An experimental killed vaccine was created from two EMCV isolates associated with zoo animal mortality in the southern United States. The vaccine was tested for safety and efficacy in eleven llamas (Llama glama). All animals received an initial vaccination and a second booster vaccination 4 wk later. Serum antibody responses were monitored at initial vaccination and at 4 wks, 8 wk, 6 mo, and 12 mo postvaccination. Eight of the 11 llamas vaccinated experienced at least a 4-fold increase in serum antibody titers to EMCV. Antibody titers of those eight animals remained elevated above prevaccination levels when measured at 12 mo. The experimental killed EMCV vaccine tested may be a useful tool to prevent EMCV infection in llamas when given in 2 doses 4 wk apart, and then revaccinated or with antibody levels monitored annually thereafter.

An oral hypnotic medication does not improve continuous positive airway pressure compliance in men with obstructive sleep apnea.

BACKGROUND: Compliance with continuous positive airway pressure (CPAP) treatment for obstructive sleep apnea (OSA) is highly variable. Recent studies suggest that the initial experience with CPAP may determine long-term success. We hypothesized that administration of a hypnotic medication to new CPAP users would facilitate acclimation and increase usage. METHODS: Seventy-two male OSA patients referred for CPAP treatment were randomized to also receive an oral hypnotic agent (zolpidem), placebo pill, or neither (standard care) for the first 14 days of CPAP treatment. CPAP usage (effective mask pressure [hours per day]) was recorded by an internal data chip. Patient symptoms were assessed with the Epworth sleepiness scale (ESS) and functional outcomes of sleep questionnaire (FOSQ). Treatment groups were matched for age, body mass index, and baseline ESS and FOSQ scores. Despite randomization, the standard care group had a higher apnea/hypopnea index than either the zolpidem or placebo pill groups (54.75 +/- 28.02 vs 32.61 +/- 25.12 vs 38.09 +/- 25.65, p = 0.012) [mean +/- SD]. Compared to placebo pill and standard care groups, the zolpidem group did not show greater CPAP usage in terms of total days used (zolpidem, 20.58 +/- 7.40 days; placebo pill, 17.83 +/- 9.33 days; standard care, 22.92 +/- 6.95 days; p = 0.198) or average time used per night (4.43 +/- 1.16 h vs 4.23 +/- 2.14 h vs 4.94 +/- 1.44 h, p = 0.361). All groups showed significant symptom improvements on both the ESS (p < 0.001) and FOSQ (p < 0.05). CONCLUSION: Administration of an oral hypnotic agent did not improve initial CPAP compliance in men with OSA.

Evolving management of upper-tract transitional-cell carcinoma at a tertiary-care center.

BACKGROUND AND PURPOSE: Traditional management of upper-tract transitional-cell carcinoma (TCC) has been open nephroureterectomy. Minimally invasive options, including laparoscopic and endoscopic techniques, are being applied with increasing frequency, however. To assess the impact of these techniques on the current management of upper-tract TCC, we reviewed our experience managing this problem over the last 3 years. PATIENTS AND METHODS: Since January 1998, 84 patients underwent definitive management of upper-tract TCC using open, laparoscopic, or endoscopic techniques. This study group includes 57 men and 27 women with a mean age of 69.9 years. RESULTS: Fifty-three patients (63.9%) were treated by laparoscopic nephroureterectomy. Twelve patients (14.5%) were treated endoscopically, with percutaneous resection in 7 patients and ureteroscopic resection in 5 patients. The indications for nephron-sparing management in these 12 patients included solitary kidneys in 6 patients, significant comorbidities in 4 patients, and bilateral disease in 1 patient. Endoscopic management was elective in one patient. Nineteen patients (22.9%) underwent open surgical procedures consisting of nephroureterectomy in 16 patients and distal ureterectomy with reimplantation in 3 patients. CONCLUSIONS: Advances in laparoscopy and endourology are significantly impacting the definitive management of upper-tract TCC. Patients with a normal contralateral kidney are currently offered laparoscopic nephroureterectomy, while those with an absent or functionally compromised contralateral kidney are generally managed with endoscopic resection. Although minimally invasive techniques have demonstrated advantages regarding postoperative pain, hospital stay, and return to regular activities, only critical long-term follow-up regarding rates of local and distant recurrence will determine the ultimate role of these techniques.

Laparoscopic partial nephrectomy for renal tumor: duplicating open surgical techniques.

PURPOSE: We describe our technique of and single institutional experience with purely laparoscopic partial nephrectomy for renal tumor, wherein the focus is to duplicate established open techniques of oncologic nephron sparing surgery. MATERIALS AND METHODS: Since August 1999 laparoscopic partial nephrectomy for renal tumor has been performed in 50 patients. Of the patients 24 (48%) had either a compromised contralateral kidney (20) or a solitary kidney (4). Mean tumor size was 3.0 cm. (range 1.4 to 7). In 9 patients (18%) the inner margin of the tumor was in close proximity to the pelvicaliceal system. Our current laparoscopic technique involves preoperative ureteral catheterization, laparoscopic renal ultrasonography, transient atraumatic clamping of the renal artery and vein, tumor excision with an approximate 0.5 cm. margin using cold endoshears and/or J-hook electrocautery, pelvicaliceal suture repair (if necessary) and suture repair of the renal parenchymal defect over surgicel bolsters. In 1 case renal surface hypothermia was achieved laparoscopically with ice slush. All suturing and knot tying were performed with free hand intracorporeal laparoscopic techniques exclusively. RESULTS: All procedures were successfully completed without open conversion. Mean surgical time was 3.0 hours (range, 0.75 to 5.8) and mean blood loss was 270.4 cc (range 40 to 1,500). Mean warm ischemia time was 23 minutes (range, 9.8 to 40). Caliceal entry in 18 cases (36%) was suture repaired in a watertight manner. Following caliceal repair, none of these 18 patients had a postoperative urine leak. Hospital stay averaged 2.2 days (range 1 to 9). Major complications occurred in 3 patients (6%) including intraoperative hemorrhage in 1, delayed hemorrhage necessitating nephrectomy in 1 and urine leak in 1. Renal cell carcinoma was confirmed on pathological examination in 34 patients (68%), and all had negative inked surgical margins for cancer. During a mean followup of 7.2 months (range 1 to 17) no patient has had local or port site recurrence or metastatic disease. CONCLUSIONS: Laparoscopic partial nephrectomy is a viable alternative for select patients with a renal tumor. The largest single institutional experience to date is presented wherein the open techniques of nephron sparing surgery have been duplicated laparoscopically.

Vaginal extraction of the intact specimen following laparoscopic radical nephrectomy.

PURPOSE: We report the detailed technique and results of transvaginal extraction of the intact laparoscopic radical nephrectomy specimen. MATERIALS AND METHODS: Since June 2000, 10 select female patients with a median age of 67 years underwent transvaginal extraction of the intact specimen after laparoscopic radical nephrectomy. In 5 patients open surgery had previously been performed on the uterus, including transabdominal hysterectomy in 2 and cesarean section in 3. Laparoscopic nephrectomy was performed via the transperitoneal and retroperitoneal approach in 5 cases each. After completion of the primary laparoscopic procedure a sponge stick was externally inserted into the sterile prepared vagina and tautly positioned in the posterior fornix. Laparoscopically a transverse posterior colpotomy was created at the apex of the tented up posterior fornix and the drawstring of the entrapped specimen was delivered into the vagina. After laparoscopic exit was completed the patient was placed in the supine lithotomy position. The specimen was extracted intact via the vagina and the posterior colpotomy incision was repaired transvaginally. Patients were mailed a linear scale analog questionnaire to assess various aspects of recovery with responses graded from 0--no pain and/or change to 10--severe pain and/or change. RESULTS: Vaginal extraction was successful in all 10 patients. Median operative time for the vaginal extraction procedure was 35 minutes. Blood loss was minimal. Median tumor size was 3.6 cm. (range 2.4 to 7.4) and median specimen weight was 327 gm. (range 152 to 484). No intraoperative complications occurred. Postoperatively blood spotting via the vagina in 1 patient resolved spontaneously. Postoperative questionnaires revealed excellent patient satisfaction and convalescence. CONCLUSIONS: Vaginal extraction is an efficacious and minimally morbid technique for removing the intact entrapped specimen after laparoscopic radical nephrectomy. It has now become our preferred technique of intact specimen extraction in appropriate female patients.