Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.

Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy.

Use of graft flow measurement and computerized tomography angiography to evaluate patency of endoscopically harvested radial artery as sequential graft in coronary artery bypass surgery.

AIM: Endoscopic radial artery (RA) graft harvesting in coronary artery bypass surgery (CABG) is attractive but concern remains regarding early graft failure. We evaluated RA graft patency via intraoperative graft flow measurements and mid-term computerized tomography angiography (CTA). METHODS: The patients who had RA harvested by endoscopic technique which was used as sequential grafts were retrospectively reviewed. Graft quality was confirmed by intraoperative transit time flow measurements. Graft stenosis was defined as stenosis >70% on CTA, 6-12 months postoperatively. RESULTS: From 2007 to 2011, 58 patients underwent endoscopic RA harvesting for sequential bypassed grafts. All received total arterialized grafts, including 22 (38%) bilateral internal thoracic arteries (ITAs), with 208 total bypassed grafts (mean: 3.59±0.52) and 128 RA bypassed grafts (mean: 2.21±0.35). Off-pump technique was performed in 43 (84%) of 51 isolated CABG patients. The pulsatility index of graft flow of the left, right ITA and sequential RA grafts with 2 or 3 targets were 1.8±0.7, 2±0.8, 1.9±0.4, and 1.7±0.7, respectively. There was no hospital mortality, and median intensive care unit and hospital stay was 2 and 8 days. Follow-up was completed in 57 patients, but 3 patients refused CTA due to lack of exertional angina. Stenosis of the left, right ITA, and RA grafts occurred in 1/54 (1.9%), 1/21 (4.8%), and 11/120 (9.2%). After a mean of 35.8±10.9 (median: 30.7) months follow-up, there was no late mortality and one documented myocardial infarction was reported. Age, diabetes, previous percutaneous coronary intervention, off-pump technique, RA target number, and graft flow or pulsatility index did not predict RA stenosis. Only RA grafts targeting the circumflex territory had an adverse impact. CONCLUSION: The RA of appropriately selected patients can be harvested safely by endoscopic technique and can be used as sequential grafts for CABG with satisfactory outcomes. Intraoperative flow measurement can assure the quality of the grafts. CTA is a valuable tool for patency follow-up.

Associations between gout tophus and polymorphisms 869T/C and -509C/T in transforming growth factor beta1 gene.

OBJECTIVES: To investigate the associations between gout tophus and polymorphisms 869T/C and -509C/T in TGF-beta1 gene. METHODS: The polymorphisms 869T/C and -509C/T were determined in 73 gout patients and 114 healthy controls among male Taiwanese using the PCR-restriction fragment length polymorphism method. Each patient was matched with 1-2 controls by age within 1-2 yrs. The tophus number was measured from all the patients' arms and legs. RESULTS: Neither 869T/C nor -509C/T showed a significant association between patients and controls in the proportions of genotypes, allele frequency or dominant and recessive models. The mean number of tophi for all patients was 1.53 +/- 3.44, showing a significant difference in distribution among the genotypes at polymorphism 869T/C (P = 0.006), but not those in polymorphism -509C/T (P > 0.05). Those carrying genotype CC at polymorphism 869T/C have a mean number of tophi 0.35 (+/- 1.11), which is significantly lower than those carrying genotype TT (3.73 +/- 4.67; P < 0.05). Those with genotype TT at polymorphism 869T/C also had 11.06 times the likelihood of having at least one tophus compared with the genotype CC after adjustment of hyperuricaemia (95% CI = 1.84, 66.36; P = 0.009). However, except for the tophus number, these two polymorphisms did not show any significant association with the clinical characteristics or biochemical markers. CONCLUSIONS: The polymorphism 869T/C in TGF-beta1 gene has a significant association with the occurrence of tophus in gout patients.

Lower serum viral loads in young patients with hepatitis-B-virus-related hepatocellular carcinoma.

Advanced age and high hepatitis B virus (HBV) DNA level are risk factors associated with the development of HBV-related hepatocellular carcinoma (HCC). However, little is known about the role of viral load in the carcinogenesis of HCC in young people. A total of 183 HBV-related HCC patients and 202 HBV carriers were therefore enrolled to compare serum viral loads in young (40 years of age) age groups. Other factors associated with the development of HCC were also analysed. The results showed that serum alanine aminotransferase (38.7 +/- 24.1 vs 58.4 +/- 65.4 IU/L, P = 0.006) and HBV DNA levels (log(10) titre: 4.20 +/- 1.33 vs 4.80 +/- 1.39, P = 0.053) were lower in young HCC patients than in old HCC patients. There was a positive correlation between age and serum HBV DNA level in HCC patients but a negative correlation in HBV carriers. Young HCC patients with HBV genotype B infection had higher viral loads than those with genotype C infection (log(10) titre: 4.79 +/- 1.34 vs 3.27 +/- 0.60, P = 0.001). By multivariate logistic regression analyses, high serum HBV DNA level was associated with the development of HCC in old patients [odds ratio (OR) 1.584, 95% confidence interval (CI) 1.075-2.333] rather than in young patients (OR 0.848, 95% CI 0.645-1.116). In conclusion, viral factors in association with the development of HBV-related HCC in young patients may be different from their old counterparts. The complicated interplay between host and virus could be responsible for the emergence and aggressive outcome of early-onset HCC.

Versatile use of extra-corporeal life support to resuscitate acute respiratory distress patients.

Extra-corporeal life support (ECLS) has been applied successfully to congenital respiratory defects but less optimally to acquired pulmonary failure. We extended this support to certain extreme complexities of patients with acute respiratory distress. From January 2003 to June 2005, 16 (nine men and seven women) patients refractory to ventilator support were treated with ECLS. Their median age was 32.4 years (1.5-70). The triggering events were pulmonary haemorrhage (n = 4), pneumonia (n = 7), aspiration (n = 2) and pancreatitis (n = 3). The indications for support were hypoxaemia in 13 and hypercapnia in three patients. Ten (63%) met the criteria of fast entry. Thirteen (81%) received veno-venous (V-V) mode support and the other three received veno-arterial mode support initially, but then converted to V-V mode after sufficient oxygenation stabilised haemodynamics. Initial pump flow was maximised to improve (mean 3250 +/- 1615 ml/min) to improve the oxygenation. Four patients with active pulmonary haemorrhage were heparin free in the first 12-24 h of support without complications. Excluding one prematurely terminated patient because of brain permanent damage, the duration of support was 162 +/- 95 h (67-363). Eleven (69%) weaned successfully from ECLS and 10 (63%) discharged and regained normal pulmonary performance in a median of 26.8 months follow-up. Pulmonary support using ECLS was feasible in selected patients with acute respiratory distress. Modification of guidelines for liberal use, early deployment before secondary organ damage and prevention of complications during support were the key to final success.

Visceral artery aneurysm: risk factor analysis and therapeutic opinion.

OBJECTIVES: To identify independent risk factors for visceral artery aneurysms. METHODS: Retrospective medical record review over 10 years. RESULTS: There were 26 men and 15 women, median age of 54 (range 22 to 85), and median follow-up was 20.6 months (range 0 to 94 months). There were 11 splenic, 17 hepatic, 8 gastroduodenal, 6 pancreatoduodenal, 5 superior mesenteric, and two inferior mesenteric artery aneurysms. Thirteen patients (13/41, 31.7%) were treated surgically without adjuvant endovascular intervention. Nineteen patients (19/41, 46.3%) were treated exclusively using endovascular procedures. Five patients (5/41, 12.2%) received second endovascular or surgical treatment as salvage procedure after the first procedure failed. Concomitant malignancy was positive predictors for in-hospital death. Renal failure, chronic lung disease, liver cirrhosis, previous abdominal surgery and concomitant malignancy were positive predictors of 2-year mortality. With the intention to treat in the whole cohort, less than 10% (2/21) of the endovascular treatments failed, compared to 18.5% (3/16) in the surgical group. Patients treated by surgery without aid of endovascular intervention, have lower 2-year mortality. In hospital-death rate was 9.8%, while overall mortality rate was 21.9%. CONCLUSIONS: The endovascular intervention provides compatible, even better early postoperative outcomes for visceral artery aneurysms to surgery. Concomitant malignancy was the major determinant of visceral artery aneurysms, both in-hospital death and survival.

A multiple channel electrospray source used to detect highly reactive ketenes from a flow pyrolyzer

This work detects protonated molecular ions of highly reactive pyrolytic products--cyclopentadienylideneketene, cyclohexadienylideneketenimine, and acetylketene--using a flow pyrolyzer connected to a multiple-channel electrospray mass spectrometry. The ketene generated in the flow pyrolyzer is directly conducted into the central channel of a seven-channel electrospray ionization source by a stream of nitrogen gas. Concurrently, a methanol solution containing 0.1% trifluoroacetic acid is electrosprayed through the outside six channels. The protonated methanol ions and the charged droplets generated from the outside six electrospray channels facilitate the ionization of the neutral ketenes through ion-molecule reactions or absorption followed by protonation.

Studies of a magnetically focused electrostatic mirror. I. Experimental test of the first order properties

When a uniform magnetic field is superimposed on a uniform electrostatic field, the combination can act as a magnetically focused mirror. This mirror is predicted to have aberrations of opposite sign to those of a magnetic lens and may therefore be useful as a corrector. We have built an electron optical system to test these ideas. The results are presented in two papers. This first paper describes the general design and the results of the measurements of the first order properties. The second paper (Tsai, F., J. Microsc. 197 (2000) 118-135) will describe the measurements of the aberration properties.

Studies of a magnetically focused electrostatic mirror. II. Aberration corrections

A magnetically focused electrostatic mirror is shown to be able to correct the spherical and chromatic aberrations of a probe forming system simultaneously. The probe forming system comprises a uniform magnetic lens and a uniform electrostatic mirror. Previous theoretical investigations showed that the spherical and chromatic aberration coefficients of these two components are the same values but with opposite sign, whose combination will therefore be free from aberrations. The experimental arrangement used a solenoid to produce a uniform magnetic field, and a series of plate electrodes to produce a uniform electrostatic field. These fields are shown to satisfy the experimental requirements. By deliberately changing the extraction voltage to defocus the electron beam, the author is able to observe correction of chromatic aberration by one order of magnitude. By deliberately changing the lens field and the mirror field, the author is able to observe the reduction of the asymmetry caused by the spherical aberration, which the author believes also indicates correction by one order of magnitude.