Industry-relevant magnetron sputtering and cathodic arc ultra-high vacuum deposition system for in situ x-ray diffraction studies of thin film growth using high energy synchrotron radiation.

We present an industry-relevant, large-scale, ultra-high vacuum (UHV) magnetron sputtering and cathodic arc deposition system purposefully designed for time-resolved in situ thin film deposition/annealing studies using high-energy (>50 keV), high photon flux (>10(12) ph/s) synchrotron radiation. The high photon flux, combined with a fast-acquisition-time (<1 s) two-dimensional (2D) detector, permits time-resolved in situ structural analysis of thin film formation processes. The high-energy synchrotron-radiation based x-rays result in small scattering angles (<11°), allowing large areas of reciprocal space to be imaged with a 2D detector. The system has been designed for use on the 1-tonne, ultra-high load, high-resolution hexapod at the P07 High Energy Materials Science beamline at PETRA III at the Deutsches Elektronen-Synchrotron in Hamburg, Germany. The deposition system includes standard features of a typical UHV deposition system plus a range of special features suited for synchrotron radiation studies and industry-relevant processes. We openly encourage the materials research community to contact us for collaborative opportunities using this unique and versatile scientific instrument.

Patterns and outcomes of aortofemoral bypass grafting in the era of endovascular interventions.

OBJECTIVES: The aim of the study is to study contemporary presentation patterns and clinical results in patients undergoing aortofemoral bypass (AFB) surgery. DESIGN: This was a retrospective comparative study. MATERIAL AND METHODS: During a 14-year period, 269 consecutive patients (mean age 65 years) underwent AFB. Indications included occlusive disease with severe intermittent claudication (IC) (n = 86), critical limb ischaemia (CLI, n = 97) and aneurysmo-occlusive disease (n = 86). RESULTS: From 2000-07 on, AFB was performed more frequently for occlusive disease with CLI than for other indications (48% vs. 31% before 2000, P = 0.009) and also in women (51% vs. 32% before 2000, P = 0.003), compared to the period before 2000. Thirty-day mortality was reduced during 2000-2007 to 2.4%, compared with 4.3% during 1993-1999, although this difference was not statistically significant (P = 0.73). Morbidity did not change substantially over the study period. Predictors of 30-day mortality included indication (CLI = 4.1% vs. claudication = 1.2% (P = 0.37)) and chronic kidney disease (CKD, serum creatinine > 1.5 mg dl⁻¹) (11.1% vs. 2.9% in normal renal function, P = 0.07), the latter being the single predictor on multivariate analysis (hazard risk 4.2, P = 0.047). Overall 5 and 10-year assisted primary and secondary patency was 95% and 88%, and 99% and 95%, respectively. Survival at 5 and 10 years was 69% and 48%, respectively. Patient age (hazard risk 1.05, P < 0.001), CKD (hazard risk 1.79, P = 0.018) and diabetes (hazard risk 1.56, P = 0.022) were independent predictors of worse long-term survival. Long-term outcome did not change over the course of the study. CONCLUSIONS: In the contemporary era, AFB is more likely to be performed for CLI and in women than in the past. Despite these changes, perioperative mortality and morbidity remain low and long-term outcome excellent.

Are the communication and professionalism competencies the new critical values in a resident's global evaluation process?

BACKGROUND: The ACGME requires the assessment of resident competency in 6 domains. Global evaluations covering all 6 competencies are routinely used. Evaluators may be overly influenced by resident affability and availability, thereby resulting in a halo effect. We hypothesized that the Interpersonal Skills and Communications (ICS) and Professionalism (PR) competencies would unduly influence other competency scores. METHODS: General surgery resident evaluations are performed by staff and peers on a rotational basis using competency-based questions. Each question is scored using a 5-point Likert scale. Mean individual composite scores for each competency were calculated and then correlated with other mean composite competency scores. Data from patient evaluations were similarly analyzed. A final correlation of competency scores to ABSITE scores, as an objective, standardized measure of a specific competency, Medical knowledge (MK) was also performed. RESULTS: Results were available for 37 residents (PGY 1-5). There was a significant association between ICS scores and higher scores in MK (r = 0.52, p = 0.004), PR (r = 0.826, p < 0.0001) and patient care (PC) (r = 0.619, p < 0.0001). No correlation, however, was found between patient evaluations of residents and their faculty/peer-based ICS scores. We found no association between ICS scores and improved patient evaluations. Lastly, we found no association between ICS or MK scores and ABSITE scores. CONCLUSIONS: It was difficult to ascertain whether residents with better ICS scores had higher PR, PC, and MK scores because of the halo effect, improper completion of evaluations, or whether those residents were truly performing better clinically. External measures of resident performance did not correlate with faculty/peer evaluations of ICS and PR. Residency programs should consider adopting a more standardized way to objectively evaluate residents.

Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy.

The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.

Changes in orientation of actin during contraction of muscle.

It is well documented that muscle contraction results from cyclic rotations of actin-bound myosin cross-bridges. The role of actin is hypothesized to be limited to accelerating phosphate release from myosin and to serving as a rigid substrate for cross-bridge rotations. To test this hypothesis, we have measured actin rotations during contraction of a skeletal muscle. Actin filaments of rabbit psoas fiber were labeled with rhodamine-phalloidin. Muscle contraction was induced by a pulse of ATP photogenerated from caged precursor. ATP induced a single turnover of cross-bridges. The rotations were measured by anisotropy of fluorescence originating from a small volume defined by a narrow aperture of a confocal microscope. The anisotropy of phalloidin-actin changed rapidly at first and was followed by a slow relaxation to a steady-state value. The kinetics of orientation changes of actin and myosin were the same. Extracting myosin abolished anisotropy changes. To test whether the rotation of actin was imposed by cross-bridges or whether it reflected hydrolytic activity of actin itself, we labeled actin with fluorescent ADP. The time-course of anisotropy change of fluorescent nucleotide was similar to that of phalloidin-actin. These results suggest that orientation changes of actin are caused by dissociation and rebinding of myosin cross-bridges, and that during contraction, nucleotide does not dissociate from actin.

Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber.

The conventional hypothesis of muscle contraction postulates that the interaction between actin and myosin involves tight coupling between the power stroke and hydrolysis of ATP. However, some in vitro experiments suggested that hydrolysis of a single molecule of ATP caused multiple mechanical cycles. To test whether the tight coupling is present in contracting muscle, we simultaneously followed mechanical and enzymatic events in a small population of cross-bridges of glycerinated rabbit psoas fibers. Such small population behaves as a single cross-bridge when muscle contraction is initiated by a sudden release of caged ATP. Mechanical events were measured by changes of orientation of probes bound to the regulatory domain of myosin. Enzymatic events were simultaneously measured from the same cross-bridge population by the release of fluorescent ADP from the active site. If the conventional view were true, ADP desorption would occur simultaneously with dissociation of cross-bridges from thin filaments and would be followed by cross-bridge rebinding to thin filaments. Such sequence of events was indeed observed in contracting muscle fibers, suggesting that mechanical and enzymatic events are tightly coupled in vivo.

Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.

The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of rotation was synchronized by a sudden release of caged ATP. The orientation of cross-bridges was measured by anisotropy of recombinant fluorescent regulatory light chains exchanged with native regulatory light chains. The orientation of actin was measured by anisotropy of phalloidin added to actin filaments. The enzymatic activity of myosin was measured by dissociation of fluorescent ADP from the active site. The onset of all three events occurred at the same time. This suggests that in contracting muscle, actin does not move independently of myosin and that ATP hydrolysis is strongly coupled to the rotation of cross-bridges.

Hypothenar hammer syndrome: an uncommon and correctable cause of digital ischemia.

Hypothenar hammer syndrome (HHS) is the rare entity of finger ischemia secondary to embolic occlusion of the digital arteries as a result of repetitive trauma to the palmar ulnar artery. We report the case of a young man found to have digital embolic complications from an ulnar artery aneurysm. This is thought to have developed as a result of palmar trauma experienced during military rifle drill exercises.

Factors affecting outcome in proximal abdominal aortic aneurysm repair.

Sixty-five consecutive patients undergoing nonemergent repair of an abdominal aortic aneurysm (AAA) originating above the visceral and/or renal arteries were studied to determine operative results and identify factors influencing outcome of proximal AAA repair. Factors associated with postoperative morbidity were analyzed using multivariate analysis. There were no postoperative deaths, paraplegia/paraparesis, or symptomatic visceral ischemia. Proximal AAA repair can be accomplished with acceptable mortality. If renal artery bypass or reimplantation is anticipated, cold renal perfusion may protect against renal dysfunction. Postoperative pulmonary dysfunction can be reduced by avoiding radial division of the diaphragm.

Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format.

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.

The 5' flanking region of the human bone morphogenetic protein-7 gene.

We describe here the cloning and characterization of the 5' flanking region of the human Bone Morphogenetic Protein-7 (BMP-7) gene from a 3.3 kb genomic DNA fragment. Functional analysis by transient transfection using the luciferase reporter gene indicated that this region had a low basal promoter activity in human Wilm's tumor derived renal (G401) and rat osteoblast (ROS 17/2.8) cell lines. Sequential deletion analysis of the promoter revealed sequences whose presence correlated with decreased expression of the reporter gene. Coexpression of transcription factors involved in epithelial/mesenchymal interactions during kidney and eye development dramatically stimulated the expression of the reporter gene from the putative BMP-7 promoter. Finally, a subset of agents that upregulated the expression of the reporter gene from the cloned promoter were also shown to increase the expression of the endogenous BMP-7 in G401 and ROS cell lines in vitro.

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.

Visceral pseudoaneurysms due to pancreatic pseudocysts: rare but lethal complications of pancreatitis.

OBJECTIVE: Erosion of pancreatic pseudocysts into adjacent vessels is a rare but highly lethal cause of intra-abdominal hemorrhage. Percutaneous angiographic embolization (PAE) of the bleeding artery has recently been advocated as the preferred therapy. This study was undertaken to survey the outcome after treatment of this complication and to make recommendations for its management. METHODS: An 11-year retrospective analysis was performed of all patients treated at a large tertiary care referral center for visceral artery pseudoaneurysms associated with pancreatic pseudocysts. RESULTS: From 1988 to 1998, 256 patients were admitted for complications of pancreatic pseudocysts. Sixteen patients (11 men and 5 women) were identified in whom a pseudocyst had eroded into a major blood vessel with hemorrhage or development of a false aneurysm. The mean age was 45 years (range, 23-67 years). Active bleeding was present in 13 patients, whereas three had evidence of recent hemorrhage. Ten of 16 patients initially underwent operative therapy, four elective and six emergency, whereas six stable patients were initially treated with PAE. Technical failures of the initial treatment or secondary complications required both therapeutic modalities in six patients, which resulted in 13 total surgical interventions and 10 PAEs. The surgical morbidity rate was 62% (8 of 13), whereas that of PAE was 50% (5 of 10). Three deaths occurred after emergency operations, two of which failed to stop the bleeding, accounting for all of the deaths in the series (3 [19%] of 16). A trend was noted toward increased death with necrotizing pancreatitis (P =.07) and emergency surgery (P =.06). Ranson's criteria were not found to be predictive of death in this series. Surgical drainage procedures were required in seven (44%) of 16 patients for infections (n = 3) or mass effect of the pseudoaneurysm (n = 3). The mean size of pseudoaneurysms that required operative intervention for secondary complications was 13.9 cm, compared with 7.7 cm for all others in the series (P =.046). Long-term follow-up was available in all 13 survivors at a mean of 44 months (range, 1-108 months). CONCLUSIONS: The management of pancreatic pseudocyst-associated pseudoaneurysms remains a challenging problem with high morbidity and death rates. Operation and PAE play complementary management roles. PAE is recommended as the initial therapy for hemodynamically stable patients. Surgery should be reserved for actively bleeding, hemodynamically unstable patients; for failed embolization; and for other secondary complications such as infection or extrinsic compression.

Phosphorus 31 nuclear magnetic resonance spectroscopy suggests a mitochondrial defect in claudicating skeletal muscle.

OBJECTIVE: Decreased oxygen supply is generally accepted as the primary cause of muscle dysfunction in patients with peripheral arterial occlusive disease (PAOD) and intermittent claudication, although reported morphologic changes in the mitochondria of claudicating muscle suggest that impaired energy utilization may also play a role. With the measurement of the phosphate-rich compounds of muscle energy metabolism (adenosinetriphosphate [ATP], adenosinediphosphate [ADP], and phosphocreatine [PCr]) and pH, phosphorus P 31 magnetic resonance spectroscopy ((31)P MRS) provides a unique, noninvasive method to investigate this hypothesis further. METHODS: Calf muscle bioenergetics were studied in 12 men with moderate claudication (ankle-brachial index >/=0.5 and .5, Pearson moment correlation). CONCLUSIONS: Phosphorus 31 MRS provides the first direct evidence of defective energy metabolism in the mitochondria of claudicating calf muscle. This defect appears to be independent of both arterial flow and the severity of occlusive disease in patients with mild to moderate claudication. Coupled with documented ultrastructural and DNA abnormalities in the mitochondria of claudicating skeletal muscle, these data provide evidence for a secondary cause of muscle dysfunction in intermittent claudication.

31P MRS studies of exercising human muscle at high temporal resolution.

Methods for measuring mitochondrial activity from 31P magnetic resonance spectroscopy data collected during and after exercise were compared in controls, weight lifters, and peripheral vascular occlusive disease (PVOD) patients. There were trends toward increasing mitochondrial activity during exercise in order from PVOD patients, moderately active controls, highly active controls, to weight lifters. Results from PVOD patients show divergence of some measures due to 1) the non-exponential nature of phosphocreatine recovery, and 2) potential breakdown of [ADP] control of the mitochondria due to lack of oxygen (for Qmax calculation). These results demonstrate the utility of obtaining and directly analyzing high time resolution data rather than assuming monoexponential behavior of metabolite recovery.

Inwardly rectifying potassium channels in lens epithelium are from the IRK1 (Kir 2.1) family.

Lens epithelial cells from many species contain inwardly rectifying K+ channels. The channels are highly selective for K+ over Na+. They have a conductance of 27-30 pS in symmetrical 150 mM K+ solutions. The conductance to inwardly flowing current depends on the external [K+], being 1/2 maximal at about 50 mM and maximal by 110-150 mM. The amino acid sequences from lens epithelium (eight different species) show at least 98% sequence homology to each other and to the potassium channel known as IRK1 (Kir 2.1). Cloned channels from chick, rabbit, and human lens epithelium all make functional channels when their cDNA is transfected into HEK-293 or tsA-201 cells. Human lens inward rectifiers when engineered as fusion proteins with green fluorescent protein (GFP) also make functional channels. In addition, their localization in the membrane and in intracellular organelles can be demonstrated by fluorescence microscopy.

Noninvasive venous testing in the diagnosis of pulmonary embolism: the impact on decisionmaking.

PURPOSE: To characterize the use and utility of lower extremity noninvasive venous testing (NIVT) in the diagnosis of pulmonary embolism (PE). METHODS: The study is a retrospective case series of consecutive patients in whom PE was suspected who were referred to a large, urban tertiary care center for NIVT. The main outcome measures of the study were the rate of positive results of NIVT, the amount of new information provided by NIVT, and the frequency of management changes that were attributable to NIVT. RESULTS: Forty-one of 450 patients (9%) had deep venous thrombosis (DVT) by NIVT. The prevalence of DVT by NIVT among patients not evaluated by ventilation/perfusion (V/Q) scanning was 8%. The prevalence of DVT by NIVT among patients with a high-probability V/Q scan result before NIVT was 39%, but no management decisions in this group were based on a positive NIVT result and only two decisions were based on negative NIVT results. The prevalence of DVT according to NIVT among patients who had a negative "diagnostic" (low, or very low probability, or normal) result of V/Q scan before NIVT was 2%. The overall frequency of management changes attributed to NIVT was only 2.5%. In the remaining 97% of patients, management was determined by the result of V/Q scanning or of subsequent pulmonary arteriography. CONCLUSIONS: In patients in whom PE is suspected, results of NIVT are usually negative for acute DVT. Management decisions are almost always based on V/Q scan or results of pulmonary arteriography and not on NIVT. The utility of NIVT to identify DVT in these patients appears limited, and a more selective approach to its application for the diagnosis of PE should be considered.

Surgical complications of transaxillary arteriography: a case-control study.

PURPOSE: The purpose of this study was to review the complications of transaxillary arteriography (TRAX), determine clinical factors associated with their occurrence, and define optimal treatment methods. METHODS: A retrospective review of 842 consecutive TRAX studies performed in a large, urban, tertiary care, academic medical center was undertaken. Patients with complications were compared with a concurrent randomized control group without complications with the use of a multivariate analysis model. Results of operative therapy for nerve injury were compared with those of nonoperative therapy. RESULTS: Nineteen (2.3%) complications were identified including 14 nerve injuries, four expanding hematomas/pseudoaneurysms without neurologic deficit, and one puncture site thrombosis. Several statistically significant or suggestive findings associated with the occurrence of complications were identified: female sex (odds ratio [OR] = 4.7), systolic blood pressure > or = 150 mm Hg at the conclusion of TRAX (OR = 9.5), periprocedural systemic heparin anticoagulation (OR = 7.9), concomitant use of intraarterial thrombolysis or percutaneous angioplasty (OR = 12.0), and duration of procedure > or = 90 minutes (OR = 4.0). Patients who underwent prompt exploration (< or = 4 hours from symptom onset) for nerve injuries were more likely to have complete resolution of their neurologic deficits (five of six patients) than those who were observed or underwent delayed operation (three of eight patients) (OR = 8.3). CONCLUSIONS: Aggressive treatment of post-TRAX hypertension, limitation of TRAX duration, delay of postprocedure anticoagulation, and use of alternative sites for arterial puncture in female patients or patients undergoing catheter-based intervention may reduce the incidence of TRAX-related complications. In patients who have neurologic deficits prompt surgical exploration of the puncture site with decompression of the involved nerve(s) may reduce the incidence of prolonged deficits.

An initiator element is required for maximal human chorionic somatomammotropin gene promoter and enhancer function.

Previous studies have indicated that cell-specific expression of the human chorionic somatomammotropin (hCS) gene may be mediated by a placental-specific enhancer (CSEn). In the current studies, we have analyzed the promoter elements that are required for enhancer and promoter function in choriocarcinoma cells (BeWo). Mutation of both hCS GHF1 sites had no effect on promoter or enhancer activity. In contrast, mutation of the Sp1 site diminished basal and CSEn-stimulated transcription by approximately 75% and approximately 56%, respectively, indicating that Sp1 was necessary but not sufficient for maximal basal and enhancer-mediated transcription. Deletion and site-specific mutation of the proximal promoter region indicated that the TATA box and an initiator site (InrE) located between nucleotides -15/+1 of the hCS promoter were required for maximal promoter and enhancer function. Mutations of the InrE were associated with reduced basal and enhancer-stimulated activities and altered transcription initiation sites. A protein of 70-kDa mass, that was preferentially expressed in human choriocarcinoma cells (BeWo and JEG-3), bound specifically to the InrE. The data suggest that an initiator present in high concentrations in placental cells contributes to the control of cell-specific hCS gene expression at the promoter level and is required for maximal enhancer function.