In-laboratory breast specimen radiography reduces tissue block utilization and improves turnaround time of pathologic examination.

BACKGROUND: This study was performed to determine whether in-laboratory specimen radiography reduces turnaround time or block utilization in surgical pathology. METHODS: Specimens processed during a 48-day trial of an in-lab cabinet radiography device (Faxitron) were compared to a control group of specimens imaged in the mammography suite during a prior 1-year period, and to a second group of specimens not undergoing imaging of any type. RESULTS: Cases imaged in the mammography suite had longer turnaround time than cases not requiring imaging (by 1.15 days for core biopsies, and 1.73 days for mastectomies; p < 0.0001). In contrast, cases imaged in-lab had turnaround time that was no longer than unimaged cases (p > 0.05 for core biopsies, lumpectomies and mastectomies). Mastectomies imaged in-lab required submission of fewer blocks than controls not undergoing any imaging (mean reduction of 10.6 blocks). CONCLUSIONS: Availability of in-lab radiography resulted in clinically meaningful improvements in turnaround time and economically meaningful reductions in block utilization.

Transvaginal fast-scanning optical-resolution photoacoustic endoscopy.

Photoacoustic endoscopy offers in vivo examination of the visceral tissue using endogenous contrast, but its typical B-scan rate is ∼10 Hz, restricted by the speed of the scanning unit and the laser pulse repetition rate. Here, we present a transvaginal fast-scanning optical-resolution photoacoustic endoscope with a 250-Hz B-scan rate over a 3-mm scanning range. Using this modality, we not only illustrated the morphological differences of vasculatures among the human ectocervix, uterine body, and sublingual mucosa but also showed the longitudinal and cross-sectional differences of cervical vasculatures in pregnant women. This technology is promising for screening the visceral pathological changes associated with angiogenesis.

BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.

Thrombin allostery.

Thrombin is a Na(+)-activated, allosteric serine protease that plays multiple functional roles in blood pathophysiology. Binding of Na(+) is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme. This review summarizes our current understanding of the molecular basis of thrombin allostery with special emphasis on the kinetic aspects of Na(+) activation. The molecular mechanism of thrombin allostery is a remarkable example of long-range communication that offers a paradigm for many other biological systems.

Rapid kinetics of Na+ binding to thrombin.

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

Crystal structure of thrombin in a self-inhibited conformation.

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

Does endograft support alter the rate of aneurysm sac shrinkage after endovascular repair?

PURPOSE: To test the hypothesis that stent-graft support influences sac shrinkage independent of endoleak rates after endovascular repair of abdominal aortic aneurysms (AAA). METHODS: Ninety AAA patients underwent treatment with bifurcated endoluminal devices at our institution between October 1996 and February 1999. Fifty-two patients were treated using a nonsupported (NS) Ancure endograft and 38 using a fully supported (FS) Excluder endograft. Computed tomographic (CT) scans were obtained during the first postoperative month and at 6, 12, and 24-month intervals. Aneurysm diameter was measured as the minor axis of the largest AAA axial slice on the CT scan. Six, 12, and 24-month sac sizes were compared to the first postoperative evaluation. RESULTS: Successful endoluminal graft placement was accomplished in all patients. The two groups were matched for age, anatomical criteria, and comorbidities except for baseline AAA size: the mean diameter was 5.4 cm in the NS group and 5.0 cm for the FS group (p<0.01). Endoleak rates were 25% (13/52) in the NS group and 18% (7/38) in the FS group (p<0.05) at 1 month. All endoleaks that did not resolve spontaneously at 6 months were treated. Initial endoleak status did not affect the sac shrinkage rates at the 12 and 24-month evaluations. At 2 years, the NS group had greater shrinkage of the sac (1.2 cm) versus the FS cohort (0.3 cm, p<0.05). In addition, more patients in the NS group had sac shrinkage >or =5 mm (83% versus 18%, p<0.05). CONCLUSIONS: Despite a higher endoleak rate, the nonsupported Dacron Ancure endografts were associated with greater sac shrinkage at up to 24 months following implantation.

Maternity policy and practice during surgery residency: how we do it.

BACKGROUND: Pregnancy during general surgery residency has traditionally been discouraged. METHODS: In 2001, using an approved protocol, we anonymously surveyed 25 residents (PGY3 level or greater) concerning their experiences working with each other during episodes of resident pregnancy and maternity leave. RESULTS: From 1995 to 2001, 13 of 59 residents in general surgery were female (22%). While training, 6 of 13 residents reported 8 pregnancies with 2 miscarriages. Five residents (39%) gave birth to 6 children and adopted 1 child. Residents worked until the day of term delivery in 5 of 6 cases; 1 pregnancy was complicated by placental abruption at 33 weeks. Residents were off work postpartum for a median of 6 weeks (range 2-6). Nursing was universal for > or = 3 months but at-work problems with privacy and stress were frequent. On survey, all resident mothers believed they had been treated very fairly, and 94% of surveyed male peers stated that the coworker's status had no effect or a positive effect on their own work life. Fatherhood was reported to occur during residency by 42% of male respondents. CONCLUSIONS: Parenthood during residency is frequent. The complexities of resident maternity can be handled with mutual safety, equity, and satisfaction by the residents and faculty of a surgical training program.