The betabellin target structure consists of <em>2</em> 3<em>2</em>-residue beta sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14<em>D</em>, the disulfide-bridged double chain. The 3<em>2</em>-residue nongenetic betabellin-14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = <em>D</em>-Ala, k = <em>D</em>-Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and beta-turn (t,r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14-residue palindromic pattern (underlined). Pairs of <em>D</em>-amino acid residues are used to favor formation of inverse-common (type-I') beta turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide-linked betabellin 14<em>D</em> is folded into a stable beta-sheet structure. Thus, folding of the covalent <em>dimer</em> beta-bellin 14<em>D</em> is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its beta-sheets is not sufficient to induce folding. Betabellin 14<em>D</em> is a very water-soluble (10 mg/mL), small (64 residues), nongenetic (1<em>2</em> <em>D</em> residues) beta-sheet protein with properties (well-dispersed proton NMR resonances; Tm = 58 degrees C and <em>delta</em> Hm = 106 kcal/mol at pH 5.5) like those of a native protein structure.

Maize (Zea mays L.) cell cultures incorporate<em>d</em> ra<em>d</em>ioactivity from [14C]cinnamate into hy<em>d</em>roxycinnamoyl-CoA <em>d</em>erivatives an<em>d</em> then into polysacchari<em>d</em>e-boun<em>d</em> feruloyl resi<em>d</em>ues. Within 5-<em>2</em>0 min, the CoA pool ha<em>d</em> lost its 14C by turnover an<em>d</em> little or no further incorporation into polysacchari<em>d</em>es then occurre<em>d</em>. The system was thus effectively a pulse-chase experiment. Kinetics of ra<em>d</em>iolabelling of <em>d</em>iferulates (also known as <em>d</em>ehy<em>d</em>ro<em>d</em>iferulates) varie<em>d</em> with culture age. In young (1-3 <em>d</em>) cultures, polysacchari<em>d</em>e-boun<em>d</em> [14C]feruloyl- an<em>d</em> [14C]<em>d</em>iferuloyl resi<em>d</em>ues were both <em>d</em>etectable within 1 min of [14C]cinnamate fee<em>d</em>ing. Thus, feruloyl resi<em>d</em>ues were <em>d</em>imerise<em>d</em> < 1 min after their attachment to polysacchari<em>d</em>es. For at least the first <em>2</em>.3 h after [14C]cinnamate fee<em>d</em>ing, polysacchari<em>d</em>e-boun<em>d</em> [14C]<em>d</em>iferuloyl resi<em>d</em>ues remaine<em>d</em> almost constant at approximately 7% of the total polysacchari<em>d</em>e-boun<em>d</em> [14C]ferulate <em>d</em>erivatives. Since feruloyl resi<em>d</em>ues are attache<em>d</em> to polysacchari<em>d</em>es < 1 min after the biosynthesis of the latter, an<em>d</em>>> 10 min before secretion, the <em>d</em>ata show that extensive feruloyl coupling occurre<em>d</em> intra-protoplasmically. Exogenous H<em>2</em>O<em>2</em> (1 mM) cause<em>d</em> little a<em>d</em><em>d</em>itional feruloyl coupling; therefore, wall-localise<em>d</em> coupling may have been peroxi<em>d</em>ase-limite<em>d</em>. In ol<em>d</em>er (e.g. 4 <em>d</em>) cultures, less intraprotoplasmic coupling occurre<em>d</em>: <em>d</em>uring the first <em>2</em>.5 h, polysacchari<em>d</em>e-boun<em>d</em> [14C]<em>d</em>iferuloyl resi<em>d</em>ues were a stea<em>d</em>y 1.4% of the total polysacchari<em>d</em>e-boun<em>d</em> [14C]ferulate <em>d</em>erivatives. In contrast to the situation in younger cultures, exogenous H<em>2</em>O<em>2</em> in<em>d</em>uce<em>d</em> a rapi<em>d</em> 4- to 6-fol<em>d</em> increase in all coupling pro<em>d</em>ucts, in<em>d</em>icating that coupling in the walls was H<em>2</em>O<em>2</em>-limite<em>d</em>. In both <em>2</em>- an<em>d</em> 4-<em>d</em>-ol<em>d</em> cultures, polysacchari<em>d</em>e-boun<em>d</em> 14C-trimers an<em>d</em> larger coupling pro<em>d</em>ucts excee<em>d</em>e<em>d</em> [14C]<em>d</em>iferulates 3- to 4-fol<em>d</em>, but followe<em>d</em> similar kinetics. Thus, although all known <em>dimers</em> of ferulate can now be in<em>d</em>ivi<em>d</em>ually quantifie<em>d</em>, it appears to be trimers an<em>d</em> larger pro<em>d</em>ucts that make the major contribution to cross-linking of wall polysacchari<em>d</em>es in culture<em>d</em> maize cells. We argue that feruloyl arabinoxylans that are cross-linke<em>d</em> before an<em>d</em> after secretion are likely to loosen an<em>d</em> tighten the cell wall, respectively. The consequences for the control of cell expansion an<em>d</em> for the response of cell walls to an oxi<em>d</em>ative burst are <em>d</em>iscusse<em>d</em>.

BACKGROUND

Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests.

METHODS

The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from <em>2</em>0 healthy volunteers taken before and <em>2</em> - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories.

RESULTS

RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected.

CONCLUSIONS

RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

OBJECTIVE

Despite a rapid spread of the technique, very little is known about the laser-tissue interaction in endovenous laser treatment (EVLT). We evaluated EVLT of the incompetent greater saphenous vein (GSV) for efficacy, treatment-related adverse effects, and putative mechanisms of action.

METHODS

Twenty-six patients with 31 limbs of clinical stages C(<em>2</em>-6), E(P), A(S,P), P(R) with incompetent GSV proven by means of duplex scanning were selected for EVLT in an outpatient setting. A 600-microm fiber was entered into the GSV via an 18-gauge needle below the knee and proceeded to the saphenofemoral junction (SFJ). After infiltration of tumescent local anesthesia, multiple laser pulses of 15 J energy and a wavelength of 940 nm were administered along the vein in a standardized fashion. <em>D</em>-<em>dimers</em> were determined in peripheral blood samples 30 minutes after completion of EVLT in 16 patients and on postoperative day 1 in <em>2</em>0 patients. One GSV that was surgically removed after EVLT was examined by means of histopathology. Additionally, an experimental in vitro set-up was constructed as a means of investigating the mechanism of laser action within a blood-filled tube.

RESULTS

A median of 80 laser pulses (range, <em>2</em><em>2</em>-116 laser pulses) were applied along the treated veins. On days 1, 7, and <em>2</em>8, all limbs except one (97%) showed a thrombotically occluded GSV. In one patient, the vessel showed incomplete occlusion. The distance of the proximal end of the thrombus to the SFJ was a median 1.1 cm (range, 0.<em>2</em>-5.9 cm) in the remaining patients. Adverse effects in all <em>2</em>6 patients were ecchymoses and palpable induration along the thrombotically occluded GSV that lasted for <em>2</em> to 3 weeks. In two limbs (6%), thrombophlebitis of a varicose tributary required oral treatment with diclofenac. <em>D</em>-<em>dimers</em> in peripheral blood were tested with normal results in 14 of 16 patients 30 minutes after completion of the procedure and elevated results in 7 of <em>2</em>0 patients at day 1 after EVLT. However, an increase of <em>D</em>-<em>dimers</em> from day 0 to day 1 was observed in 15 of the 16 patients undergoing tests 30 minutes after EVLT and on day 1. The 940-nm laser was demonstrated by means of in vitro experiments and the histopathological examination of one explanted GSV to act by means of indirect heat damage of the inner vein wall.

CONCLUSIONS

EVLT of the GSV with a 940-nm diode laser is effective in inducing thrombotic vessel occlusion and is associated with only minor adverse effects. Laser-induced indirect local heat injury of the inner vein wall by steam bubbles originating from boiling blood is proposed as the pathophysiological mechanism of action of EVLT.

Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate C(<em>2</em>0) geranylgeranyl pyrophosphate, which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether-linked lipid. For short-chain trans-prenyltransferases that synthesize C(10)-C(<em>2</em>5) products, bulky amino acid residues generally occupy the fourth or fifth position upstream from the first <em>D</em><em>D</em>XX<em>D</em> motif to block further elongation of the final products. However, the short-chain type-III GGPPs in eukaryotes lack any large amino acid at these positions. In this study, the first structure of type-III GGPPs from Saccharomyces cerevisiae has been determined to 1.98 A resolution. The structure is composed entirely of 15 alpha-helices joined by connecting loops and is arranged with alpha-helices around a large central cavity. <em>D</em>istinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight <em>dimer</em> formation. <em>D</em>eletion of the first 9 or 17 amino acids caused the dissociation of <em>dimer</em> into monomer, and the <em>Delta</em>(1-17) mutant showed abolished enzyme activity. In each subunit, an elongated hydrophobic crevice surrounded by <em>D</em>, F, G, H, and I alpha-helices contains two <em>D</em><em>D</em>XX<em>D</em> motifs at the top for substrate binding with one Mg(<em>2</em>+) coordinated by Asp(75), Asp(79), and four water molecules. It is sealed at the bottom with three large residues of Tyr(107), Phe(108), and His(139). Compared with the major product C(30) synthesized by mutant H139A, the products generated by mutant Y107A and F108A are predominantly C(40) and C(30), respectively, suggesting the most important role of Tyr(107) in determining the product chain length.

<AbstractText>The emerging infection of the <em>2</em>019 novel coronavirus (<em>2</em>019-nCoV) in late <em>D</em>ecember, <em>2</em>019 in Wuhan, China, has caused an extreme health concern, with many patients having progressed to acute respiratory disease or other complications in a short period. Meanwhile, the risk factors associated with the disease progression still remain elusive.</AbstractText><AbstractText>A cohort of 17 patients with laboratory-confirmed <em>2</em>019-nCoV infections who were admitted to the Ninth Hospital of Nanchang between January <em>2</em>8 and February 6, <em>2</em>0<em>2</em>0, were enrolled in this study. All the patients received standardized treatment. The disease progression was evaluated every 7 days after admission. The clinical, radiologic, and laboratory characteristics were retrospectively analyzed, and the factors associated with the disease progression were screened by binary logistic regression analysis.</AbstractText><AbstractText>The cohort comprised 11 women (64.7%) and 6 men (35.3%) between the ages of 18 to 70 years old. All patients had a reported history of contact with infection-confirmed patients. Fever (11/64.7%) and cough (8/47.1%) were the most common symptoms, whereas dyspnea (<em>2</em>/11.8%) and fatigue (3/17.6%) were rare, and there was no patient with diarrhea symptoms. There were 5 patients with aggravated disease at the first disease progression evaluation, and no patient received mechanical ventilation, transferred to the intensive care unit (ICU), or progressed to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, coagulation dysfunction, or death. Based on the disease progression, patients were divided into the non-aggravation group (1<em>2</em> cases) and the aggravation group (5 cases). There were no significant differences between the <em>2</em> groups with respect to their clinical characteristics. Chest computed tomography (CT) on admission revealed there were 8 patients (47.1%) with invasive lesions found bilaterally on the lungs on multiple lobes, 4 patients (<em>2</em>3.5%) with invasive lesions on 1 lobe, and 5 patients (<em>2</em>9.4%) with normal chest CT. The aggravation group had1 patient (<em>2</em>0.0%) with invasive lesions on one lobe, 3 (60.0%) with invasive lesions on multiple lobes, bilaterally, and 1 (<em>2</em>0.0%) with normal chest CT; meanwhile, the nonaggravation group had 3 patients (<em>2</em>5.0%) with invasive lesions on one lobe, 5 (41.7%) with invasive lesions on multiple lobes, bilaterally, and 4 (33.3%) with normal chest CT. No significant difference was found between the <em>2</em> groups. In the aggravation group, the total lymphocyte counts significantly decreased in comparison to that in the non-aggravation group. Further analysis showed that the C<em>D</em>4+ T cell count but not the C<em>D</em>8+ T cell count of the aggravation group was significantly lower than that of the non-aggravation group. Correlation analysis indicated total lymphocyte count was positively correlated with C<em>D</em>4+ T cell count, and no significant differences were found between the <em>2</em> groups in other laboratory measurements, including those of white blood cell (WBC) count, C-reactive protein (CRP), albumin, lactate dehydrogenase (L<em>D</em>H), and <em>D</em>-<em>dimer</em>. Finally, a binary logistic regression model was used to identify the factors associated with the disease progression. It was found that total lymphocyte count was a risk factor associated with disease progression in patients infected with <em>2</em>019-nCoV.</AbstractText><AbstractText>A higher cell count of total lymphocytes may indicate a better outcome of the disease, and immune response may be a vital factor for directing disease progression in the early stage of <em>2</em>019-nCoV infection.</AbstractText>

Patients who are hospitalized for medical illness remain at risk for venous thromboembolism after discharge, but the role of extended thromboprophylaxis in the treatment of such patients is a subject of controversy.

In this ran<em>d</em>omize<em>d</em>, <em>d</em>ouble-blin<em>d</em> trial, me<em>d</em>ically ill patients who were at increase<em>d</em> risk for venous thromboembolism on the basis of a mo<em>d</em>ifie<em>d</em> International Me<em>d</em>ical Prevention Registry on Venous Thromboembolism (IMPROVE) score of 4 or higher (scores range from 0 to 10, with higher scores in<em>d</em>icating a higher risk of venous thromboembolism) or a score of <em>2</em> or 3 plus a plasma <em>d</em>-<em>dimer</em> level of more than twice the upper limit of the normal range (<em>d</em>efine<em>d</em> accor<em>d</em>ing to local laboratory criteria) were assigne<em>d</em> at hospital <em>d</em>ischarge to either once-<em>d</em>aily rivaroxaban at a <em>d</em>ose of 10 mg (with the <em>d</em>ose a<em>d</em>juste<em>d</em> for renal insufficiency) or placebo for 45 <em>d</em>ays. The primary efficacy outcome was a composite of symptomatic venous thromboembolism or <em>d</em>eath <em>d</em>ue to venous thromboembolism. The principal safety outcome was major blee<em>d</em>ing.

Of the 1<em>2</em>,0<em>2</em>4 patients who un<em>d</em>erwent ran<em>d</em>omization, 1<em>2</em>,019 were inclu<em>d</em>e<em>d</em> in the intention-to-treat analysis. The primary efficacy outcome occurre<em>d</em> in 50 of 6007 patients (0.83%) who were given rivaroxaban an<em>d</em> in 66 of 601<em>2</em> patients (1.10%) who were given placebo (hazar<em>d</em> ratio, 0.76; 95% confi<em>d</em>ence interval [CI], 0.5<em>2</em> to 1.09; P=0.14). The prespecifie<em>d</em> secon<em>d</em>ary outcome of symptomatic nonfatal venous thromboembolism occurre<em>d</em> in 0.18% of patients in the rivaroxaban group an<em>d</em> 0.4<em>2</em>% of patients in the placebo group (hazar<em>d</em> ratio, 0.44; 95% CI, 0.<em>2</em><em>2</em> to 0.89). Major blee<em>d</em>ing occurre<em>d</em> in 17 of 598<em>2</em> patients (0.<em>2</em>8%) in the rivaroxaban group an<em>d</em> in 9 of 5980 patients (0.15%) in the placebo group (hazar<em>d</em> ratio, 1.88; 95% CI, 0.84 to 4.<em>2</em>3).

Rivaroxaban, given to me<em>d</em>ical patients for 45 <em>d</em>ays after hospital <em>d</em>ischarge, was not associate<em>d</em> with a significantly lower risk of symptomatic venous thromboembolism an<em>d</em> <em>d</em>eath <em>d</em>ue to venous thromboembolism than placebo. The inci<em>d</em>ence of major blee<em>d</em>ing was low. (Fun<em>d</em>e<em>d</em> by Janssen Research an<em>d</em> Development; MARINER ClinicalTrials.gov number, NCT0<em>2</em>111564 .).

The aim of this study is to search the most powerful prognostic factor from routine blood test for esophageal squamous cell cancer (ESCC) patients. Multiple laboratory tests were evaluated including those reflecting red blood cell parameters (hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (R<em>D</em>W)), platelet morphological parameters (mean platelet volume (MPV) and platelet count (PLT)), blood coagulation status (<em>D</em>-<em>dimer</em>), and tumor biomarker (CA19-9). Known inflammatory indices (NLR and PLR) were also calculated. A total of 468 patients who were diagnosed with ESCC between <em>D</em>ecember <em>2</em>005 and <em>D</em>ecember <em>2</em>008 were retrospectively analyzed in this study. By utilizing univariate and multivariate Cox proportional hazard analyses, we found that PLT and MPV were significantly associated with overall survival (OS) and disease-free survival (<em>D</em>FS) of ESCC patients, with optimal cutoff values of <em>2</em>1<em>2</em> and 10.6, respectively. Moreover, the combination of the preoperative PLT and MPV (COP-MPV) was calculated as follows: patients with both PLT (≥<em>2</em>1<em>2</em> × 10(9) L(-1)) and MPV (≥10.6 fL) elevation were assigned a score of <em>2</em>, and patients with one or neither were assigned a score of 1 and 0. The COP-MPV was an independent prognostic factor for OS (hazard ratio (HR) 0.378, 95 % confidence interval (CI) 0.<em>2</em>41 to 0.593, P < 0.001, 0/<em>2</em>) and <em>D</em>FS (HR 0.341, 95 % CI 0.<em>2</em>18 to 0.534, P < 0.001, 0/<em>2</em>) in multivariate analyses. In subgroup analyses for early (stages I and II) and locally (stage III) advanced stage patients, COP-MPV was found significantly associated with OS and <em>D</em>FS in each group (P = 0.0<em>2</em>5 and P = 0.018 for OS and P = 0.0<em>2</em>9 and P = 0.00<em>2</em> for <em>D</em>FS). In conclusion, we considered that COP-MPV is a promising predictor for postoperative survival in ESCC patients.

<em>D</em>evelopment and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. <em>2</em>0 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(<em>2</em>)-antiplasmin complexes (PAPs) and <em>D</em>-<em>dimers</em> release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and <em>D</em>-<em>dimer</em> plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and <em>D</em>-<em>dimers</em>. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and <em>D</em>-<em>dimers</em> found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.

Xeroderma pigmentosum factor <em>D</em> (XP<em>D</em>) is a 5'-3' superfamily <em>2</em> helicase and the founding member of a family of <em>D</em>NA helicases with iron-sulphur cluster domains. As a component of transcription factor II H (TFIIH), XP<em>D</em> is involved in <em>D</em>NA unwinding during nucleotide excision repair (NER). Archaeal XP<em>D</em> is closely related in sequence to the eukaryal enzyme and the crystal structure of the archaeal enzyme has provided a molecular understanding of mutations causing xeroderma pigmentosum and trichothiodystrophy in humans. Consistent with a role in NER, we show that archaeal XP<em>D</em> can initiate unwinding from a <em>D</em>NA bubble structure, differentiating it from the related helicases FancJ and <em>D</em>inG. XP<em>D</em> was not stalled by substrates containing extrahelical fluorescein adducts, abasic sites nor a cyclobutane pyrimidine <em>dimer</em>, regardless of whether these modifications were placed on either the displaced or translocated strands. This suggests that <em>D</em>NA lesions repaired by NER may not present a barrier to XP<em>D</em> translocation in vivo, in contrast to some predictions. Preferential binding of a fluorescein-adducted oligonucleotide was observed, and XP<em>D</em> helicase activity was readily inhibited by both single- and double-stranded <em>D</em>NA binding proteins. These observations have several implications for the current understanding of the NER pathway.

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of <em>dimers</em> (around 75%) contains A(<em>2</em>)pm(3) → A(<em>2</em>)pm(3) (A(<em>2</em>)pm, <em>2</em>,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(<em>2</em>)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of <em>D</em>-Ala(4). Two L,<em>D</em>-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd<em>2</em>), were inactivated. The inactivation of either ldt(cd1) or ldt(cd<em>2</em>) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd<em>2</em>)-encoded proteins have a redundant L,<em>D</em>-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,<em>D</em>-transpeptidation pathway in C. difficile.

BACKGROUND

The revised Geneva score is a fully standardized clinical decision rule (CDR) in the diagnostic workup of patients with suspected pulmonary embolism (PE). The variables of the decision rule have different weights, which could lead to miscalculations in an acute setting. We have validated a simplified version of the revised Geneva score.

METHODS

<em>D</em>ata from 1049 patients from <em>2</em> large prospective diagnostic trials that included patients with suspected PE were used and combined to validate the simplified revised Geneva score. We constructed the simplified C<em>D</em>R by attributing 1 point to each item of the original C<em>D</em>R and compared the diagnostic accuracy of the <em>2</em> versions by a receiver operating characteristic curve analysis. We also assessed the clinical utility of the simplified C<em>D</em>R by evaluating the safety of ruling out PE on the basis of the combination of either a low-intermediate clinical probability (using a 3-level scheme) or a "PE unlikely" assessment (using a dichotomized rule) with a normal result on a highly sensitive <em>D</em>-<em>dimer</em> test.

RESULTS

The complete study population had an overall prevalence of venous thromboembolism of <em>2</em>3%. The diagnostic accuracy between the <em>2</em> C<em>D</em>Rs did not differ (area under the curve for the revised Geneva score was 0.75 [95% confidence interval, 0.71-0.78] vs 0.74 [0.70-0.77] for the simplified revised Geneva score). <em>D</em>uring 3 months of follow-up, no patient with a combination of either a low (0%; 95% confidence interval, 0.0%-1.7%) or intermediate (0%; 0.0%-<em>2</em>.8%) clinical probability, or a "PE unlikely" assessment (0%; 0.0%-1.<em>2</em>%) with the simplified score and a normal result of a <em>D</em>-<em>dimer</em> test was diagnosed as having venous thromboembolism.

CONCLUSIONS

This study suggests that simplification of the revised Geneva score does not lead to a decrease in diagnostic accuracy and clinical utility, which should be confirmed in a prospective study.

OBJECTIVE

The aim of the study was to determine whether C-reactive protein (CRP), procalcitonin (PCT), and d-dimer (DD) are markers of mortality in patients admitted to the emergency department (ED) with suspected infection and sepsis.

METHODS

We conducted a prospective cohort in a university hospital in Medellín, Colombia. Patients were admitted between August 1, 2007, and January 30, 2009. Clinical and demographic data and Acute Physiology and Chronic Health Evaluation II and Sepsis Organ Failure Assessment scores as well as blood samples for CRP, PCT, and DD were collected within the first 24 hours of admission. Survival was determined on day 28 to establish its association with the proposed biomarkers using logistic regression and receiver operating characteristic curves.

RESULTS

We analyzed 684 patients. The median Acute Physiology and Chronic Health Evaluation II and Sepsis Organ Failure Assessment scores were 10 (interquartile range [IQR], 6-15) and 2 (IQR, 1-4), respectively. The median CRP was 9.6 mg/dL (IQR, 3.5-20.4 mg/dL); PCT, 0.36 ng/mL (IQR, 0.1-3.7 ng/mL); and DD, 1612 ng/mL (IQR, 986-2801 ng/mL). The median DD in survivors was 1475 ng/mL (IQR, 955-2627 ng/mL) vs 2489 ng/mL (IQR, 1698-4573 ng/mL) in nonsurvivors (P=.0001). The discriminatory ability showed area under the curve-receiver operating characteristic for DD, 0.68; CRP, 0.55; and PCT, 0.59. After multivariate analysis, the only biomarker with a linear relation with mortality was DD, with an odds ratio of 2.07 (95% confidence interval, 0.93-4.62) for values more than 1180 and less than 2409 ng/mL and an odds ratio of 3.03 (95% confidence interval, 1.38-6.62) for values more than 2409 ng/mL.

CONCLUSIONS

Our results suggest that high levels of DD are associated with 28-day mortality in patients with infection or sepsis identified in the emergency department.

Pharmacokinetics and systemic effects of recombinant tissue-type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 1<em>2</em> acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in <em>2</em> minutes resulting in a peak plasma concentration (mean +/- S<em>D</em>) of 3310 +/- 950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of <em>2</em><em>2</em>10 +/- 470 ng/ml and 930 +/- <em>2</em>00 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380 +/- 74 ml/min, t1/<em>2</em> alpha was 3.6 +/- 0.9 min and t1/<em>2</em> beta was 16 +/- 5.4 min. After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 5<em>2</em>%, alpha <em>2</em>-antiplasmin to <em>2</em>5%, alpha <em>2</em>-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin <em>D</em>-<em>dimer</em> concentrations increased from 0.40 to <em>2</em>.7 micrograms/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.

An ethidium homo<em>dimer</em> and acridine ethidium hetero<em>dimer</em> have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to <em>D</em>NA has been studied. We show that these <em>dimers</em> intercalate only one of their chromophores in <em>D</em>NA. At high salt concentration (Na+ greater than 1 M) only a single type of <em>D</em>NA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. <em>D</em>., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the <em>dimers</em> cover four base pairs when bound to <em>D</em>NA. Binding affinities have been deduced from competition experiments in 0.<em>2</em> M Na+ and are in agreement with the extrapolated values determined from direct <em>D</em>NA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these <em>dimers</em> is considerably larger than the affinity of the monomer (ethidium <em>dimer</em> K = <em>2</em> X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.<em>2</em> M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium hetero<em>dimer</em> when bound to <em>D</em>NA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to <em>D</em>NA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the <em>D</em>NA binding constant of ligands of high binding affinity such as bifunctional intercalators.

Ly49A, an inhibitory C-type lectin-like mouse natural killer cell receptor, functions through interaction with the major histocompatibility complex class I molecule, H-<em>2</em>D(<em>d</em>). The x-ray crystal structure of the Ly49A.H-<em>2</em>D(<em>d</em>) complex reveale<em>d</em> that homo<em>dimer</em>ic Ly49A interacts at two <em>d</em>istinct sites of H-<em>2</em>D(<em>d</em>): Site 1, spanning one si<em>d</em>e of the alpha1 an<em>d</em> alpha<em>2</em> helices, an<em>d</em> Site <em>2</em>, involving the alpha1, alpha<em>2</em>, alpha3, an<em>d</em> beta(<em>2</em>)m <em>d</em>omains. Mutants of Ly49A, H-<em>2</em>D(<em>d</em>), an<em>d</em> beta(<em>2</em>)-microglobulin at intermolecular contacts an<em>d</em> the Ly49A <em>dimer</em> interface were examine<em>d</em> for bin<em>d</em>ing affinity an<em>d</em> kinetics. Although mutations at Site 1 ha<em>d</em> little affect, several at Site <em>2</em> an<em>d</em> at the <em>dimer</em> interface hampere<em>d</em> the Ly49A.H-<em>2</em>D(<em>d</em>) interaction, with no effect on gross structure or T cell receptor interaction. The region surroun<em>d</em>ing the most critical resi<em>d</em>ues (in H-<em>2</em>D(<em>d</em>), Asp(1<em>2</em><em>2</em>); in Ly49A, Asp(<em>2</em><em>2</em>9), Ser(<em>2</em>36), Thr(<em>2</em>38), Arg(<em>2</em>39), an<em>d</em> Asp(<em>2</em>41); an<em>d</em> in beta(<em>2</em>)-microglobulin, Gln(<em>2</em>9) an<em>d</em> Lys(58)) of the Ly49A.H-<em>2</em>D(<em>d</em>) interface at Site <em>2</em> inclu<em>d</em>es a network of water molecules, suggesting a molecular basis for allelic specificity in natural killer cell recognition.

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (S<em>D</em>S-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MAL<em>D</em>I-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only <em>dimers</em>, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (<em>2</em>-<em>D</em>) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent S<em>D</em>S-PAGE step.

HER-<em>2</em>/neu (erbB-<em>2</em>) encodes an 185-k<em>D</em>a orphan receptor tyrosine kinase that is constitutively active as a <em>dimer</em> and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of approximately 68 k<em>D</em>a, designated herstatin, as the product of an alternative HER-<em>2</em> transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-<em>2</em> followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to HER-<em>2</em>-transfected cells with a K(<em>D</em>) of approximately 14 nM and was chemically crosslinked to p185HER-<em>2</em>, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-<em>2</em> mRNA in carcinoma cells that contain an amplified HER-<em>2</em> gene. Herstatin appears to be an inhibitor of p185HER-<em>2</em>, because it disrupts <em>dimers</em>, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that overexpress HER-<em>2</em>.

A prothrombotic state may contribute to the elevated cardiovascular risk in patients with obstructive sleep apnea (OSA). We investigated the relationship between apnea severity and hemostasis factors and effect of continuous positive airway pressure (CPAP) treatment on hemostatic activity. We performed full overnight polysomnography in 44 OSA patients (mean age 47+/-10 years), yielding apnea-hypopnea index (AHI) and mean nighttime oxyhemoglobin saturation (SpO<em>2</em>) as indices of apnea severity. For treatment, subjects were double-blind randomized to <em>2</em> weeks of either therapeutic CPAP (n = 18), 3 l/min supplemental nocturnal oxygen (n = 16) or placebo-CPAP (<1 cm H<em>2</em>O) (n = 10). Levels of von Willebrand factor antigen (VWF:Ag), soluble tissue factor (sTF), <em>D</em>-<em>dimer</em>, and plasminogen activator inhibitor (PAI)-1 antigen were measured in plasma pre- and posttreatment. Before treatment, PAI-1 was significantly correlated with AHI (r = 0.47, p = 0.001) and mean nighttime SpO<em>2</em> (r = -0.3<em>2</em>, p = 0.035), but these OSA measures were not significantly related with VWF:Ag, sTF, and <em>D</em>-<em>dimer</em>. AHI was a significant predictor of PAI-1 (R<em>2</em> = 0.<em>2</em>19, standardized beta = 0.47, p = 0.001), independent of mean nighttime SpO<em>2</em>, body mass index (BMI), and age. A weak time-by-treatment interaction for PAI-1 was observed (p = 0.041), even after adjusting for age, BMI, pre-treatment AHI, and mean SpO<em>2</em> (p = 0.046). Post hoc analyses suggested that only CPAP treatment was associated with a decrease in PAI-1 (p = 0.039); there were no changes in VWF:Ag, sTF, and <em>D</em>-<em>dimer</em> associated with treatment with placebo-CPAP or with nocturnal oxygen. Apnea severity may be associated with impairment in the fibrinolytic capacity. To the extent that our sample size was limited, the observation that CPAP treatment led to a decrease in PAI-1 in OSA must be regarded as tentative.

No single noninvasive test for pulmonary embolism is both sensitive and specific. Some tests are good for "ruling in" pulmonary embolism (e.g., helical CT) and some tests are good for "ruling out" pulmonary embolism (e.g., <em>D</em>-<em>dimer</em>); others are able to do both but are often nondiagnostic (e.g., ventilation-perfusion lung scanning). For optimal efficiency, choice of the initial diagnostic test should be guided by clinical assessment of the probability of pulmonary embolism and by patient characteristics that may influence test accuracy. This selective approach to testing enables pulmonary embolism to be diagnosed or excluded in a minimum number of steps. However, even with the appropriate use of combinations of noninvasive tests, it is often not possible to definitively diagnose or exclude pulmonary embolism at initial presentation. Most of these patients can be managed safely without treatment or pulmonary angiography by repeating ultrasound testing of the proximal veins after one and <em>2</em> weeks to detect evolving deep vein thrombosis. Helical CT and MRI are rapidly improving as diagnostic tests for pulmonary embolism and are expected to become central to its evaluation.

OBJECTIVE

The purpose of this study was to evaluate the prognostic value of plasma von Willebrand factor (vWF) levels and fibrin d-dimer in a large cohort of anticoagulated permanent atrial fibrillation (AF) patients.

BACKGROUND

In nonanticoagulated AF patients, plasma vWF levels have been related to stroke and vascular events. There are limited data on the prognostic role of biomarkers in anticoagulated AF patients in relation to adverse events (including thromboembolism), mortality, and major bleeding.

METHODS

We studied 829 patients (50% male; median age 76 years) with permanent AF who were stabilized (for at least 6 months) on oral anticoagulation therapy (international normalized ratio: 2.0 to 3.0). Plasma d-dimer and vWF levels were quantified by enzyme-linked immunosorbent assay. Patients were followed for 2 years, and adverse events (thrombotic and vascular events, mortality, and major bleeding) were recorded.

RESULTS

Patients were followed for a median of 828 days (range 18 to 1,085 days). On multivariate analysis, age 75 years and older, previous stroke, heart failure, and high plasma vWF levels (≥ 221 IU/dl) were associated with future adverse cardiovascular events (all p values <0.05). High plasma vWF levels, elderly patients, diabetes, hypercholesterolemia, and current smoking were associated with mortality (all p values <0.05). High plasma vWF levels were also an independent predictor of major bleeding (hazard ratio: 4.47, 95% confidence interval: 1.86 to 10.75; p < 0.001). High plasma vWF levels were able to refine clinical risk stratification schema for stroke (CHADS₂ [Congestive heart failure, Hypertension, Age ≥ 75, Diabetes mellitus, and prior Stroke or transient ischemic attack (doubled)], CHA₂DS₂-VASc [Congestive heart failure, Hypertension, Age ≥ 75 years, Diabetes mellitus, Stroke, Vascular disease, Age 65 to 74 years, Sex category]) and bleeding (HAS-BLED [Hypertension, Abnormal renal/liver function, Stroke, Bleeding history or predisposition, Labile International Normalized Ratio, Elderly, Drugs/alcohol concomitantly]). d-dimer did not show any significant impact on adverse events.

CONCLUSIONS

High plasma vWF levels (≥221 IU/dl) are an independent risk factor for adverse events in anticoagulated permanent AF patients. This biomarker may potentially be used to refine stroke and bleeding clinical risk stratification in AF.

This report <em>d</em>escribes the evaluation of bio<em>d</em>istribution properties of three ra<em>d</em>iotracers, [(99m)Tc(SQ168)(EDDA)], [(99m)Tc(SQ168)(tricine)(PDA)], an<em>d</em> [(99m)Tc(SQ168)(tricine)(TPPTS)] (SQ168 = [<em>2</em>-[[[5-[carboonyl]-<em>2</em>-pyri<em>d</em>inyl]hy<em>d</em>razono]methyl]benzenesulfonic aci<em>d</em>]-Glu(cyclo{Lys-Arg-Gly-Asp-<em>d</em>-Phe})-cyclo{Lys-Arg-Gly-Asp-<em>d</em>-Phe}; EDDA = ethylene<em>d</em>iamine-N,N'-<em>d</em>iacetic aci<em>d</em>; PDA = <em>2</em>,5-pyri<em>d</em>ine<em>d</em>icarboxylic aci<em>d</em>; TPPTS = triso<em>d</em>ium triphenylphosphine-3,3',3' '-trisulfonate), an<em>d</em> their potential to image the glioma integrin alpha(v)beta(3) expression in BALB/c nu<em>d</em>e mice bearing the U87MG human glioma xenografts. It was foun<em>d</em> that all three ra<em>d</em>iotracers were able to localize in glioma tumors with a relatively high tumor uptake an<em>d</em> long tumor retention time by bin<em>d</em>ing to the integrin alpha(v)beta(3) expresse<em>d</em> on both tumor cells an<em>d</em> en<em>d</em>othelial cells of tumor neovasculature. It seems that the coligan<em>d</em> has minimal effect on integrin alpha(v)beta(3) targeting capability of the (99m)Tc-labele<em>d</em> RGDfK <em>dimer</em>, but it has a significant impact on their bio<em>d</em>istribution properties. For example, the complex [(99m)Tc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake an<em>d</em> the highest metabolic stability in normal BALB/c nu<em>d</em>e mice. Results from SPECT imaging stu<em>d</em>ies show that the glioma tumors can be clearly visualize<em>d</em> with all three ra<em>d</em>iotracers at 4 h postinjection. Among the three ra<em>d</em>iotracers evaluate<em>d</em> in this stu<em>d</em>y, [(99m)Tc(SQ168)(tricine)(TPPTS)] has the best imaging quality an<em>d</em> is a promising can<em>d</em>i<em>d</em>ate for more preclinical evaluations in the future.

Titration calorimetry measurements of the binding of methyl alpha-<em>D</em>-mannopyranoside (Me alpha Man), <em>D</em>-mannopyranoside (Man), methyl alpha-<em>D</em>-glucopyranoside (Me alpha Glu), and <em>D</em>-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at <em>2</em>81 and <em>2</em>9<em>2</em> K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+<em>2</em> and Ca+<em>2</em> ions. The site binding enthalpies, <em>delta</em> H, are the same at both temperatures and range from -<em>2</em>8.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -<em>2</em>6.<em>2</em> +/- 1.1 (Me alpha Man) to -1<em>2</em>.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.<em>2</em> kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at <em>2</em>81.<em>2</em> K) to <em>2</em>30 +/- <em>2</em>0 M-1 (Glu to lentil lectin at <em>2</em>9<em>2</em>.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of <em>2</em>1:4:5:1, while the corresponding ratio is 5:<em>2</em>:1.5:1 for pea lectin and 4:<em>2</em>:<em>2</em>:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that <em>delta</em> Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.1<em>2</em>)T(K) <em>delta</em> Sb (J mol-1K-1). <em>D</em>ifferential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin <em>dimers</em> dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.

To clarify whether the formation of thrombi could be induced by atrial fibrillation itself or by factors predisposing to atrial fibrillation such as mitral stenosis, plasma <em>D</em>-<em>dimer</em> levels (cross-linked fibrin degradation products) were measured in 73 patients without atrial fibrillation (Group <em>2</em>). In Group 1, 49 of the 73 patients had factors predisposing to atrial fibrillation such as valvular heart disease, and the remaining <em>2</em>4 had lone atrial fibrillation. In Group <em>2</em>, 16 patients had organic heart disease and the remaining 5 had a chest pain syndrome. The plasma <em>D</em>-<em>dimer</em> level was significantly higher in Group 1 (150 +/- 19 ng/ml) than in Group <em>2</em> (61 +/- 3 ng/ml) (p less than 0.01, mean +/- standard error of the mean). In both groups, there were no significant differences in plasma <em>D</em>-<em>dimer</em> level between patients with and without organic heart disease (146 +/- 18 versus 156 +/- 46 ng/ml in Group 1; 61 +/- 4 versus 59 +/- 10 ng/ml in Group <em>2</em>). These findings indicate that atrial fibrillation itself may be more important than factors predisposing to atrial fibrillation in the development of intracardiovascular clotting.