Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (13<em>2</em> kDa), epsilon (<em>2</em>7 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (5<em>2</em> kDa), <em>delta</em> (35 kDa), <em>delta</em>' (33 kDa), chi (15 kDa), and psi (1<em>2</em> kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel <em>dimer</em>ic form which may provide the foundation for the <em>dimer</em>ic nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a <em>dimer</em>ic core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg<em>2</em>+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta <em>dimers</em> in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma <em>2</em> <em>delta</em> <em>delta</em>' chi psi complex (gamma complex), beta, and tau retained the core, one beta <em>dimer</em>, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric <em>dimer</em>ic particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.

1,<em>2</em>5-<em>D</em>ihydroxyvitamin <em>D</em>(3) (1,<em>2</em>5(OH)(<em>2</em>)<em>D</em>(3)) is the biologically active ligand for the vitamin <em>D</em> receptor (V<em>D</em>R). V<em>D</em>R(-/-) mice have a hair follicle-cycling defect resulting in alopecia. However, mice lacking <em>2</em>5-hydroxyvitamin <em>D</em>(3) 1alpha-hydroxylase (CYP<em>2</em>7B1(-/-)), and having no circulating 1,<em>2</em>5(OH)(<em>2</em>)<em>D</em>(3), have normal follicular function. These mouse models indicate that V<em>D</em>R functions independently of 1,<em>2</em>5(OH)(<em>2</em>)<em>D</em>(3) in regulating hair-follicle cycling. Here, we show that V<em>D</em>R(-/-) mice rapidly develop chemically induced skin tumors, whereas CYP<em>2</em>7B1(-/-) and wild-type mice do not, indicating that V<em>D</em>R, and not the 1,<em>2</em>5(OH)(<em>2</em>)<em>D</em>(3) ligand, is essential for protection against skin tumorigenesis. Because the majority of human skin cancer results from exposure to UV, the susceptibility of V<em>D</em>R(-/-) mice to this carcinogen was also evaluated. V<em>D</em>R(-/-) mice developed UV-induced tumors more rapidly and with greater penetrance than did V<em>D</em>R(+/+) mice. p53 protein levels were upregulated at similar rates in UV-treated keratinocytes of V<em>D</em>R(-/-) and V<em>D</em>R(+/+) mice. However, rates of thymine-<em>dimer</em> repair and UV-induced apoptosis were significantly lower in V<em>D</em>R(-/-) epidermis compared with the wild type epidermis. UV-induced epidermal thickening was also attenuated in V<em>D</em>R(-/-) skin, indicating that V<em>D</em>R plays a critical role in the repair and removal of severely damaged keratinocytes and adaptation of the skin to chronic UV exposure.

OBJECTIVE

The pathogenesis of cerebral small vessel disease (SVD) is poorly understood, but endothelial activation and dysfunction may play a causal role. Cross-sectional studies have found increased circulating markers of endothelial activation, but this study design cannot exclude causality from secondary elevations. Confluent white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI) appear to represent asymptomatic cerebral SVD. In a prospective study, we determined whether circulating markers of endothelial activation predicted progression of WMH.

METHODS

In the community-based Austrian Stroke Prevention Study, MRI was performed at baseline in <em>2</em>96 subjects and repeated at 3 and 6 years. The following were measured on baseline plasma samples: intercellular adhesion molecule (ICAM), thrombomodulin, tissue factor plasma inhibitor, prothrombin fragments 1 and <em>2</em>, and <em>D</em>-<em>dimers</em>.

RESULTS

ICAM was associated with age- and gender-adjusted WMH lesion progression at both 3 and 6 years, respectively; (odds ratio [OR], 1.007; 95% confidence interval [CI], 1.00<em>2</em> to 1.01<em>2</em>; P=0.004; and OR, 1.004; 95% CI, 1.000 to 1.009 per ng/mL; P=0.057). After multivariate analysis controlling for other cardiovascular risk factors and C-reactive protein, 3-year OR was 1.010 (95% CI, 1.004 to 1.017; P=0.001) and 6-year OR was 1.008 (1.00<em>2</em> to 1.014 per ng/mL; P=0.006). Baseline log lesion volume was a strong independent predictor of progression but associations remained after controlling for this (3-year OR, 1.011; 95% CI, 1.00<em>2</em> to 1.0<em>2</em>0; P=0.013; and 6-year OR, 1.009; 95% CI, 1.000 to 1.017; P=0.039 per ng/mL). There was no association between WMH progression and other markers.

CONCLUSIONS

ICAM levels are related to progression of WMH on MRI. The prospective study design increases the likelihood that this association is causal and supports a role of endothelial cell activation in disease pathogenesis. In contrast, we found no evidence for coagulation activation being important.

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PS<em>D</em>-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-<em>D</em>-aspartate (NM<em>D</em>A) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel <em>dimer</em>ic inhibitor, Tat-NPEG4(IET<em>D</em>V)(<em>2</em>) (Tat-N-<em>dimer</em>), which binds the tandem P<em>D</em>Z1-<em>2</em> domain of PS<em>D</em>-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between <em>dimer</em>ic inhibitor and P<em>D</em>Z1-<em>2</em>, and also provided a dynamic model of the conformational changes of P<em>D</em>Z1-<em>2</em> induced by the <em>dimer</em>ic inhibitor. A single intravenous injection of Tat-N-<em>dimer</em> (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-<em>dimer</em> is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

The tyrosine-sensitive 3-deoxy-<em>D</em>-arabino-heptulosonate 7-phosphate synthase (7-phospho-<em>2</em>-keto-3-deoxy-<em>D</em>-arabino-heptonate <em>D</em>-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.<em>2</em>.1.15) was purified to homogeneity from extracts of Escherichia coli K1<em>2</em>. A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at <em>2</em>3<em>2</em> nm, was developed. The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-<em>2</em>00, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This suggests either a rapid monomer-<em>dimer</em> equilibrium, or a very asymmetric shape for the native enzyme. The enzyme shows a narrow pH optimum around pH 7.0. The enzyme is stable for several months when stored at -<em>2</em>0 degrees in phosphate buffer containing phosphoenol-pyruvate. Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction. Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex. The feedback inhibitor tyrosine at concentrations above 10 muM exhibits noncompetitive inhibition with respect to erythrose-4-P, and competitive inhibition with respect to the other substrate, P-enolpyruvate. In addition, tyrosine at concentrations of at least 10 muM causes an alteration of one or more than one kinetic parameter of the enzyme.

BACKGROUND

Native glucagon-like peptide-1 increases insulin secretion, decreases glucagon secretion, and reduces appetite but is rapidly inactivated by dipeptidyl peptidase-4. Albiglutide is a novel dipeptidyl peptidase-4-resistant glucagon-like peptide-1 dimer fused to human albumin designed to have sustained efficacy in vivo.

OBJECTIVE

The objectives were to investigate pharmacodynamics, pharmacokinetics, safety, and tolerability of albiglutide in type 2 diabetes subjects.

METHODS

In a single-blind dose-escalation study, 54 subjects were randomized to receive placebo or 9-, 16-, or 32-mg albiglutide on d 1 and 8. In a complementary study, 46 subjects were randomized to a single dose (16 or 64 mg) of albiglutide to the arm, leg, or abdomen.

RESULTS

Significant dose-dependent reductions in 24-h mean weighted glucose [area under the curve((0-24 h))] were observed, with placebo-adjusted least squares means difference values in the 32-mg cohort of -34.8 and -56.4 mg/dl [95% confidence interval (-54.1, -15.5) and (-82.2, -30.5)] for d 2 and 9, respectively. Placebo-adjusted fasting plasma glucose decreased by -26.7 and -50.7 mg/dl [95% confidence interval (-46.3, -7.06) and (-75.4, -26.0)] on d 2 and 9, respectively. Postprandial glucose was also reduced. No hypoglycemic episodes were detected in the albiglutide cohorts. The frequency and severity of the most common adverse events, headache and nausea, were comparable with placebo controls. Albiglutide half-life ranged between 6 and 7 d. The pharmacokinetics or pharmacodynamic of albiglutide was unaffected by injection site.

CONCLUSIONS

Albiglutide improved fasting plasma glucose and postprandial glucose with a favorable safety profile in subjects with type 2 diabetes. Albiglutide's long half-life may allow for once-weekly or less frequent dosing.

The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homo<em>dimer</em>ic ATPase <em>delta</em>(<em>2</em>) (ParA) and binding of omega(<em>2</em>) (ParB) to its cognate parS DNA. The 1.83 A resolution crystal structure of <em>delta</em>(<em>2</em>) in a complex with non-hydrolyzable ATPgammaS reveals a unique ParA <em>dimer</em> assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro, <em>delta</em>(<em>2</em>) had minimal ATPase activity in the absence of omega(<em>2</em>) and parS DNA. However, stoichiometric amounts of omega(<em>2</em>) and parS DNA stimulated the <em>delta</em>(<em>2</em>) ATPase activity and mediated plasmid pairing, whereas at high (4:1) omega(<em>2</em>) : <em>delta</em>(<em>2</em>) ratios, stimulation of the ATPase activity was reduced and <em>delta</em>(<em>2</em>) polymerized onto DNA. Stimulation of the <em>delta</em>(<em>2</em>) ATPase activity and its polymerization on DNA required ability of omega(<em>2</em>) to bind parS DNA and its N-terminus. In vivo experiments showed that <em>delta</em>(<em>2</em>) alone associated with the nucleoid, and in the presence of omega(<em>2</em>) and parS DNA, <em>delta</em>(<em>2</em>) oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar omega(<em>2</em>) : <em>delta</em>(<em>2</em>) ratio regulates the polymerization properties of (<em>delta</em>*ATP*Mg(<em>2</em>+))(<em>2</em>) on and depolymerization from parS DNA, thereby controlling the temporal and spatial segregation of pSM19035 before cell division.

The novel coronavirus infection (COVID-19) is cause<em>d</em> by the new coronavirus SARS-CoV-<em>2</em> an<em>d</em> is characterize<em>d</em> by an exaggerate<em>d</em> inflammatory response that can lea<em>d</em> to severe manifestations such as a<em>d</em>ult respiratory syn<em>d</em>rome, sepsis, coagulopathy, an<em>d</em> <em>d</em>eath in a proportion of patients. Among other factors an<em>d</em> <em>d</em>irect viral effects, the increase in the vasoconstrictor angiotensin II, the <em>d</em>ecrease in the vaso<em>d</em>ilator angiotensin, an<em>d</em> the sepsis-in<em>d</em>uce<em>d</em> release of cytokines can trigger a coagulopathy in COVID-19. A coagulopathy has been reporte<em>d</em> in up to 50% of patients with severe COVID-19 manifestations. An increase in <em>d</em>-<em>dimer</em> is the most significant change in coagulation parameters in severe COVID-19 patients, an<em>d</em> progressively increasing values can be use<em>d</em> as a prognostic parameter in<em>d</em>icating a worse outcome. Limite<em>d</em> <em>d</em>ata suggest a high inci<em>d</em>ence of <em>d</em>eep vein thrombosis an<em>d</em> pulmonary embolism in up to 40% of patients, <em>d</em>espite the use of a stan<em>d</em>ar<em>d</em> <em>d</em>ose of low-molecular-weight heparin (LMWH) in most cases. In a<em>d</em><em>d</em>ition, pulmonary microvascular thrombosis has been reporte<em>d</em> an<em>d</em> may play a role in progressive lung failure. Prophylactic LMWH has been recommen<em>d</em>e<em>d</em> by the International Society on Thrombosis an<em>d</em> Haemostasis (ISTH) an<em>d</em> the American Society of Hematology (ASH), but the best effective <em>d</em>osage is uncertain. A<em>d</em>apte<em>d</em> to the in<em>d</em>ivi<em>d</em>ual risk of thrombosis an<em>d</em> the <em>d</em>-<em>dimer</em> value, higher <em>d</em>oses can be consi<em>d</em>ere<em>d</em>, especially since blee<em>d</em>ing events in COVID-19 are rare. Besi<em>d</em>es the anticoagulant effect of LMWH, nonanticoagulant properties such as the re<em>d</em>uction in interleukin 6 release have been shown to improve the complex picture of coagulopathy in patients with COVID-19.

Keywords: COVID-19; anticoagulation; thrombosis.

Prolonged survival in HIV infection is accompanied by an increased frequency of non-HIV-related comorbidities. A number of age-related comorbidities occur earlier in HIV-infected patients than in individuals without HIV infection. This "accelerated aging" appears to be largely related to chronic inflammation, chronic immune activation, and immunosenescence in HIV infection. Levels of markers of inflammation and coagulopathy are elevated in HIV-infected patients, and elevations in markers such as high-sensitivity C-reactive protein, <em>D</em>-<em>dimer</em>, and interleukin 6 (IL-6) have been associated with increased risk for cardiovascular disease, opportunistic conditions, or all-cause mortality. In both HIV infection and aging, immunosenescence is marked by an increased proportion of C<em>D</em><em>2</em>8-, C<em>D</em>57+ memory C<em>D</em>8+ T cells with reduced capacity to produce interleukin <em>2</em> (IL-<em>2</em>), increased production of IL-6, resistance to apoptosis, and shortened telomeres. A number of AI<em>D</em>S Clinical Trials Group studies are under way to examine treatment aimed at reducing chronic inflammation and immune activation in HIV infection. This article summarizes a presentation by Judith A. Aberg, M<em>D</em>, at the IAS-USA live continuing medical education course held in New York City in October <em>2</em>011.

<strong class="sub-title"> Objectives: </strong> The aim of this study was to investigate whether right ventricular longitudinal strain (RVLS) was independently predictive of higher mortality in patients with coronavirus disease <em>2</em>019 (COVID-19).

<strong class="sub-title"> Background: </strong> RVLS obtained from <em>2</em>-dimensional speckle-tracking echocardiography has been recently demonstrated to be a more accurate and sensitive tool to estimate right ventricular (RV) function. The prognostic value of RVLS in patients with COVID-19 remains unknown.

<strong class="sub-title"> Methods: </strong> One hundred twenty consecutive patients with COVID-19 who underwent echocardiographic examinations were enrolled in our study. Conventional RV functional parameters, including RV fractional area change, tricuspid annular plane systolic excursion, and tricuspid tissue Doppler annular velocity, were obtained. RVLS was determined using <em>2</em>-dimensional speckle-tracking echocardiography. RV function was categorized in tertiles of RVLS.

<strong class="sub-title"> Results: </strong> Compared with patients in the highest RVLS tertile, those in the lowest tertile were more likely to have higher heart rate; elevated levels of D-dimer and C-reactive protein; more high-flow oxygen and invasive mechanical ventilation therapy; higher incidence of acute heart injury, acute respiratory distress syndrome, and deep vein thrombosis; and higher mortality. After a median follow-up period of 51 days, 18 patients died. Compared with survivors, nonsurvivors displayed enlarged right heart chambers, diminished RV function, and elevated pulmonary artery systolic pressure. Male sex, acute respiratory distress syndrome, RVLS, RV fractional area change, and tricuspid annular plane systolic excursion were significant univariate predictors of higher risk for mortality (p < 0.05 for all). A Cox model using RVLS (hazard ratio: 1.33; 95% confidence interval [CI]: 1.15 to 1.53; p < 0.001; Akaike information criterion = 1<em>2</em>9; C-index = 0.89) was found to predict higher mortality more accurately than a model with RV fractional area change (Akaike information criterion = 14<em>2</em>, C-index = 0.84) and tricuspid annular plane systolic excursion (Akaike information criterion = 144, C-index = 0.83). The best cutoff value of RVLS for prediction of outcome was -<em>2</em>3% (AUC: 0.87; p < 0.001; sensitivity, 94.4%; specificity, 64.7%).

Conclusions: RVLS is a powerful predictor of higher mortality in patients with COVID-19. These results support the application of RVLS to identify higher risk patients with COVID-19.

<strong class="sub-title"> Keywords: </strong> COVID-19; SARS-CoV-<em>2</em>; right ventricular function; speckle tracking echocardiography; strain.

Patients hospitalized for Covid-19 severe infection are more prone to excessive coagulation activation leading to thrombotic events. Tang et al.1 discussed the importance of high <em>D</em>-<em>dimer</em> and Fibrin degradation product level to determine the patient prognostic and the risk of thrombosis. However they did not look at lupus anticoagulant (LAC). Zhang et al. described, three cases of thrombosis associated with antiphospholipid antibodies represented by anticardiolipin (aCL) and anti-β<em>2</em>-glycoprotein I (aβ<em>2</em>GPI)<em>2</em>. No lupus anticoagulant was detected in any of the patients.

BACKGROUND

Meningococcal septic shock in children results in high mortality and morbidity, and decreased protein C levels in these patients are associated with a poor outcome. We carried out a randomized, double-blinded, placebo-controlled study by supplying protein C concentrate. This phase <em>2</em> study was designed to assess the activation process of protein C and to study the dosing regimen of protein C concentrate in children with purpura fulminans and meningococcal septic shock in the perspective of a possible phase 3 trial.

METHODS

Forty children were randomized to receive placebo or protein C concentrate (<em>2</em>00 IU/kg, 400 IU/kg, or 600 IU/kg), for a maximum of 7 days. Clinical and laboratory data, including plasma levels of protein C and activated protein C (APC), were collected at various time points. All patients received standard therapy for septic shock, including antibiotics, inotropic/vasoactive drugs, and blood products.

RESULTS

Increased APC levels relative to baseline were observed for the <em>2</em>7 of <em>2</em>8 patients treated with protein C concentrate, and the areas under the curve of protein C and APC were correlated with the dosage of protein C concentrate administered. Activation of coagulation, as evidenced by d-dimer levels, as well as the ratio of thrombin vs. APC normalized significantly faster with increasing dosages of protein C concentrate. No adverse reactions related to protein C concentrate were observed. Nine of the 40 (<em>2</em>3%) patients died, and five survivors required amputations, with no differences in these rates among the randomized groups. Baseline APC levels were positively correlated with sequential organ failure assessment and pediatric risk of mortality scores and with d-dimers, tumor necrosis factor-alpha, interleukin-1, interleukin-6, interleukin-8, plasminogen activator inhibitor-1, TAT complexes, and PAP complexes.

CONCLUSIONS

Treatment with protein C concentrate is safe in children with purpura fulminans and meningococcal septic shock and leads to dose-related increases of plasma APC and resolution of coagulation imbalances.

We have developed a host cell reactivation assay of <em>D</em>NA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged <em>D</em>NA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV<em>2</em>cat, 50<em>2</em>0 base pairs; pSV<em>2</em>catSVgpt, 7<em>2</em>68 base pairs; and pRSVcat, 50<em>2</em>7 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV<em>2</em>catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and <em>D</em> lines (<em>D</em>0 = 56 J X m-<em>2</em>) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(<em>D</em>0 = 680 J X m-<em>2</em>)(<em>D</em>0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The <em>D</em>0 of the CAT inactivation curve was 50 J X m-<em>2</em> for pSV<em>2</em>cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the <em>D</em>0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine <em>dimer</em> formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine <em>dimer</em> inactivates a target of about <em>2</em> kilobases, close to the size of the putative CAT mRNA.

BACKGROUND

Pulmonary embolism (PE) continues to be a major challenge in terms of diagnosis, as evidenced by the fact that many patients die undiagnosed and/or untreated. The aim of this multicenter study was to determine the accuracy of thorax ultrasound (TUS) in the diagnosis of PE (TUSPE).

METHODS

From January <em>2</em>00<em>2</em> through September <em>2</em>003, 35<em>2</em> patients with suspecte<em>d</em> PE were examine<em>d</em> in seven clinics. The patients were investigate<em>d</em> prospectively by TUS accor<em>d</em>ing to the following criteria: (1) PE confirme<em>d</em>: two or more typical triangular or roun<em>d</em>e<em>d</em> pleural-base<em>d</em> lesions; (<em>2</em>) PE probable: one typical lesion with pleural effusion; (3) PE possible: small (< 5 mm) subpleural lesions or a single pleural effusion alone; or (4) normal TUS fin<em>d</em>ings. In all cases, CT pulmonary angiography (CTPA) was use<em>d</em> as the reference metho<em>d</em>. In the event of <em>d</em>iscrepant fin<em>d</em>ings, a combination of <em>d</em>uplex sonography of the leg veins, echocar<em>d</em>iography, ventilation/perfusion scintigraphy, an<em>d</em> a quantitative enzyme-linke<em>d</em> immunosorbent assay or latex <em>d</em>-<em>dimer</em>, or a biopsy/autopsy was performe<em>d</em>.

RESULTS

PE was <em>d</em>iagnose<em>d</em> in 194 patients. On TUS, 144 patients ha<em>d</em> a total of 333 subpleural lesions (mean, <em>2</em>.3 lesions per patient) averaging 15.5 x 1<em>2</em>.4 mm in size. A<em>d</em><em>d</em>itionally, a narrow pleural effusion was foun<em>d</em> in 49% of the patients. TUS yiel<em>d</em>e<em>d</em> the following results un<em>d</em>er application of the strict criteria 1 an<em>d</em> <em>2</em>: PE true-positive, n = 144; PE false-positive, n = 8; PE true-negative, n = 150; an<em>d</em> PE false-negative, n = 50. The sensitivity was 74%, specificity was 95%, positive pre<em>d</em>ictive value was 95%, negative pre<em>d</em>ictive was value 75%, an<em>d</em> accuracy was 84%, at a prevalence of 55%. The sensitivity in patients with criterion 1 was 43% an<em>d</em> a specificity of 99%.

CONCLUSIONS

TUS is a noninvasive method to diagnose peripheral PE. In the absence of CTPA, TUS is a suitable tool to demonstrate a PE at the bedside and in the emergency setting.

BACKGROUND

The hemolytic-uremic syndrome is a thrombotic complication of Escherichia coli O157:H7 infection. It is not known whether the coagulation abnormalities precede, and potentially cause, this disorder.

METHODS

In 53 children infected with E. coli O157:H7, we measured a panel of markers indicating activation of the clotting cascade and renal function within four days after the onset of illness. These markers were measured again in as many as possible of the 16 children in whom the hemolytic-uremic syndrome developed.

RESULTS

The children in whom the hemolytic-uremic syndrome subsequently developed had significantly higher median plasma concentrations of prothrombin fragment 1+<em>2</em>, tissue plasminogen activator (t-PA) antigen, t-PA-plasminogen-activator inhibitor type 1 (PAI-1) complex, and <em>D</em>-<em>dimer</em> than children with uncomplicated infection. These abnormalities preceded the development of azotemia and thrombocytopenia. When the hemolytic-uremic syndrome developed, the urinary concentrations of beta<em>2</em>-microglobulin and N-acetyl-beta-glucosaminidase rose significantly (P=0.03 for both increases); the plasma concentrations of t-PA antigen, t-PA-PAI-1 complex, <em>D</em>-<em>dimer</em>, and plasmin-antiplasmin complex also increased significantly. The concentration of t-PA antigen correlated with that of the t-PA-PAI-1 complex in a linear regression model (squared correlation coefficient, 0.80; P<0.001).

CONCLUSIONS

In the hemolytic-uremic syndrome, thrombin generation (probably due to accelerated thrombogenesis) and inhibition of fibrinolysis precede renal injury and may be the cause of such injury.

Objective: To evaluate association between biomarkers and outcomes in COVID-19 hospitalised patients. COVID-19 pandemic has been a challenge. Biomarkers have always played an important role in clinical decision making in various infectious diseases. It is crucial to assess the role of biomarkers in evaluating severity of disease and appropriate allocation of resources.

<strong class="sub-title"> Design and setting: </strong> Systematic review and meta-analysis. English full text observational studies describing the laboratory findings and outcomes of COVID-19 hospitalised patients were identified searching PubMed, Web of Science, Scopus, medRxiv using Medical Subject Headings (MeSH) terms COVID-19 OR coronavirus OR SARS-CoV-<em>2</em> OR <em>2</em>019-nCoV from 1 December <em>2</em>019 to 15 August <em>2</em>0<em>2</em>0 following Meta-analyses Of Observational Studies in Epidemiology (MOOSE) guidelines.

Participants: Studies having biomarkers, including lymphocyte, platelets, D-dimer, lactate dehydrogenase (LDH), C reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, procalcitonin (PCT) and creatine kinase (CK), and describing outcomes were selected with the consensus of three independent reviewers.

Main outcome measures: Composite poor outcomes include intensive care unit admission, oxygen saturation <90%, invasive mechanical ventilation utilisation, severe disease, in-hospital admission and mortality. The OR and 95% CI were obtained and forest plots were created using random-effects models. Publication bias and heterogeneity were assessed by sensitivity analysis.

<strong class="sub-title"> Results: </strong> 3<em>2</em> studies with 10 491 confirmed COVID-19 patients were included. We found that lymphopenia (pooled-OR: 3.33 (95% CI: <em>2</em>.51-4.41); p<0.00001), thrombocytopenia (<em>2</em>.36 (1.64-3.40); p<0.00001), elevated D-dimer (3.39 (<em>2</em>.66-4.33); p<0.00001), elevated CRP (4.37 (3.37-5.68); p<0.00001), elevated PCT (6.33 (4.<em>2</em>4-9.45); p<0.00001), elevated CK (<em>2</em>.4<em>2</em> (1.35-4.3<em>2</em>); p=0.003), elevated AST (<em>2</em>.75 (<em>2</em>.30-3.<em>2</em>9); p<0.00001), elevated ALT (1.71 (1.3<em>2</em>-<em>2</em>.<em>2</em>0); p<0.00001), elevated creatinine (<em>2</em>.84 (1.80-4.46); p<0.00001) and LDH (5.48 (3.89-7.71); p<0.00001) were independently associated with higher risk of poor outcomes.

Conclusion: Our study found a significant association between lymphopenia, thrombocytopenia and elevated levels of CRP, PCT, LDH, D-dimer and COVID-19 severity. The results have the potential to be used as an early biomarker to improve the management of COVID-19 patients, by identification of high-risk patients and appropriate allocation of healthcare resources in the pandemic.

Keywords: critical care; evidence-based practice; global health; infectious disease medicine; prognosis.

OBJECTIVE

To evaluate the performance of procalcitonin (PCT), interleukin-6 (IL-6), C-reactive protein, leukocyte count, D-dimer, and antithrombin III at onset of septic episode and 24 h later in prediction of hospital mortality in critically ill patients with suspected sepsis.

METHODS

Prospective, cohort study in two university hospital intensive care units.

METHODS

61 critically ill patients with suspected sepsis.

RESULTS

The outcome measure was hospital mortality. Hospital survivors ( n=41) and nonsurvivors ( n=20) differed statistically significantly on day 1 (admission) in PCT, IL-6, SOFA score, and APACHE II score, and 24 h later in PCT, IL-6, and D-dimer values. AT III, CRP, and leukocyte count did not differ. The areas under receiver operating curves showed reasonable discriminative power (>0.75) in predicting hospital mortality only for day 2 IL-6 (0.799) and day 2 PCT (0.777) values which were comparable to that of APACHE II (0.786), and which remained the only independent predictor of mortality.

CONCLUSIONS

Admission and day 2 IL-6, and day 2 PCT, and day 2 D-dimer values differed significantly between hospital survivors and nonsurvivors among critically ill patients with suspected sepsis. However, in prediction of hospital mortality, only the discriminative power of day 2 PCT and IL-6 values, and APACHE II was reasonable as judged by AUC analysis (>0.75).

A number of cellular processes use both microtubules an<em>d</em> actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be eluci<em>d</em>ate<em>d</em> in <em>d</em>etail. Formins are actin-bin<em>d</em>ing proteins that have multiple effects on actin <em>d</em>ynamics, an<em>d</em> one formin, mDia<em>2</em>, has been shown to bin<em>d</em> an<em>d</em> stabilize microtubules through its formin homology <em>2</em> (FH<em>2</em>) <em>d</em>omain. Here we show that three formins, INF<em>2</em>, mDia1, an<em>d</em> mDia<em>2</em>, <em>d</em>isplay important <em>d</em>ifferences in their interactions with microtubules an<em>d</em> actin. Constructs containing FH1, FH<em>2</em>, an<em>d</em> C-terminal <em>d</em>omains of all three formins bin<em>d</em> microtubules with high affinity (K(<em>d</em>) < 100 nM). However, only mDia<em>2</em> bin<em>d</em>s microtubules at 1:1 stoichiometry, with INF<em>2</em> an<em>d</em> mDia1 showing saturating bin<em>d</em>ing at approximately 1:3 (formin <em>dimer</em>:tubulin <em>dimer</em>). INF<em>2</em>-FH1FH<em>2</em>C is a potent microtubule-bun<em>d</em>ling protein, an effect that results in a large re<em>d</em>uction in catastrophe rate. In contrast, neither mDia1 nor mDia<em>2</em> is a potent microtubule bun<em>d</em>ler. The C-termini of mDia<em>2</em> an<em>d</em> INF<em>2</em> have <em>d</em>ifferent functions in microtubule interaction, with mDia<em>2</em>'s C-terminus require<em>d</em> for high-affinity bin<em>d</em>ing an<em>d</em> INF<em>2</em>'s C-terminus require<em>d</em> for bun<em>d</em>ling. mDia<em>2</em>'s C-terminus <em>d</em>irectly bin<em>d</em>s microtubules with submicromolar affinity. These formins also <em>d</em>iffer in their abilities to bin<em>d</em> actin an<em>d</em> microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia<em>2</em>, whereas they mo<em>d</em>erately inhibit mDia1 an<em>d</em> have no effect on INF<em>2</em>. Conversely, actin monomers inhibit microtubule bin<em>d</em>ing/bun<em>d</em>ling by INF<em>2</em> but <em>d</em>o not affect mDia1 or mDia<em>2</em>. These <em>d</em>ifferences in interactions with microtubules an<em>d</em> actin suggest <em>d</em>ifferential function in cellular processes requiring both cytoskeletal elements.

<strong class="sub-title"> Background: </strong> In our institute in Marseille, France, we initiated early and massive screening for coronavirus disease <em>2</em>019 (COVI<em>D</em>-19). Hospitalization and early treatment with hydroxychloroquine and azithromycin (HCQ-AZ) was proposed for the positive cases.

<strong class="sub-title"> Methods: </strong> We retrospectively report the clinical management of 3,737 screened patients, including 3,119 (83.5%) treated with HCQ-AZ (<em>2</em>00 mg of oral HCQ, three times daily for ten days and 500 mg of oral AZ on day 1 followed by <em>2</em>50 mg daily for the next four days, respectively) for at least three days and 618 (16.5%) patients treated with other regimen ("others"). Outcomes were death, transfer to the intensive care unit (ICU), ≥10 days of hospitalization and viral shedding.

<strong class="sub-title"> Results: </strong> The patients' mean age was 45 (sd 17) years, 45% were male, and the case fatality rate was 0.9%. We performed <em>2</em>,065 low-dose computed tomography (CT) scans highlighting lung lesions in 59<em>2</em> of the 991 (59.7%) patients with minimal clinical symptoms (NEWS score = 0). A discrepancy between spontaneous dyspnoea, hypoxemia and lung lesions was observed. Clinical factors (age, comorbidities, NEWS-<em>2</em> score), biological factors (lymphocytopenia; eosinopenia; decrease in blood zinc; and increase in <em>D</em>-<em>dimers</em>, lactate dehydrogenase, creatinine phosphokinase, and C-reactive protein) and moderate and severe lesions detected in low-dose CT scans were associated with poor clinical outcome. Treatment with HCQ-AZ was associated with a decreased risk of transfer to ICU or death (Hazard ratio (HR) 0.18 0.11-0.<em>2</em>7), decreased risk of hospitalization ≥10 days (odds ratios 95% CI 0.38 0.<em>2</em>7-0.54) and shorter duration of viral shedding (time to negative PCR: HR 1.<em>2</em>9 1.17-1.4<em>2</em>). QTc prolongation (>60 ms) was observed in <em>2</em>5 patients (0.67%) leading to the cessation of treatment in 1<em>2</em> cases including 3 cases with QTc> 500 ms. No cases of torsade de pointe or sudden death were observed.

Conclusion: Although this is a retrospective analysis, results suggest that early diagnosis, early isolation and early treatment of COVID-19 patients, with at least 3 days of HCQ-AZ lead to a significantly better clinical outcome and a faster viral load reduction than other treatments.

<strong class="sub-title"> Keywords: </strong> Azithromycin; COVI<em>D</em>-19; Hydroxychloroquine; SARS-CoV-<em>2</em>.

BACKGROUND

Traditional atherosclerotic risk factors predict long-term cardiovascular disease events but are poor predictors of near-term events.

OBJECTIVE

To determine whether elevated levels of D-dimer and biomarkers of inflammation were more closely associated with near-term than long-term mortality in patients with lower-extremity peripheral arterial disease (PAD) and whether greater increases in biomarker levels were associated with higher mortality rates during the first year after the increase than during later years.

METHODS

Prospective cohort study with a mean follow-up of 3.4 years.

METHODS

Academic medical center.

METHODS

377 men and women with PAD.

METHODS

Mortality within 1 year after biomarker measurement, 1 to 2 years after biomarker measurement, and 2 to 3 years after biomarker measurement. Cox regression analyses were used to evaluate associations of biomarkers levels and changes in biomarkers with cardiovascular and all-cause mortality. Hazard ratios were calculated for each 1-unit increase in log1.5(biomarker level). Analyses were adjusted for age, sex, race, comorbid conditions, ankle-brachial index, and other confounders.

RESULTS

Seventy-six patients (20%) died during follow-up. Higher levels of D-dimer, C-reactive protein, and serum amyloid A were associated with higher all-cause mortality among patients who died within 1 year after biomarker measurement (hazard ratio, 1.20 [95% CI, 1.08 to 1.33], 1.13 [CI, 1.05 to 1.21], and 1.12 [CI, 1.04 to 1.20], respectively; P < 0.001, P < 0.001, and P = 0.003) and among patients who died 1 to 2 years after biomarker measurement (hazard ratio, 1.14 [CI, 1.02 to 1.27], 1.15 [CI, 1.06 to 1.24], and 1.13 [CI, 1.04 to 1.24]; P = 0.022, P = 0.001, and P = 0.005]). However, higher levels of each biomarker were not associated with all-cause mortality for deaths occurring 2 to 3 years after biomarker measurement. Similar results were observed for cardiovascular mortality. Greater increases in each biomarker were associated with higher all-cause and cardiovascular mortality during the following year.

CONCLUSIONS

The small number of deaths limited the statistical power of the analyses.

CONCLUSIONS

Among persons with PAD, circulating levels of D-dimer and inflammatory markers are higher in the 1 to 2 years before death than in periods more remote from death. Increasing levels of D-dimer and inflammatory biomarkers are independently associated with higher mortality in persons with PAD.

A basic phospholipase A<em>2</em> (PLA<em>2</em>) enzyme, TFV PL-X (pI 9.<em>2</em>) an<em>d</em> two aci<em>d</em>ic PLA<em>2</em> enzymes, TFV PL-Ia (pI 4.9) an<em>d</em> TFV PL-Ib (pI 4.5) were purifie<em>d</em> from Trimeresurus flavoviri<em>d</em>is venom on CM-Sepha<em>d</em>ex C-<em>2</em>5 an<em>d</em> QAE-Sepha<em>d</em>ex A-<em>2</em>5 columns, respectively. The basic enzyme exists as a monomer, whereas the aci<em>d</em>ic enzymes are <em>dimers</em>. These enzymes <em>d</em>iffer in properties such as molecular weight, Km, optimum pH an<em>d</em> temperature an<em>d</em> pharmacological properties. The basic enzyme hy<em>d</em>rolyse<em>d</em> purifie<em>d</em> phospholipi<em>d</em>s in the or<em>d</em>er of PC greater than PE greater than PS greater than PI = 0, while for TFV PL-Ia an<em>d</em> TFV PL-Ib the or<em>d</em>er was PC greater than PE greater than PS = PI = 0. TFV PL-X was comparatively more toxic, with an LD50 value of 4.<em>2</em> micrograms/g (i.p.), while the aci<em>d</em>ic PLA<em>2</em> enzymes ha<em>d</em> LD50 values above 8 micrograms/g (i.p.). All three enzymes in<em>d</em>uce<em>d</em> e<em>d</em>ema when injecte<em>d</em> into the mouse foot pa<em>d</em>. Aristolochic aci<em>d</em>, an alkaloi<em>d</em> (8-methoxy-6-nitrophenanthro(3,4-<em>d</em>)-1,3-<em>d</em>ioxole-5-carboxylic aci<em>d</em>) from the me<em>d</em>icinal plant Aristolochia ra<em>d</em>ix, interacts with these PLA<em>2</em> enzymes. It is a competitive inhibitor with varying affinity when PC is use<em>d</em> as substrate. Aristolochic aci<em>d</em> inhibits <em>d</em>irect an<em>d</em> in<em>d</em>irect hemolytic activity, as well as e<em>d</em>ema-in<em>d</em>ucing activity, of TFV PL-X, but fails to neutralize the lethal potency of the enzyme. On the other han<em>d</em>, it inhibits <em>d</em>irect an<em>d</em> in<em>d</em>irect lytic activity of TFV PL-Ia an<em>d</em> TFV PL-Ib only at 10-fol<em>d</em> higher concentrations an<em>d</em> it enhances the e<em>d</em>ema-in<em>d</em>ucing activity of these enzymes. Such effects of aristolochic aci<em>d</em> in<em>d</em>icates that (1) <em>d</em>ifferent mechanisms may be involve<em>d</em> in the e<em>d</em>ema-in<em>d</em>ucing activity of PLA<em>2</em> enzymes an<em>d</em> (<em>2</em>) catalytic an<em>d</em> pharmacological sites are separate on the PLA<em>2</em> molecule.

The use of non-crystallographic symmetry restraints in the refinement of the haemocyanin hexamer from Panulirus interruptus at 3.<em>2</em> A resolution has resulted in a final model with a very reasonable geometry and a crystallographic R-factor of <em>2</em>0.1%, using 59,193 observed structure factor amplitudes between 8.0 and 3.<em>2</em> A. The mean co-ordinate error is approximately 0.35 A. The six subunits appear to be related by symmetry operations that differ slightly from 3<em>2</em> point group symmetry. The six subunits have essentially maintained the same structure. The hexamer, with point group 3<em>2</em>, is best described as a trimer of "tight <em>dimers</em>". The contacts between the subunits in such a <em>dimer</em> are more numerous, and better conserved during evolution than contacts in a trimer. The interface of a tight <em>dimer</em> is separated by an internal cavity into two "contact areas". The contact area nearest to the centre of the hexamer is most extensive and consists mainly of residues that are quite conserved among arthropodan haemocyanins. All these residues are provided by the second domain of each subunit. Hence, this second domain may play a crucial role in the allosteric functioning of this oxygen transport protein. The dinuclear copper oxygen-binding site resides in the centre of domain <em>2</em>. This oxygen-binding centre is not fully accessible from the solvent. Three large cavities occur, however, within each subunit at the interfaces of the three domains. All three cavities contain ordered water molecules, and two of them are accessible from the surrounding solvent. These cavities may play a role in facilitating fast movement of dioxygen towards the binding site, which is situated in a highly conserved, rather hydrophobic core. A detailed definition of the geometry of the copper site is, of course, not possible at the limited resolution of 3.<em>2</em> A. Nevertheless, it is possible to conclude that each copper is co-ordinated by two, more or less tightly bound, histidine ligands and one more distant histidine residue. The six histidine residues utilize their N epsilon atoms for copper co-ordination, while their N <em>delta</em> atoms are engaged in hydrogen bonds with conserved residues or water molecules. The two distant histidine ligands are located in apical positions and are on opposite sides with respect to the plane approximately defined by the four more tightly bound histidine ligands and the two copper ions. The copper-to-copper distance is 3.5 to 3.6 A in four of the subunits, but this distance deviates considerably in two others.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

The optimal duration of anticoagulation for a first episode of unprovoked venous thromboembolism (VTE) is uncertain. Methods for predicting risk for recurrence may identify low-risk patients who are less likely to benefit from prolonged anticoagulation.

OBJECTIVE

To synthesize evidence evaluating the value of D-dimer as a predictor of recurrent disease in patients who have stopped anticoagulant therapy after a first unprovoked VTE.

METHODS

The MEDLINE, EMBASE, CINAHL, and Cochrane databases were searched until March 2008 without language restrictions. The strategy was supplemented with manual review of reference lists and contact with content experts.

METHODS

Randomized, controlled trials or prospective cohort studies that measured D-dimer after anticoagulant therapy in patients who received at least 3 months of anticoagulant treatment of unprovoked VTE.

METHODS

Two authors independently reviewed articles and extracted data.

RESULTS

Seven studies, totaling 1888 patients with a first unprovoked VTE, were eligible for analysis. During 4500 person-years of follow up, annual rates of recurrent VTE differed statistically significantly: 8.9% (95% CI, 5.8% to 11.9%) in patients with positive D-dimer results and 3.5% (CI, 2.7% to 4.3%) in patients with negative D-dimer results.

CONCLUSIONS

The duration of anticoagulation, timing of D-dimer testing, and D-dimer assay varied across studies.

CONCLUSIONS

In patients who have completed at least 3 months of anticoagulation for a first episode of unprovoked VTE and after approximately 2 years of follow-up, a negative D-dimer result was associated with a 3.5% annual risk for recurrent disease, whereas a positive D-dimer result was associated with an 8.9% annual risk for recurrence. These rates should inform decisions about the balance of risks and benefits of prolonging anticoagulation.

BACKGROUND

In patients with unprovoked venous thromboembolism (VTE), the optimal duration of anticoagulation is anchored on estimating the risk of disease recurrence.

OBJECTIVE

We aimed to develop a score that could predict the recurrence risk following a first episode of unprovoked VTE, pooling individual patient data from seven prospective studies.

METHODS

One thousand eight hundred and eighteen cases with unprovoked VTE treated for at least 3 months with a vitamin K antagonist were available for analysis. Optimism-corrected Cox regression coefficients were used to develop a recurrence score that was subsequently internally validated by bootstrap analysis.

RESULTS

Abnormal <em>D</em>-<em>dimer</em> after stopping anticoagulation, age <50 years, male sex and VTE not associated with hormonal therapy (in women) were the main predictors of recurrence and were used to derive a prognostic recurrence score (<em>D</em>ASH, <em>D</em>-<em>dimer</em>, Age, Sex, Hormonal therapy) showing a satisfactory predictive capability (ROC area =0.71). The annualized recurrence risk was 3.1% (95% confidence interval [CI], <em>2</em>.3-3.9) for a score ≤ 1, 6.4% (95% CI, 4.8-7.9) for a score=<em>2</em> and 1<em>2</em>.3% (95% CI, 9.9-14.7) for a score ≥ 3. By considering at low recurrence risk those patients with a score ≤ 1, life-long anticoagulation might be avoided in about half of patients with unprovoked VTE.

CONCLUSIONS

The DASH prediction rule appears to predict recurrence risk in patients with a first unprovoked VTE and may be useful to decide whether anticoagulant therapy should be continued indefinitely or stopped after an initial treatment period of at least 3 months.