Nectins are Ca(<em>2</em>+)-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions. Nectins have three Ig-like domains in the extracellular region, and the first one is essential in cell-cell adhesion and plays a central role in the interaction with the envelope glycoprotein <em>D</em> of several viruses. Five Nectin-like molecules (Necl-1 through -5) with similar domain structures to those of Nectins have been identified. Necl-1 is specifically expressed in neural tissue, has Ca(<em>2</em>+)-independent homophilic and heterophilic cell-cell adhesion activity, and plays an important role in the formation of synapses, axon bundles, and myelinated axons. Here we report the first crystal structure of its N-terminal Ig-like V domain at <em>2</em>.4 A, providing insight into trans-cellular recognition mediated by Necl-1. The protein crystallized as a <em>dimer</em>, and the <em>dimer</em>ic form was confirmed by size-exclusion chromatography and chemical cross-linking experiments, indicating this V domain is sufficient for homophilic interaction. Mutagenesis work demonstrated that Phe(8<em>2</em>) is a key residue for the adhesion activity of Necl-1. A model for homophilic adhesion of Necl-1 at synapses is proposed based on its structure and previous studies.

The kinetic mechanism of the nonclaret <em>d</em>isjunctional protein (Nc<em>d</em>) motor was investigate<em>d</em> using the <em>dimer</em> terme<em>d</em> MC1 (resi<em>d</em>ues <em>2</em>09-700), which has been shown to exhibit negative-en<em>d</em> <em>d</em>irecte<em>d</em> motility (Chan<em>d</em>ra et al., 1993). The kinetic properties are similar to those of the monomeric Nc<em>d</em> motor <em>d</em>omain (Pechatnikova an<em>d</em> Taylor, 1997). The maximum stea<em>d</em>y-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. <em>D</em>issociation constants in the presence of nucleoti<em>d</em>es showe<em>d</em> the same tren<em>d</em> but with approximately a two-fol<em>d</em> <em>d</em>ecrease in the values: K(<em>d</em>) values are 1.0 microM for A<em>D</em>P-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for A<em>D</em>P, an<em>d</em> 10 microM for A<em>D</em>P-vana<em>d</em>ate (in <em>2</em>5 mM NaCl, <em>2</em><em>2</em> <em>d</em>egrees C). The apparent secon<em>d</em>-or<em>d</em>er rate constants for the bin<em>d</em>ing of ATP an<em>d</em> A<em>D</em>P to the microtubule-motor complex (MtMC1) are <em>2</em> microM(-1) s(-1). Base<em>d</em> on measurements at high microtubule concentrations the kinetic steps were fitte<em>d</em> to the scheme,[see text] where N refers to one hea<em>d</em> of the <em>dimer</em> an<em>d</em> T, <em>D</em>, an<em>d</em> P stan<em>d</em> for ATP, A<em>D</em>P, an<em>d</em> inorganic phosphate. k(1) an<em>d</em> k(-4) are the first-or<em>d</em>er rate constants of the transition in<em>d</em>uce<em>d</em> by the bin<em>d</em>ing of mant ATP an<em>d</em> mant A<em>D</em>P respectively. A<em>D</em>P release is the main rate-limiting step in the MtMC1 mechanism. The bin<em>d</em>ing of the MC1-mant A<em>D</em>P complex to microtubules release<em>d</em> less than half of the mant A<em>D</em>P (alternating site reactivity). The secon<em>d</em> mant A<em>D</em>P is only release<em>d</em> by the bin<em>d</em>ing of nucleoti<em>d</em>es that <em>d</em>issociate the MtMC1 complex (ATP an<em>d</em> A<em>D</em>P but not AMPPNP). The apparent rate constant for <em>d</em>issociation of the secon<em>d</em> mant A<em>D</em>P is four times smaller than the first an<em>d</em> much smaller than the rate of <em>d</em>issociation of MtMC1 by ATP or A<em>D</em>P. These results are explaine<em>d</em> by a mo<em>d</em>el in which MC1.A<em>D</em>P is first <em>d</em>issociate<em>d</em> from the microtubule by ATP, followe<em>d</em> by rebin<em>d</em>ing to the microtubule by the A<em>D</em>P-containing hea<em>d</em>. Nc<em>d</em> may follow a <em>d</em>ifferent reaction pathway than <em>d</em>oes kinesin, but the <em>d</em>ifferences in rate constants <em>d</em>o not explain the opposite <em>d</em>irection of motion. The kinetic evi<em>d</em>ence an<em>d</em> the high ratio of motile velocity to ATPase support a nonprocessive, low <em>d</em>uty cycle mechanism for the Nc<em>d</em> motor.

The structure of the chromomycin-<em>D</em>NA complex at the <em>d</em>eoxyoctanucleoti<em>d</em>e <em>d</em>uplex level has been <em>d</em>etermine<em>d</em> from one- an<em>d</em> two-<em>d</em>imensional proton NMR stu<em>d</em>ies in Mg-containing aqueous solution. The NMR results <em>d</em>emonstrate that the antitumor agent bin<em>d</em>s as a symmetrical <em>dimer</em> to the self-complementary <em>d</em>[T-T-G-G-C-C-A-A] <em>d</em>uplex with retention of the <em>2</em>-fol<em>d</em> symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the <em>dimer</em> share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hy<em>d</em>rophilic e<em>d</em>ge of the chromophore is locate<em>d</em> next to the G-G.C-C half-site an<em>d</em> each C-<em>D</em>-E trisacchari<em>d</em>e chain exten<em>d</em>s towar<em>d</em> the 3'-<em>d</em>irection of the octanucleoti<em>d</em>e <em>d</em>uplex. In a<em>d</em><em>d</em>ition, the A-B <em>d</em>isacchari<em>d</em>e segment an<em>d</em> the hy<em>d</em>rophilic si<em>d</em>e chain of the antitumor agent are <em>d</em>irecte<em>d</em> towar<em>d</em> the phosphate backbone. The observe<em>d</em> changes in nucleic aci<em>d</em> NOEs an<em>d</em> coupling patterns on complex formation establish a transition to a wi<em>d</em>er an<em>d</em> shallower minor groove at the central G-G-C-C.G-G-C-C segment require<em>d</em> for accommo<em>d</em>ating the chromomycin <em>dimer</em>. The present <em>d</em>emonstration that chromomycin bin<em>d</em>s as a <em>dimer</em> an<em>d</em> switches the conformation of the <em>D</em>NA at its G.C-rich minor groove bin<em>d</em>ing site provi<em>d</em>es new insights into antitumor agent <em>d</em>esign an<em>d</em> the sequence specificity of antitumor agent-<em>D</em>NA recognition.

Enolase, a glycolytic enzyme that catalyzes the <em>d</em>ehy<em>d</em>ration of <em>2</em>-phospho-<em>d</em>-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homo<em>dimer</em> in all eukaryotes an<em>d</em> many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase an<em>d</em> an equilibrium mixture of PGA an<em>d</em> PEP. The structure has been refine<em>d</em> using <em>2</em>9 854 reflections with an F/sigma(F) of>>/=3 to an R of 0.137 with average <em>d</em>eviations of bon<em>d</em> lengths an<em>d</em> bon<em>d</em> angles from i<em>d</em>eal values of 0.013 A an<em>d</em> 3.1 <em>d</em>egrees , respectively. In this structure, the <em>dimer</em> constitutes the crystallographic asymmetric unit. The two subunits are similar, an<em>d</em> their superposition gives a rms <em>d</em>istance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits <em>d</em>iffer by up to 4 A an<em>d</em> the loop Ser<em>2</em>50-Gln<em>2</em>77, which follows the catalytic loop Val153-Phe169. In the first subunit, the imi<em>d</em>azole si<em>d</em>e chain of His159 is in contact with the phosphate group of the substrate/pro<em>d</em>uct molecule; in the other it is separate<em>d</em> by water molecules. A series of hy<em>d</em>rogen bon<em>d</em>s lea<em>d</em>ing to a neighboring enolase <em>dimer</em> can be i<em>d</em>entifie<em>d</em> as being responsible for or<em>d</em>ering an<em>d</em> stabilization of the conformationally <em>d</em>ifferent subunits in the crystal lattice. The electron <em>d</em>ensity present in the active site suggests that in the active site with the <em>d</em>irect ligan<em>d</em>-His159 hy<em>d</em>rogen bon<em>d</em> PGA is pre<em>d</em>ominantly boun<em>d</em> while in the active site where water molecules separate His159 from the ligan<em>d</em> the bin<em>d</em>ing of PEP <em>d</em>ominates. The structure in<em>d</em>icates that the water molecule hy<em>d</em>rating carbon-3 of PEP in the PEP ->> PGA reaction is activate<em>d</em> by the carboxylates of Glu168 an<em>d</em> Glu<em>2</em>11. The crystals are unique because they have resolve<em>d</em> two interme<em>d</em>iates on the opposite si<em>d</em>es of the transition state.

In vertebrate smooth muscles, phosphorylation of the regulatory light chain appears to be necessary for actin activation of the Mg-ATPase activity and for the in vitro assembly of myosin into filaments. From a correlation between the degree of phosphorylation and enzymatic activity, it was suggested that both myosin heads must be phosphorylated before either head could be activated by actin, and that phosphorylation of filamentous myosin occurred in a negatively cooperative manner (Persechini, A., and Hartshorne, <em>D</em>. J. (1981) Science <em>2</em>13, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (198<em>2</em>) J. Biochem. (Tokyo) 91, 1809-181<em>2</em>; Sellers, J. R., Chock, P. B., and Adelstein, R. S. (1983) J. Biol. Chem. <em>2</em>58, 14181-14188). Here we have determined the mechanism of phosphorylation by separating dephosphorylated and phosphorylated myosin species based on their different structural properties in the minifilament buffer system (5 mM citrate, <em>2</em><em>2</em> mM Tris). Fully phosphorylated myosin remained assembled as minifilaments in 1 mM Mg-ATP, but dephosphorylated myosin dissociated to a mixture of folded monomers and <em>dimers</em>. Gel filtration was used to separate these two structures. At intermediate levels of phosphorylation, the relative amount of myosin that formed minifilament and <em>dimer</em> and the degree of phosphorylation of the separated species relative to the initial level of phosphorylation was measured. From these data, it was possible to deduce that singly and doubly phosphorylated myosin remained assembled in the presence of nucleotide. Myosin molecules with 0, 1, or <em>2</em> heads phosphorylated could also be separated by nondenaturing gel electrophoresis. The amount of myosin which formed each species was quantitated as a function of phosphorylation. Results from the combined approaches are consistent with a model in which light chain kinase randomly phosphorylates myosin, independent of the state of aggregation of the myosin.

The X-ray crystallographic structures of the metal-activated enzyme xylose isomerase from Streptomyces olivochromogenes with the substrates <em>D</em>-glucose, 3-O-methyl-<em>D</em>-glucose and in the absence of substrate were determined to 1.96-, <em>2</em>.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose isomerase catalyzes the interconversion between glucose and fructose (xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts xylose isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-<em>2</em> was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O<em>2</em> and O4 and by forming hydrogen bonds with His53 through O5 and Lys18<em>2</em> through O1. Mg-<em>2</em> has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O<em>2</em> and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible <em>dimers</em>, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this xylose isomerase.

The diagnosis of pulmonary embolism (PE) is usually established by a combination of clinical assessment, <em>D</em>-<em>dimer</em> testing, and imaging with either pulmonary ventilation-perfusion (V/Q) scintigraphy or pulmonary multidetector CT (M<em>D</em>CT) angiography. Both V/Q SPECT and M<em>D</em>CT angiography seem to have high diagnostic accuracy. However, only limited data directly comparing these <em>2</em> modalities are available. Hybrid gamma-camera/M<em>D</em>CT systems have been introduced and allow simultaneous 3-dimensional lung V/Q SPECT and M<em>D</em>CT angiography, suitable for diagnosing PE. The aim of our study was to compare, in a prospective design, the diagnostic ability of V/Q SPECT, V/Q SPECT combined with low-dose CT, and pulmonary M<em>D</em>CT angiography obtained simultaneously using a combined SPECT/M<em>D</em>CT scanner in patients suspected of having PE.

METHODS

Consecutive patients from June <em>2</em>006 to February <em>2</em>008 suspected of having acute PE were referred to the <em>D</em>epartment of Nuclear Medicine at Rigshospitalet or Frederiksberg Hospital, <em>D</em>enmark, for V/Q SPECT as a first-line imaging procedure. The number of eligible patients was 196. Patients with positive <em>D</em>-<em>dimer</em> results (>0.5 mmol/mL) or a clinical assessment with a Wells score greater than <em>2</em> were included and underwent V/Q SPECT, low-dose CT, and pulmonary M<em>D</em>CT angiography in a single session. Patient follow-up was 6 mo.

RESULTS

A total of 81 simultaneous studies were available for analysis, of which 38% were from patients with PE. V/Q SPECT had a sensitivity of 97% and a specificity of 88%. When low-dose CT was added, the sensitivity was still 97% and the specificity increased to 100%. Perfusion SPECT with low-dose CT had a sensitivity of 93% and a specificity of 51%. MDCT angiography alone had a sensitivity of 68% and a specificity of 100%.

CONCLUSIONS

We conclude that V/Q SPECT in combination with low-dose CT without contrast enhancement has an excellent diagnostic performance and should therefore probably be considered first-line imaging in the work-up of PE in most cases.

Electron transfer from the Rieske iron-sulfur protein to cytochrome c(1) (cyt c(1)) in the Rhodobacter sphaeroides cytochrome bc(1) complex was studied using a ruthenium <em>dimer</em> complex, Ru(<em>2</em>)<em>D</em>. Laser flash photolysis of a solution containing reduced cyt bc(1), Ru(<em>2</em>)<em>D</em>, and a sacrificial electron acceptor results in oxidation of cyt c(1) within 1 micros, followed by electron transfer from the iron-sulfur center (<em>2</em>Fe-<em>2</em>S) to cyt c(1) with a rate constant of 80,000 s(-1). Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of +17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of <em>2</em>Fe-<em>2</em>S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of <em>2</em>Fe-<em>2</em>S by 6<em>2</em>, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from <em>2</em>Fe-<em>2</em>S to cyt c(1) is controlled by conformational gating.

BACKGROUND

Previous studies have suggested that statins exert beneficial effects beyond their favorable lipid lowering effect. Particularly, the modification of thrombus formation and degradation, alteration in inflammatory response, plaque stabilization and improved endothelial function are thought to be responsible for additional reduction of morbidity and mortality due to cardiovascular events. To date, however, it is still unclear whether these effects are elicited by all statins.

RESULTS

We set out to compare in a controlle<em>d</em>, ran<em>d</em>omize<em>d</em>, <em>d</em>ouble-blin<em>d</em> stu<em>d</em>y <em>d</em>esign the effects of almost equieffective cholesterol lowering <em>d</em>oses of three chemically an<em>d</em> pharmacokinetically <em>d</em>ifferent statins (atorvastatin, simvastatin, pravastatin) on hemostatic an<em>d</em> inflammatory markers in 99 hypercholesterolemic patients. At entry an<em>d</em> 3 months after onset of statin therapy plasma cholesterol an<em>d</em> von Willebran<em>d</em> factor antigen (vWf-Ag), fibrinogen, <em>d</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em> (F1.<em>2</em>) an<em>d</em> C-reactive protein (CRP) were measure<em>d</em>. The effect on plasma values of F1.<em>2</em>, vWf-Ag, <em>d</em>-<em>dimer</em> an<em>d</em> CRP was not significantly <em>d</em>ifferent between the three treatment groups. The effect of simvastatin on fibrinogen (p = 0.005) was more pronounce<em>d</em> than the effects of atorvastatin (p = 0.48 n.s.) an<em>d</em> pravastatin (p = 0.15 n.s.). Plasma levels of F1.<em>2</em> an<em>d</em> vWf-Ag (when <em>d</em>ata of all statins were poole<em>d</em>) were significantly re<em>d</em>uce<em>d</em> by 7% an<em>d</em> 10% versus baseline, respectively. No significant re<em>d</em>uction was observe<em>d</em> for <em>d</em>-<em>dimer</em> (p = 0.<em>2</em>6) an<em>d</em> CRP (p = 0.5). Total plasma cholesterol levels <em>d</em>ecrease<em>d</em> significantly (p < 0.0001 in all groups) between <em>2</em><em>2</em>% an<em>d</em> <em>2</em>9% compare<em>d</em> to baseline.

CONCLUSIONS

The present study shows similar short-term (3 months) effects of atorvastatin, simvastatin and pravastatin on selected hemostatic and inflammatory parameters in plasma in patients with hypercholesterolemia. Thus, chemical and pharmacological differences between statins appear to exert no major influence on these parameters.

The optimal duration of anticoagulation in patients with venous thromboembolism (VTE) is uncertain. We investigated whether persistently negative <em>D</em>-<em>dimers</em> in patients with vein recanalization or stable thrombotic burden can identify subjects at low recurrence risk. Outpatients with a first VTE (unprovoked or associated with weak risk factors) were eligible after at least 3 months (1<em>2</em> in those with residual thrombosis) of anticoagulation. They received serial <em>D</em>-<em>dimer</em> measurements using commercial assays with predefined age/sex-specific cutoffs and were followed for up to <em>2</em> years. Of 1010 patients, anticoagulation was stopped in 5<em>2</em>8 (5<em>2</em>.3%) with persistently negative <em>D</em>-<em>dimer</em> who subsequently experienced <em>2</em>5 recurrences (3.0% pt-y; 95% confidence interval [CI], <em>2</em>.0-4.4%). Of the remaining 48<em>2</em> patients, 373 resumed anticoagulation and 109 refused it. Recurrent VTE developed in 15 patients (8.8% pt-y; 95% CI, 5.0-14.1) of the latter group and in 4 of the former (0.7% pt-y; 95% CI, 0.<em>2</em>-1.7; hazard ratio = <em>2</em>.9<em>2</em>; 95% CI, 1.87-9.7<em>2</em>; P = .0006). Major bleeding occurred in 14 patients (<em>2</em>.3% pt-y; 95% CI, 1.3-3.9) who resumed anticoagulation. Serial <em>D</em>-<em>dimer</em> measurement is suitable in clinical practice for the identification of VTE patients in whom anticoagulation can be safely discontinued. This study was registered at clinicaltrials.gov as #NCT00954395.

<strong class="sub-title"> Background: </strong> The COVID-19 pandemic, caused by SARS-CoV-<em>2</em> virus, has resulted in over 100,000 deaths in the USA. Our institution has treated over <em>2</em>000 COVID-19 patients during the pandemic in New York City. The pandemic directly impacted cancer patients and the organization of cancer care. Mount Sinai Hospital has a large and diverse multiple myeloma (MM) population. Herein, we report the characteristics of COVID-19 infection and serological response in MM patients in a large tertiary care institution in New York.

<strong class="sub-title"> Methods: </strong> We performed a retrospective study on a cohort of 58 patients with a plasma-cell disorder (54 MM, 4 smoldering MM) who developed COVID-19 between March 1, <em>2</em>0<em>2</em>0, and April 30, <em>2</em>0<em>2</em>0. We report epidemiological, clinical, and laboratory characteristics including the persistence of viral detection by polymerase chain reaction (PCR) and anti-SARS-CoV-<em>2</em> antibody testing, treatments initiated, and outcomes.

<strong class="sub-title"> Results: </strong> Of the 58 patients diagnosed with COVID-19, 36 were hospitalized and <em>2</em><em>2</em> were managed at home. The median age was 67 years; 5<em>2</em>% of patients were male and 63% were non-White. Hypertension (64%), hyperlipidemia (6<em>2</em>%), obesity (37%), diabetes mellitus (<em>2</em>8%), chronic kidney disease (<em>2</em>4%), and lung disease (<em>2</em>1%) were the most common comorbidities. In the total cohort, 14 patients (<em>2</em>4%) died. Older age (> 70 years), male sex, cardiovascular risk, and patients not in complete remission (CR) or stringent CR were significantly (p < 0.05) associated with hospitalization. Among hospitalized patients, laboratory findings demonstrated elevation of traditional inflammatory markers (CRP, ferritin, D-dimer) and a significant (p < 0.05) association between elevated inflammatory markers, severe hypogammaglobulinemia, non-White race, and mortality. Ninety-six percent (<em>2</em><em>2</em>/<em>2</em>3) of patients developed antibodies to SARS-CoV-<em>2</em> at a median of 3<em>2</em> days after initial diagnosis. The median time to PCR negativity was 43 (range 19-68) days from initial positive PCR.

<strong class="sub-title"> Conclusions: </strong> Drug exposure and MM disease status at the time of contracting COVID-19 had no bearing on mortality. Mounting a severe inflammatory response to SARS-CoV-<em>2</em> and severe hypogammaglobulinemia was associated with higher mortality. The majority of patients mounted an antibody response to SARS-CoV-<em>2</em>. These findings pave a path to the identification of vulnerable MM patients who need early intervention to improve outcomes in future outbreaks of COVID-19.

<strong class="sub-title"> Keywords: </strong> COVID-19; Multiple myeloma; New York; Pandemic; SARS; SARS-Cov-<em>2</em>; Smoldering multiple myeloma.

Concomitant coagulation disorder can occur in severe patients withCOVI<em>D</em>-19, but in-depth studies are limited. This study aimed to describe the parameters of coagulation function of patients with COVI<em>D</em>-19 and reveal the risk factors of developing severe disease. This study retrospectively analyzed 113patients with SARS-CoV-<em>2</em> infection in Taizhou Public Health Center. Clinical characteristics and indexes of coagulation function were collected. A multivariate Cox analysis was performed to identify potential biomarkers for predicting disease progression. Based on the results of multivariate Cox analysis, a Nomogram was built and the predictive accuracy was evaluated through the calibration curve, decision curve, clinical impact curve, and Kaplan-Meier analysis. Sensitivity, specificity, predictive values were calculated to assess the clinical value. The data showed that Fibrinogen, FAR, and <em>D</em>-<em>dimer</em> were higher in the severe patients, while PLTcount, Alb were much lower. Multivariate Cox analysis revealed that FAR and PLT count were independent risk factors for disease progression. The optimal cutoff values for FAR and PLT count were 0.0883 and 135*10⁹/L, respectively. The C-index [0.71<em>2</em> (95% CI = 0.610-0.814)], decision curve, clinical impact curve showed that Nomogram could be used to predict the disease progression. In addition, the Kaplan-Meier analysis revealed that potential risk decreased in patients with FAR<0.0883 and PLT count>135*10⁹/L.The model showed a good negative predictive value [(0.9474 (95%CI = 0.845-0.986)].This study revealed that FAR and PLT count were independent risk factors for severe illness and the severity of COVI<em>D</em>-19 might be excluded when FAR<0.0883 and PLT count>135*10⁹/L.

Rapid attachment to actin of the detached motor domain of myosin <em>dimers</em> with one motor domain already attached has been hypothesized to explain the stretch-induced changes in X-ray interference and stiffness of active muscle. Here, using half-sarcomere mechanics in single frog muscle fibres (<em>2</em>.15 microm sarcomere length and 4 degrees C), we show that: (1) an increase in stiffness of the half-sarcomere under stretch is specific to isometric contraction and does not occur in rigor, indicating that the mechanism of stiffness increase is an increase in the number of attached motors; (<em>2</em>) <em>2</em> ms after 100 micros stretches (amplitude <em>2</em>-8 nm per half-sarcomere) imposed during an isometric tetanus, the stiffness of the array of myosin motors in each half-sarcomere (e(m)) increases above the isometric value (e(m0)); (3) e(m) has a sigmoidal dependence on the distortion of the motor domains (<em>Delta</em> z) attached in isometric contraction, with a maximum approximately <em>2</em> e(m0) for a distortion of approximately 6 nm; e(m) is influenced by detachment of motors at z>> 6 nm; (4) at the end of the 100 micros stretch the relation between e(m)/e(m0) and <em>Delta</em> z lies slightly but not significantly above that at <em>2</em> ms. These results support the idea that stretch-induced sliding of the actin filament distorts the actin-attached motor domain of the myosin <em>dimers</em> away from the centre of the sarcomere, providing the steric conditions for rapid attachment of the second motor domain. The rate of new motor attachment must be as high as 7.5 x 10(4) s(1) and explains the rapid and efficient increase of the resistance of active muscle to stretch.

TtgV modulates the expression of the ttgGHI operon, which encodes an efflux pump that extrudes a wide variety of chemicals including mono- and binuclear aromatic hydrocarbons, aliphatic alcohols, and antibiotics of dissimilar chemical structure. Using a 'lacZ fusion to the ttgG promoter, we show that the most efficient in vivo inducers were 1-naphthol, <em>2</em>,3-dihydroxynaphthalene, 4-nitrotoluene, benzonitrile, and indole. The thermodynamic parameters for the binding of different effector molecules to purified TtgV were determined by isothermal titration calorimetry. For the majority of effectors, the interaction was enthalpy-driven and counterbalance by unfavorable entropy changes. The TtgV-effector dissociation constants were found to vary between <em>2</em> and 890 mum. There was a relationship between TtgV affinity for the different effectors and their potential to induce gene expression in vivo, indicating that the effector binding constant is a major determinant for efficient efflux pump gene expression. Equilibrium dialysis and isothermal titration calorimetry studies indicated that a TtgV <em>dimer</em> binds one effector molecule. No evidence for the simultaneous binding of multiple effectors to TtgV was obtained. The binding of TtgV to a 63-bp <em>D</em>NA fragment containing its cognate operator was tight and entropy-driven (K(<em>D</em>) = <em>2</em>.4 +/- 0.35 nm, <em>D</em>eltaH = 5.5 +/- 0.04 kcal/mol). The TtgV-<em>D</em>NA complex was shown to bind 1-napthol with an affinity comparable with the free soluble TtgV protein, K(<em>D</em>) = 4.8 +/- 0.19 and 3.0 +/- 0.15 mum, respectively. The biological relevance of this finding is discussed.

Although coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) predominantly disrupts the respiratory system, there is accumulating experience that the disease, particularly in its more severe manifestations, also affects the cardiovascular system. Cardiovascular risk factors and chronic cardiovascular conditions are prevalent among patients affected by COVI<em>D</em>-19 and associated with adverse outcomes. However, whether pre-existing cardiovascular disease is an independent determinant of higher mortality risk with COVI<em>D</em>-19 remains uncertain. Acute cardiac injury, manifest by increased blood levels of cardiac troponin, electrocardiographic abnormalities, or myocardial dysfunction, occurs in up to ~60% of hospitalized patients with severe COVI<em>D</em>-19. Potential contributors to acute cardiac injury in the setting of COVI<em>D</em>-19 include (1) acute changes in myocardial demand and supply due to tachycardia, hypotension, and hypoxemia resulting in type <em>2</em> myocardial infarction; (<em>2</em>) acute coronary syndrome due to acute atherothrombosis in a virally induced thrombotic and inflammatory milieu; (3) microvascular dysfunction due to diffuse microthrombi or vascular injury; (4) stress-related cardiomyopathy (Takotsubo syndrome); (5) nonischemic myocardial injury due to a hyperinflammatory cytokine storm; or (6) direct viral cardiomyocyte toxicity and myocarditis. <em>D</em>iffuse thrombosis is emerging as an important contributor to adverse outcomes in patients with COVI<em>D</em>-19. Practitioners should be vigilant for cardiovascular complications of COVI<em>D</em>-19. Monitoring may include serial cardiac troponin and natriuretic peptides, along with fibrinogen, <em>D</em>-<em>dimer</em>, and inflammatory biomarkers. Management decisions should rely on the clinical assessment for the probability of ongoing myocardial ischemia, as well as alternative nonischemic causes of injury, integrating the level of suspicion for COVI<em>D</em>-19.

OBJECTIVE

To evaluate whether patients with Cushing's syndrome (CS) had i) changes in coagulative and fibrinolytic parameters associated with CS activity and ii) higher prevalence of venous thromboembolic events (VTE).

METHODS

Prospective study conducted on patients with CS evaluated at diagnosis and 1<em>2</em> months after surgery.

METHODS

Forty patients with active CS (36 with Cushing's disease (CD) and 4 with an adrenal adenoma) were evaluated. Forty normal subjects and 70 patients with non-ACTH-secreting pituitary adenomas served as controls. All patients and controls underwent an assessment of coagulation and fibrinolysis indexes before and after surgery.

RESULTS

CS patients at baseline had a hypercoagulative phenotype when compared with normal subjects (activated partial thromboplastin time (aPTT), fibrinogen, <em>D</em>-<em>Dimer</em>, von Willebrand factor (VWF), plasminogen activator inhibitor 1 (PAI-1 or SERPINE1), antithrombin III (ATIII or SERPINC1), P<0.0001, α(<em>2</em>) antiplasmin, P=0.0004, thrombin-antithrombin complex (TAT), P=0.01, factor IX (F9), P=0.03). Patients with still active disease after surgery had higher coagulative parameters than those in remission (VWF (P<0.0001), PAI-1 (P=0.004), TAT (P=0.0001), ATIII (P=0.000<em>2</em>) and α(<em>2</em>) antiplasmin (or SERPINF<em>2</em>; P=0.006)), whereas aPTT levels (P=0.007) were significantly reduced. VTE occurred in three patients with C<em>D</em> (7.5%): one had a pulmonary embolism and two patients had a deep venous thrombosis; no patients submitted to transsphenoidal surgery for non-Cushing's pituitary adenoma had VTE (P=0.04).

CONCLUSIONS

Patients with CS have a procoagulative phenotype due to cortisol-associated changes in haemostatic and fibrinolytic markers, leading to increased incidence of VTE. Thromboprophylaxis seems to be appropriated in patients with active disease, particularly in the postoperative period.

UV radiation (UVR) is essential for formation of vitamin <em>D</em>(3), which can be hydroxylated locally in the skin to 1α,<em>2</em>5-dihydroxyvitamin <em>D</em>(3) [1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3)]. Recent studies implicate 1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3) in reduction of UVR-induced <em>D</em>NA damage, particularly thymine <em>dimers</em>. There is evidence that photoprotection occurs through the steroid nongenomic pathway for 1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3) action. In the current study, we tested the involvement of the classical vitamin <em>D</em> receptor (V<em>D</em>R) and the endoplasmic reticulum stress protein 57 (ERp57), in the mechanisms of photoprotection. The protective effects of 1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3) against thymine <em>dimers</em> were abolished in fibroblasts from patients with hereditary vitamin <em>D</em>-resistant rickets that expressed no V<em>D</em>R protein, indicating that the V<em>D</em>R is essential for photoprotection. Photoprotection remained in hereditary vitamin <em>D</em>-resistant rickets fibroblasts expressing a V<em>D</em>R with a defective <em>D</em>NA-binding domain or a mutation in helix H1 of the classical ligand-binding domain, both defects resulting in a failure to mediate genomic responses, implicating nongenomic responses for photoprotection. Ab099, a neutralizing antibody to ERp57, and ERp57 small interfering RNA completely blocked protection against thymine <em>dimers</em> in normal fibroblasts. Co-IP studies showed that the V<em>D</em>R and ERp57 interact in nonnuclear extracts of fibroblasts. 1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3) up-regulated expression of the tumor suppressor p53 in normal fibroblasts. This up-regulation of p53, however, was observed in all mutant fibroblasts, including those with no V<em>D</em>R, and with Ab099; therefore, V<em>D</em>R and ERp57 are not essential for p53 regulation. The data implicate the V<em>D</em>R and ERp57 as critical components for actions of 1,<em>2</em>5-(OH)(<em>2</em>)<em>D</em>(3) against <em>D</em>NA damage, but the V<em>D</em>R does not require normal <em>D</em>NA binding or classical ligand binding to mediate photoprotection.

The Escherichia coli histone-like HU protein pool is composed of three <em>dimer</em>ic forms: two homo<em>dimers</em>, EcHUalpha(<em>2</em>) and EcHUbeta(<em>2</em>), and a hetero<em>dimer</em>, EcHUalphabeta. The relative abundance of these <em>dimer</em>ic forms varies during cell growth and in response to environmental changes, suggesting that each <em>dimer</em> plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (C<em>D</em>) were used to study the thermal stability of the three E.coli HU <em>dimers</em> and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(<em>2</em>)<->><em>2</em><em>D</em>), this present thermodynamic study shows that the three E.coli <em>dimers</em> melt according to a two-step mechanism (N(<em>2</em>)<->>I(<em>2</em>)<->><em>2</em><em>D</em>). The native <em>dimer</em>, N(<em>2</em>), melts partially into a <em>dimer</em>ic intermediate, I(<em>2</em>), which in turn yields the unfolded monomers, <em>D</em>. In addition, the crystal structure of the EcHUalpha(<em>2</em>) <em>dimer</em> has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(<em>2</em>) and the HU homo<em>dimer</em> from Bacillus stearothermophilus suggests that the E.coli <em>dimer</em> is constituted by two subdomains of different energetic properties. The C<em>D</em> study indicates that the intermediate, I(<em>2</em>), corresponds to an HU <em>dimer</em> having partly lost its alpha-helices. The partially unfolded <em>dimer</em> I(<em>2</em>) is unable to complex with high-affinity, single-stranded break-containing <em>D</em>NA. These structural, thermodynamic and functional results suggest that the N(<em>2</em>)<->>I(<em>2</em>) equilibrium plays a central role in the physiology of E.coli HU. The I(<em>2</em>) molecular species seems to be the EcHUbeta(<em>2</em>) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(<em>2</em>) might be required in E.coli for the HU chain exchange, which allows the hetero<em>dimer</em> formation from homo<em>dimers</em>.

The pathology of coronavirus disease 2019 (COVID-19) is exacerbated by the progression of thrombosis, and disseminated intravascular coagulation (DIC), and cytokine storms. The most frequently reported coagulation/fibrinolytic abnormality in COVID-19 is the increase in D-dimer, and its relationship with prognosis has been discussed. However, limits exist to the utility of evaluation by D-dimer alone. In addition, since the coagulation/fibrinolytic condition sometimes fluctuates within a short period of time, regular examinations in recognition of the significance of the examination are desirable. The pathophysiology of disseminated intravascular coagulation (DIC) associated with COVID-19 is very different from that of septic DIC, and both thrombotic and hemorrhagic pathologies should be noted. COVID-19 thrombosis includes macro- and microthrombosis, with diagnosis of the latter depending on markers of coagulation and fibrinolysis. Treatment of COVID-19 is classified into antiviral treatment, cytokine storm treatment, and thrombosis treatment. Rather than providing uniform treatment, the treatment method most suitable for the severity and stage should be selected. Combination therapy with heparin and nafamostat is expected to develop in the future. Fibrinolytic therapy and adsorption therapy require further study.

Keywords: COVID-19; Cytokine storm; DIC; Disseminated intravascular coagulation; SARS-CoV-2; Thrombosis.

The shikimate pathway, responsible for the biosynthesis of aromatic compoun<em>d</em>s, is essential for the growth of Mycobacterium tuberculosis an<em>d</em> is a potential target for the <em>d</em>esign of new anti-tuberculosis <em>d</em>rugs. The first step of this pathway is catalyze<em>d</em> by 3-<em>d</em>eoxy-<em>d</em>-arabino-heptulosonate 7-phosphate synthase (DAH7PS). The DAH7PSs have been classifie<em>d</em> into two apparently unrelate<em>d</em> types an<em>d</em>, whereas structural <em>d</em>ata have been obtaine<em>d</em> for the type I DAH7PSs, no structural information is available for their type II counterparts. The type II DAH7PS from M.tuberculosis has been expresse<em>d</em> in Escherichia coli, purifie<em>d</em>, functionally characterize<em>d</em> an<em>d</em> crystallize<em>d</em>. It is foun<em>d</em> to be metal ion-<em>d</em>epen<em>d</em>ent an<em>d</em> subject to fee<em>d</em>back inhibition by phenylalanine, tryptophan, tyrosine an<em>d</em> chorismate, with a significant synergistic effect when tryptophan is use<em>d</em> in combination with phenylalanine. The crystal structure of M.tuberculosis DAH7PS has been <em>d</em>etermine<em>d</em> by single-wavelength anomalous <em>d</em>iffraction an<em>d</em> refine<em>d</em> at <em>2</em>.3A in complex with substrate phosphoenolpyruvate an<em>d</em> Mn(<em>2</em>+). The structure reveals a tightly associate<em>d</em> <em>dimer</em> of (beta/alpha)(8) TIM barrels. The monomer fol<em>d</em>, the arrangement of key resi<em>d</em>ues in the active site, an<em>d</em> the bin<em>d</em>ing mo<em>d</em>es of PEP an<em>d</em> Mn(<em>2</em>+), all match those of the type I enzymes, an<em>d</em> in<em>d</em>icate a common ancestry for the type I an<em>d</em> type II DAH7PSs, <em>d</em>espite their minimal sequence i<em>d</em>entity. In contrast, the structural elements that <em>d</em>ecorate the core (beta/alpha)(8) fol<em>d</em> <em>d</em>iffer from those in the type I enzymes, consistent with their <em>d</em>ifferent regulatory an<em>d</em> oligomeric properties.

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised 113 patients with chronic AF (70.<em>2</em> +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of prothrombin fragment 1+<em>2</em> (F1+<em>2</em>), thrombin-antithrombin complexes (TAT), <em>D</em>-<em>dimer</em>, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR <em>2</em>.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F1+<em>2</em> by 67%,TAT by 30% and <em>D</em>-<em>dimer</em> by 48% (all p <0.001). <em>D</em>uring an average follow-up of 44 months <em>2</em><em>2</em>/111 (<em>2</em>0%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of <em>D</em>-<em>dimer</em> at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs 18 ng/mL, p = 0.00<em>2</em>), and of t-PA antigen at entry (14.3 vs 10.9 ng/mL, p = 0.0<em>2</em>) and during OAT (15.0 vs 11.<em>2</em> ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio <em>2</em>.91; 95% CI 1.17-7.<em>2</em>6; p = 0.0<em>2</em>), high levels of <em>D</em>-<em>dimer</em> on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI 1.39-16.41; p = 0.01) and t-PA antigen levels (continuous variable) (hazard ratio 1.09; 95% CI 1.01-1.17; p = 0.0<em>2</em>) were significantly associated with combined cardiovascular events. In conclusion, high levels of <em>D</em>-<em>dimer</em> and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.

OBJECTIVE

The aim of the present study was to evaluate whether elevated D-dimer levels can predict subsequent thromboembolic and cardiovascular events in patients with atrial fibrillation during oral anticoagulant therapy.

BACKGROUND

Atrial fibrillation is associated with hemostatic abnormalities even during oral anticoagulant therapy. D-dimer levels reflect a pro-thrombogenic state and thus might serve as a marker of thromboembolic and cardiovascular events.

METHODS

This was a single-center, prospective, observational study. Patients with atrial fibrillation (269 patients, age 74 +/- 9 years, 160 paroxysmal atrial fibrillation) treated with warfarin (target prothrombin time-international normalized ratio: 1.5 to 3.0) were included. D-dimer levels were measured to assess the relationship of this parameter with subsequent thromboembolic and cardiovascular events. End points were thromboembolic events and combined cardiovascular events (thromboembolic events, cerebral hemorrhage, myocardial infarction, cardiovascular death).

RESULTS

D-dimer levels were elevated >> or =0.5 microg/ml) in 63 (23%) patients. During an average follow-up period of 756 +/- 221 days, 10 (1.8%/year) thromboembolic events (8 ischemic strokes, 1 transient ischemic attack, and 1 peripheral embolism) and 27 (4.8%/year) combined cardiovascular events (10 thromboembolisms, 9 deaths from heart failure, 3 sudden deaths, 2 myocardial infarctions, and 3 cerebral hemorrhages) occurred. Patients with elevated D-dimer levels experienced higher thromboembolic and combined cardiovascular events. Cox proportional hazard model revealed that elevated D-dimer levels were associated with both thromboembolic (p < 0.01, hazard ratio: 15.8; 95% confidence interval: 3.33 to 75.5) and combined cardiovascular (p < 0.01, hazard ratio: 7.64; 95% confidence interval: 3.42 to 17.1) events.

CONCLUSIONS

D-dimer might be a useful marker of both thromboembolic and cardiovascular events in patients with atrial fibrillation during oral anticoagulant therapy.

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)<em>2</em>10 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll <em>dimer</em> (P*) transfers an electron to a bacteriopheophytin (HL) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- <em>2</em>, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from HL- to a quinone (QA) with a time constant of 0.16 ns in wild-type RCs, 0.<em>2</em>4 ns in the Phe mutant, and 0.<em>2</em>0 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 pcs in the Trp mutant. Most of the electron transfer to HL occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (Em) of the P/P+ redox couple is measured by an electrochemical technique; the Em values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 55<em>2</em> +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (<em>delta</em> G degrees) associated with formation of the P+HL-radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from HL- to QA. The free energy of P+HL- is elevated by an amount comparable to that calculated from the increase in the Em of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute painful crisis is a common sequela that can cause significant morbi<em>d</em>ity an<em>d</em> negatively impact the quality of life of patients with sickle cell <em>d</em>isease (SCD). Plasma levels of several chemokines an<em>d</em> cytokines inclu<em>d</em>ing tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), an<em>d</em> interferon γ (IFN-γ) in patients with SCD showe<em>d</em> a <em>d</em>istinct an<em>d</em> statistically significant rise either <em>d</em>uring painful crisis or at stea<em>d</em>y state. Plasma levels of various growth factors, inclu<em>d</em>ing human vascular en<em>d</em>othelial growth factor (VEGF), human basic fibroblast growth factor (FGF), an<em>d</em> human hepatocyte growth factor (HGF), showe<em>d</em> a sustaine<em>d</em> <em>2</em>- to 3-fol<em>d</em> increase either <em>d</em>uring painful crisis or at stea<em>d</em>y state in patients with SCD. Furthermore, plasma levels of the biomarker <em>d</em>-<em>Dimer</em>, a marker of hypercoagulation, showe<em>d</em> a <em>2</em>- to 3-fol<em>d</em> increase either <em>d</em>uring painful crisis or at stea<em>d</em>y state in patients with SCD as compare<em>d</em> to that in healthy participants, suggesting an increase<em>d</em> risk of thrombosis.