The structures of <em>D</em>-xylose isomerase from Arthrobacter strain B37<em>2</em>8 containing the polyol inhibitors xylitol and <em>D</em>-sorbitol have been solved at <em>2</em>.5 A and <em>2</em>.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the <em>D</em>-sorbitol (xylitol) bound molecules, for 43,615 (3<em>2</em>,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a <em>dimer</em>, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu<em>2</em>16, Asp<em>2</em>44 and Asp<em>2</em>9<em>2</em>. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-<em>2</em> and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a <em>dimer</em> of "active" <em>dimers</em>, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp<em>2</em>3 and Arg139, appears to play a key role in stabilizing the active <em>dimer</em> and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATE<em>D</em> AT 400 WOR<em>D</em>S)

<strong class="sub-title"> Purpose: </strong> An ongoing outbreak of coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) emerged in Wuhan since <em>D</em>ecember <em>2</em>019 and spread globally. However, information about critically ill patients with COVI<em>D</em>-19 is still limited. We aimed to describe the clinical characteristics and outcomes of critically ill patients with COVI<em>D</em>-19 and figure out the risk factors of mortality.

<strong class="sub-title"> Methods: </strong> We extracted data retrospectively regarding 733 critically ill adult patients with laboratory-confirmed COVI<em>D</em>-19 from 19 hospitals in China through January 1 to February <em>2</em>9, <em>2</em>0<em>2</em>0. <em>D</em>emographic data, symptoms, laboratory values, comorbidities, treatments, and clinical outcomes were collected. The primary outcome was <em>2</em>8-day mortality. <em>D</em>ata were compared between survivors and non-survivors.

<strong class="sub-title"> Results: </strong> Of the 733 patients included in the study, the median (IQR) age was 65 (56-73) years and <em>2</em>56 (34.9%) were female. Among these patients, the median (IQR) APACHE II score was 10 (7 to 14) and <em>2</em>8-day mortality was 53.8%. Respiratory failure was the most common organ failure (597 [81.5%]), followed by shock (<em>2</em>0%), thrombocytopenia (18.8%), central nervous system (8.6%) and renal dysfunction (8%). Multivariate Cox regression analysis showed that older age, malignancies, high APACHE II score, high <em>D</em>-<em>dimer</em> level, low PaO_<em>2</em>/FiO_<em>2</em> level, high creatinine level, high hscTnI level and low albumin level were independent risk factors of <em>2</em>8-day mortality in critically ill patients with COVI<em>D</em>-19.

<strong class="sub-title"> Conclusion: </strong> In this case series of critically ill patients with COVI<em>D</em>-19 who were admitted into the ICU, more than half patients died at day <em>2</em>8. The higher percentage of organ failure in these patients indicated a significant demand for critical care resources.

Keywords: COVID-19; Critically ill; Mortality; Organ failure.

<AbstractText>Since the pan<em>d</em>emic outbreak of COVID-19, limite<em>d</em> information is available on <em>d</em>iabetic patients with COVID-19.</AbstractText><AbstractText>We retrospectively analyse<em>d</em> 166 COVID-19 patients at Tongji Hospital (Wuhan) from February 8 to March <em>2</em>1, <em>2</em>0<em>2</em>0. Clinical characteristics an<em>d</em> outcomes (as of April 4, <em>2</em>0<em>2</em>0) were compare<em>d</em> among control (group 1), secon<em>d</em>ary hyperglycaemia (group <em>2</em>: no <em>d</em>iabetes history, FPG levels ≥7.0 mmol/L once an<em>d</em> HbA1c values <6.5%) an<em>d</em> <em>d</em>iabetic (group 3) patients.</AbstractText><AbstractText>Compare<em>d</em> to group 1, groups <em>2</em> an<em>d</em> 3 ha<em>d</em> higher rates of leukocytosis, neutrophilia, lymphocytopenia, eosinopenia, an<em>d</em> levels of sCRP, ferritin an<em>d</em> <em>d</em>-<em>dimer</em> (P < 0.05 for all). Group <em>2</em> patients have higher levels of LDH, prevalence of liver <em>d</em>ysfunction an<em>d</em> increase<em>d</em> IL-8 than those in group 1, a higher prevalence of increase<em>d</em> IL-8 was foun<em>d</em> in group <em>2</em> than in group 3 (P < 0.05 for all). The proportions of critical patients in groups <em>2</em> an<em>d</em> 3 were significantly higher compare<em>d</em> to group 1 (38.1%, 3<em>2</em>.8% vs. 9.5%, P < 0.05 for both). Groups <em>2</em> an<em>d</em> 3 ha<em>d</em> significantly longer hospital stays than group 1, which was nearly one week longer. The composite outcomes risks were 5.47 (1.56-19.8<em>2</em>) an<em>d</em> <em>2</em>.61 (0.86-7.88) times greater in group <em>2</em> an<em>d</em> 3 than in group 1.</AbstractText><AbstractText>Hyperglycemia in both <em>d</em>iabetes an<em>d</em> secon<em>d</em>ary hyperglycemia patients with COVID-19 may in<em>d</em>icate poor prognoses. There were <em>d</em>ifferences between secon<em>d</em>ary hyperglycemia an<em>d</em> <em>d</em>iabetes patients. We recommen<em>d</em> that clinicians pay more attention to the bloo<em>d</em> glucose status of COVID-19 patients, even those not <em>d</em>iagnose<em>d</em> with <em>d</em>iabetes before a<em>d</em>mission.</AbstractText>

<strong class="sub-title"> Objective: </strong> To explore whether at-admission hyperglycemia is associated with worse outcomes in patients hospitalized for coronavirus disease <em>2</em>019 (COVID-19).

<strong class="sub-title"> Research design and methods: </strong> Hospitalized COVID-19 patients (<i>N</i> = <em>2</em>71) were subdivided based on at-admission glycemic status: <i>1</i>) glucose levels <7.78 mmol/L (NG) (<i>N</i> = 149 [55.0%]; median glucose 5.99 mmol/L [range 5.38-6.7<em>2</em>]), <i><em>2</em></i>) known diabetes mellitus (DM) (<i>N</i> = 56 [<em>2</em>0.7%]; 9.18 mmol/L [7.67-1<em>2</em>.71]), and <i>3</i>) no diabetes and glucose levels ≥7.78 mmol/L (HG) (<i>N</i> = 66 [<em>2</em>4.3%]; 8.57 mmol/L [8.18-10.47]).

<strong class="sub-title"> Results: </strong> Neutrophils were higher and lymphocytes and PaO_<em>2</em>/FiO_<em>2</em> lower in HG than in DM and NG patients. DM and HG patients had higher D-dimer and worse inflammatory profile. Mortality was greater in HG (39.4% vs. 16.8%; unadjusted hazard ratio [HR] <em>2</em>.<em>2</em>0, 95% CI 1.<em>2</em>7-3.81, <i>P</i> = 0.005) than in NG (16.8%) and marginally so in DM (<em>2</em>8.6%; 1.73, 0.9<em>2</em>-3.<em>2</em>5, <i>P</i> = 0.086) patients. Upon multiple adjustments, only HG remained an independent predictor (HR 1.80, 95% CI 1.03-3.15, <i>P</i> = 0.04). After stratification by quintile of glucose levels, mortality was higher in quintile 4 (Q4) (3.57, 1.46-8.76, <i>P</i> = 0.005) and marginally in Q5 (<em>2</em>9.6%) (<em>2</em>.3<em>2</em>, 0.91-5.96, <i>P</i> = 0.079) vs. Q1.

Conclusions: Hyperglycemia is an independent factor associated with severe prognosis in people hospitalized for COVID-19.

<strong class="sub-title"> Importance: </strong> Coronavirus disease <em>2</em>019 (COVID-19) is an acute respiratory illness with a high rate of hospitalization and mortality. Biomarkers are urgently needed for patient risk stratification. Red blood cell distribution width (RDW), a component of complete blood counts that reflects cellular volume variation, has been shown to be associated with elevated risk for morbidity and mortality in a wide range of diseases.

Objective: To investigate whether an association between mortality risk and elevated RDW at hospital admission and during hospitalization exists in patients with COVID-19.

<strong class="sub-title"> Design, setting, and participants: </strong> This cohort study included adults diagnosed with SARS-CoV-<em>2</em> infection and admitted to 1 of 4 hospitals in the Boston, Massachusetts area (Massachusetts General Hospital, Brigham and Women's Hospital, North Shore Medical Center, and Newton-Wellesley Hospital) between March 4, <em>2</em>0<em>2</em>0, and April <em>2</em>8, <em>2</em>0<em>2</em>0.

Main outcomes and measures: The main outcome was patient survival during hospitalization. Measures included RDW at admission and during hospitalization, with an elevated RDW defined as greater than 14.5%. Relative risk (RR) of mortality was estimated by dividing the mortality of those with an elevated RDW by the mortality of those without an elevated RDW. Mortality hazard ratios (HRs) and 95% CIs were estimated using a Cox proportional hazards model.

<strong class="sub-title"> Results: </strong> A total of 1641 patients were included in the study (mean [SD] age, 6<em>2</em>[18] years; 886 men [54%]; 740 White individuals [45%] and 497 Hispanic individuals [30%]; <em>2</em>76 nonsurvivors [17%]). Elevated RDW (>14.5%) was associated with an increased mortality risk in patients of all ages. The RR for the entire cohort was <em>2</em>.73, with a mortality rate of 11% in patients with normal RDW (1173) and 31% in those with an elevated RDW (468). The RR in patients younger than 50 years was 5.<em>2</em>5 (normal RDW, 1% [n = 341]; elevated RDW, 8% [n = 65]); <em>2</em>.90 in the 50- to 59-year age group (normal RDW, 8% [n = <em>2</em>56]; elevated RDW, <em>2</em>4% [n = 63]); 3.96 in the 60- to 69-year age group (normal RDW, 8% [n = <em>2</em><em>2</em>6]; elevated RDW, 30% [104]); 1.45 in the 70- to 79-year age group (normal RDW, <em>2</em>3% [n = 18<em>2</em>]; elevated RDW, 33% [n = 113]); and 1.59 in those ≥80 years (normal RDW, <em>2</em>9% [n = 168]; elevated RDW, 46% [n = 1<em>2</em>3]). RDW was associated with mortality risk in Cox proportional hazards models adjusted for age, D-dimer (dimerized plasmin fragment D) level, absolute lymphocyte count, and common comorbidities such as diabetes and hypertension (hazard ratio of 1.09 per 0.5% RDW increase and <em>2</em>.01 for an RDW >14.5% vs ≤14.5%; P < .001). Patients whose RDW increased during hospitalization had higher mortality compared with those whose RDW did not change; for those with normal RDW, mortality increased from 6% to <em>2</em>4%, and for those with an elevated RDW at admission, mortality increased from <em>2</em><em>2</em>% to 40%.

Conclusions and relevance: Elevated RDW at the time of hospital admission and an increase in RDW during hospitalization were associated with increased mortality risk for patients with COVID-19 who received treatment at 4 hospitals in a large academic medical center network.

BACKGROUND

Ambient particulate matter (PM) is associated with cardiovascular morbidity and mortality. It has been proposed that PM induces a pro-thrombotic process, increasing the risk of cardiovascular events, with some support from epidemiological and laboratory-based models. Diesel exhaust is a major contributor to urban PM, and we conducted a controlled human exposure of diesel exhaust in healthy subjects.

OBJECTIVE

To evaluate diesel exhaust exposure effects on fibrinolytic burden (D-dimer), platelet number, and endothelial injury (von Willebrand's factor, VWF), inhibition of the fibrinolytic pathway (plasminogen activator inhibitor-1 [PAI-1]), and inflammation (C-reactive protein, CRP).

METHODS

Randomized, crossover, double-blinded design, with 13 healthy participants exposed on three different days >>or=2 weeks washout) to diesel exhaust at 0 (filtered air), 100 microg PM(2.5)/m(3) and 200 microg PM(2.5)/m(3). We assessed diesel exhaust-associated changes in D-dimer, VWF, PAI-1 and platelets at 3, 6 and 22 h, and CRP at 22 h, after exposure initiation.

RESULTS

Significant changes did not occur in any primary endpoints. Among secondary endpoints, diesel exhaust (200 microg PM(2.5)/m(3)) effect on PAI-1 levels at 22 h was of borderline significance, with a 1.32-fold decrease after exposure to diesel exhaust (200 microg PM(2.5)/m(3)), relative to filtered air (CI 1.00 to 1.54). Diurnal patterns in D-dimer and PAI-1 were observed.

CONCLUSIONS

In healthy individuals, exposure to 200 microg PM(2.5)/m(3) diesel exhaust did not affect primary pro-thrombotic endpoints. Thus, these data do not support a diesel exhaust-induced pro-thrombotic phenomenon. Replication of these studies should be carried out to ascertain whether or not they inform our mechanistic understanding of air pollution's cardiovascular effects.

OBJECTIVE

To review the evidence on the diagnostic accuracy of the currently available point of care D-dimer tests for excluding venous thromboembolism.

METHODS

Systematic review of research on the accuracy of point of care D-dimer tests, using bivariate regression to examine sources of variation and to estimate sensitivity and specificity.

METHODS

Studies on the diagnostic accuracy of point of care D-dimer tests published between January 1995 and September 2008 and available in either Medline or Embase. Review methods The analysis included studies that compared point of care D-dimer tests with predefined reference criteria for venous thromboembolism, enrolled consecutive outpatients, and allowed for construction of a 2x2 table.

RESULTS

23 studies (total number of patients 13 959, range in mean age 38-65 years, range of venous thromboembolism prevalence 4-51%) were included in the meta-analysis. The studies reported two qualitative point of care D-dimer tests (SimpliRED D-dimer (n=12) and Clearview Simplify D-dimer (n=7)) and two quantitative point of care D-dimer tests (Cardiac D-dimer (n=4) and Triage D-dimer (n=2)). Overall sensitivity ranged from 0.85 (95% confidence interval 0.78 to 0.90) to 0.96 (0.91 to 0.98) and overall specificity from 0.48 (0.33 to 0.62) to 0.74 (0.69 to 0.78). The two quantitative tests Cardiac D-dimer and Triage D-dimer scored most favourably.

CONCLUSIONS

In outpatients suspected of venous thromboembolism, point of care D-dimer tests can contribute important information and guide patient management, notably in low risk patients (that is, those patients with a low score on a clinical decision rule).

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully <em>d</em>eciphere<em>d</em>. Recent stu<em>d</em>ies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggeste<em>d</em> that selection may be me<em>d</em>iate<em>d</em> by an RNA switch mechanism, in which conserve<em>d</em> UCUG elements that are sequestere<em>d</em> by base-pairing in the monomeric RNA become expose<em>d</em> upon <em>dimer</em>ization to allow bin<em>d</em>ing to the cognate nucleocapsi<em>d</em> (NC) <em>d</em>omains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslate<em>d</em> region that contains all resi<em>d</em>ues necessary for efficient RNA packaging (Psi(WT); resi<em>d</em>ues 147-6<em>2</em>3) also exhibits a <em>dimer</em>ization-<em>d</em>epen<em>d</em>ent affinity for NC, with the native <em>dimer</em> ([Psi(WT)](<em>2</em>)) bin<em>d</em>ing 1<em>2</em>+/-<em>2</em> NC molecules with high affinity (K(<em>d</em>)=17+/-7 nM) an<em>d</em> with the monomer, stabilize<em>d</em> by substitution of <em>dimer</em>-promoting loop resi<em>d</em>ues with hairpin-stabilizing sequences (Psi(M)), bin<em>d</em>ing 1-<em>2</em> NC molecules. I<em>d</em>entical <em>dimer</em>-inhibiting mutations in MoMuLV-base<em>d</em> vectors significantly inhibit genome packaging in vivo (approximately 100-fol<em>d</em> <em>d</em>ecrease), whereas a large <em>d</em>eletion of nearly <em>2</em>00 nucleoti<em>d</em>es just upstream of the gag start co<em>d</em>on has minimal effects. Our fin<em>d</em>ings support the propose<em>d</em> RNA switch mechanism an<em>d</em> further suggest that virus assembly may be initiate<em>d</em> by a complex comprising as few as 1<em>2</em> Gag molecules boun<em>d</em> to a <em>dimer</em>ic packaging signal.

Ristocetin in aqueous solution dimerizes with an equilibrium dissociation constant of 5.0 x 10(-4) M, i.e. approximately 1.1 mg ml-1 (Waltho, J.P., and Williams, <em>D</em>. H. (1989) J. Am. Chem. Soc. 111, <em>2</em>475-<em>2</em>480). At concentrations of about 1.0 mg ml-1 ristocetin flocculates many proteins, lyses platelets and, in the presence of von Willebrand factor, agglutinates both fresh and formalin-fixed platelets. Because ristocetin exists as both monomeric and dimeric species, we sought to determine which of these forms flocculates proteins and agglutinates platelets. We found that: 1) the initial rate of flocculation of certain proteins, <em>2</em>) the initial rate of agglutination of formalin-fixed platelets, and 3) the binding of ristocetin to formalin-fixed platelets are higher order solely with respect to the concentration of ristocetin <em>dimers</em>. As to the operative mechanism, it appears that bifunctional <em>dimers</em> cross-link proteins that possess multiple copies of a common recognition site. Preliminary evidence indicates that a recognition site is a beta-turn of the form X-P-G-X'.

The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha <em>2</em> epsilon <em>2</em> theta <em>2</em> tau <em>2</em> gamma <em>2</em> <em>delta</em> 1 <em>delta</em>' 1 chi 1 psi 1. The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a <em>dimer</em> of tau and one gamma complex clamp loader (gamma <em>2</em> <em>delta</em> 1 <em>delta</em>' 1 chi 1 psi 1) for loading beta onto DNA. At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the <em>delta</em>, <em>delta</em>', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.

Cell surface expression of class II major histocompatibility complex-enco<em>d</em>e<em>d</em> (Ia) molecules <em>d</em>epen<em>d</em>s on association of the component alpha an<em>d</em> beta chains into a stable hetero<em>d</em>imer. In the mouse, two isotypes of class II molecules have been i<em>d</em>entifie<em>d</em>, A beta A alpha an<em>d</em> E beta E alpha. However, experiments from this laboratory have shown that, following DNA-me<em>d</em>iate<em>d</em> gene transfer into murine L cells, an A beta E alpha-mixe<em>d</em>-isotype molecule can be assemble<em>d</em> an<em>d</em> expresse<em>d</em> at the cell surface. In the present stu<em>d</em>y, we have investigate<em>d</em> the structural features of the beta chain that control the extent of association an<em>d</em> level of membrane expression of A beta E alpha interisotypic pairs. The use of intact allelic A beta genes <em>d</em>emonstrate<em>d</em> that only A beta <em>d</em> chains, but not A beta b or A beta k chains, can be coexpresse<em>d</em> on the surface membrane with E alpha chains. Transfection of recombinant A beta genes that enco<em>d</em>e all or half of the beta 1 <em>d</em>omain from one allele an<em>d</em> the rest of the chain from another allele reveale<em>d</em> that the 5-7 polymorphic resi<em>d</em>ues in the amino-terminal 50 resi<em>d</em>ues of the A beta chain completely controlle<em>d</em> this variation in expression with E alpha. Isotypically mixe<em>d</em> beta genes enco<em>d</em>ing the A beta 1 <em>d</em>omain of either A beta <em>d</em> or A beta k chains an<em>d</em> the beta <em>2</em>, transmembrane, an<em>d</em> intracytoplasmic portions of E beta chains were use<em>d</em> to assess the role of isotypically conserve<em>d</em> structures in alpha beta pairing an<em>d</em> expression. In marke<em>d</em> contrast to the major alterations in expression accompanying changes in the amino-terminal polymorphic resi<em>d</em>ues, exchange of these carboxyl-terminal isotypic segments ha<em>d</em> no <em>d</em>etectable influence on the efficiency of expression with either A alpha or E alpha chains. These results argue strongly that variations in the efficiency with which <em>d</em>istinct Ia alpha beta <em>dimers</em> assemble an<em>d</em> are transporte<em>d</em> to the membrane is <em>d</em>etermine<em>d</em> almost exclusively by a critical chain interaction involving the amino-terminal <em>d</em>omains of the molecules.

Ben<em>d</em>ing flexibility of the six tetrameric <em>d</em>uplexes was investigate<em>d</em> <em>d</em>(AAAA):<em>d</em>(TTTT), <em>d</em>(AATT)<em>2</em>, <em>d</em>(TTAA)<em>2</em>, <em>d</em>(GGGG):<em>d</em>(CCCC), <em>d</em>(GGCC)<em>2</em> an<em>d</em> <em>d</em>(CCGG)<em>2</em>,. The tetramers were exten<em>d</em>e<em>d</em> in the both <em>d</em>irections by regular <em>d</em>ouble helices. The stiffness of the B-DNA <em>d</em>ouble helix when bent into the both grooves prove<em>d</em> to be less than that in the perpen<em>d</em>icular <em>d</em>irection by an or<em>d</em>er of magnitu<em>d</em>e. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculate<em>d</em> fluctuations of the DNA ben<em>d</em>ing along the <em>d</em>ya<em>d</em> axis, 5-7 <em>d</em>egree, are in agreement with experimental value of the DNA persistence length. Anisotropy of the <em>d</em>ouble helix is sequence-<em>d</em>epen<em>d</em>ent: most easily bent into the minor groove are the tetramers with purine-pyrimi<em>d</em>ine <em>dimer</em> (RY) in the mi<em>d</em><em>d</em>le. In contrast, YR <em>d</em>inucleoti<em>d</em>es prefer ben<em>d</em>ing into the major groove. Moreover, they have an equilibrium ben<em>d</em> of 6-1<em>2</em> <em>d</em>egree into this groove. The above inequality is cause<em>d</em> by stacking interaction of the bases. The ben<em>d</em> in the central <em>dimer</em> is <em>d</em>istribute<em>d</em> to some extent between the a<em>d</em>jacent links, though the main fraction of the ben<em>d</em> remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by <em>2</em>0 <em>d</em>egree. the backbone <em>d</em>ihe<em>d</em>ral angles vary by no more than 15 <em>d</em>egree. The obtaine<em>d</em> results are in accor<em>d</em> with x-ray structure of the B-DNA <em>d</em>o<em>d</em>ecamer; they further substantiate our early mo<em>d</em>el of DNA wrapping in the nucleosome by means of "mini-kinks" separate<em>d</em> by a half-pitch of the <em>d</em>ouble helix, i.e. by 5-6 b.p. Sequence-<em>d</em>epen<em>d</em>ent anisotropy of DNA presumably <em>d</em>ictates the three-<em>d</em>imensional structure of DNA in solution as well. We have foun<em>d</em> that nonran<em>d</em>om allocation of YR <em>dimers</em> lea<em>d</em>s to the systematic ben<em>d</em>s in equilibrium structure of certain DNA fragments.

The glycosyltransferase UrdGT<em>2</em> from Streptomyces fradiae catalyzes the formation of a glycosidic C-C bond between a polyketide aglycone and <em>D</em>-olivose. The enyzme was expressed in Escherichia coli, purified and crystallized. Its structure was established by X-ray diffraction at 1.9 A resolution. It is the first structure of a C-glycosyltransferase. UrdGT<em>2</em> belongs to the structural family GT-B of the glycosyltransferases and is likely to form a C(<em>2</em>)-symmetric <em>dimer</em> in solution. The binding structures of donor and acceptor substrates in five structurally homologous enzymes provided a clear and consistent guide for the substrate-binding structure in UrdGT<em>2</em>. The modeled substrate locations suggest the deeply buried Asp137 as the activator for C-C bond formation and explain the reaction. The putative model can be used to design mutations that change the substrate specificity. Such mutants are of great interest in overcoming the increasing danger of antibiotic resistance.

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as '<em>D</em>', all of which are <em>dimers</em> of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 49<em>2</em>, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and '<em>D</em>' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are <em>2</em> mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and '<em>D</em>' represent two different Yb homo<em>dimers</em>, which we refer to here as Y1bY1b and Y<em>2</em>bY<em>2</em>b respectively. GSH transferase C is the corresponding heterodimer Y1bY<em>2</em>b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase '<em>D</em>' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. <em>2</em>57, 9909-991<em>2</em>] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and '<em>D</em>' have considerable homology since there are <em>2</em>3 peptides common to both, 1<em>2</em> peptides unique to A and 8 peptides unique to '<em>D</em>'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y<em>2</em>b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (198<em>2</em>) Eur. J. Biochem. 1<em>2</em>6, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.

BACKGROUND

Clinical trials of postmenopausal hormone therapy (HT) have shown increased risk of coronary heart disease (CHD) in the first few years after initiation of therapy and no overall benefit.

METHODS

This nested case-control study evaluates a range of inflammatory, lipid, thrombotic, and genetic markers for their association with CHD in the 4 years after randomization and assesses whether any of these markers modified or mediated the initially increased risk associated with HT in postmenopausal women aged 50 to 79 years at baseline. Conjugated equine estrogens, 0.625 mg/d, or placebo was given to 10 739 hysterectomized women, and the same estrogen plus medroxyprogesterone acetate, 2.5 mg/d, was given to 16 608 women with an intact uterus.

RESULTS

In multivariate-adjusted analyses of 359 cases and 820 controls in the combined trials, baseline levels of 12 of the 23 biomarkers studied were associated with CHD events: interleukin 6, matrix metalloproteinase 9, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, total cholesterol, triglycerides, D-dimer, factor VIII, von Willebrand factor, leukocyte count, homocysteine, and fasting insulin. Biomarkers tended to be more strongly associated with CHD in the initial 2 years after randomization. The genetic polymorphism glycoprotein IIIa leu33pro was significantly associated with CHD. Baseline low-density lipoprotein cholesterol interacted significantly with HT so that women with higher levels were at higher risk for CHD when given HT (P = .03 for interaction). The levels of several biomarkers were changed by HT, but these changes did not seem to be associated with future CHD events.

CONCLUSIONS

Several thrombotic, inflammatory, and lipid biomarkers were associated with CHD events in postmenopausal women, but only low-density lipoprotein cholesterol modified the effect of HT. Further research is needed to identify the mechanisms by which HT increases the risk of CHD.

BACKGROUND

clinicaltrials.gov Identifier: NCT00000611.

In a prospective clinical study of 50 patients with <em>D</em>engue Shock Syndrome (<em>D</em>SS), we investigated the association of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), and IL-6 with activation markers of coagulation (F1+<em>2</em> and TATc) and fibrinolysis (t-PA, PAPc, and <em>D</em>-<em>dimer</em>). We found that TNF-alpha, IL-1beta and Il-1Ra, but not IL-6, concentrations were elevated in the circulation during the early stage of infection and at discharge from hospital. TNF-alpha was significantly associated with <em>D</em>-<em>dimer</em>, an activation marker of fibrinolysis (p < 0.003), but not with activation markers of coagulation. IL-1beta was significantly associated with t-PA (p < 0.03). IL-1Ra was significantly associated with F1+<em>2</em>, TATc (p < 0.04 and p < 0.0<em>2</em>, respectively), whereas IL-6 was significantly associated with both, activation markers of coagulation (F1+<em>2</em>; p < 0.03) and fibrinolysis (PAPc; p = 0.00<em>2</em>). Our data are in line with studies in bacterial sepsis. In severe dengue virus infection the same cytokines are involved in the onset and regulation of hemostasis.

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P<em>2</em><em>2</em> is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s(<em>2</em>0,w) of 9.3S, a <em>D</em>(<em>2</em>0,w) of 4.3 x 10(-7) cm(<em>2</em>)/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a <em>dimer</em> of gp9 and each phage containing six of these <em>dimers</em>. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage "tailed" with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (<em>2</em>.5% residual activity). From these results we propose that the glycosidic activity of the P<em>2</em><em>2</em> tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.

<strong class="sub-title">Objectives:</strong> The aim of this study was to determine the frequency of venous thromboembolism in critically ill coronavirus disease <em>2</em>019 patients and associate a degree of inflammatory marker elevation to venous thromboembolism development.

<strong class="sub-title">Design:</strong> An observational study that identified patients with severe coronavirus disease <em>2</em>019 between March 1<em>2</em>, <em>2</em>0<em>2</em>0, and March 31, <em>2</em>0<em>2</em>0. Data reported are those available through May 6, <em>2</em>0<em>2</em>0.

Setting: A multicenter study including three Indianapolis area academic hospitals.

<strong class="sub-title">Patients:</strong> Two-hundred forty consecutive patients with confirmed severe acute respiratory syndrome coronavirus <em>2</em> infection were admitted to one of three hospitals. One-hundred nine critically ill coronavirus disease <em>2</em>019 patients admitted to the ICU were included in the analysis.

Interventions: All patients received routine subcutaneous chemical venous thromboembolism prophylaxis.

<strong class="sub-title">Measurements and main results:</strong> The primary outcome of this study was to determine the frequency of venous thromboembolism and the degree of inflammatory and coagulation marker elevation associated with venous thromboembolism development. Descriptive statistics outlined the frequency of venous thromboembolism at any time during severe coronavirus disease <em>2</em>019. Clinical course and laboratory metrics were compared between patients that developed venous thromboembolism and patients that did not develop venous thromboembolism. Hypercoagulable thromboelastography was defined as two or more hypercoagulable parameters.

<strong class="sub-title">Main results:</strong> One-hundred nine patients developed severe coronavirus disease <em>2</em>019 requiring ICU care. The mean (± SD) age was 61 ± 16 years and 57% were male. Seventy-five patients (69%) were discharged home, 7 patients (6%) remain in the hospital, and <em>2</em>7 patients (<em>2</em>5%) died. Venous thromboembolism was diagnosed in 31 patients (<em>2</em>8%) 8 ± 7 days after hospital admission, including two patients diagnosed with venous thromboembolism at presentation to the hospital. Elevated admission D-dimer and peak D-dimer were associated with venous thromboembolism development (p < 0.05). D-dimer greater than <em>2</em>,600 ng/mL predicted venous thromboembolism with an area under the receiver operating characteristic curve of 0.760 (95% CI, 0.661-0.858; p < 0.0001), sensitivity of 89.7%, and specificity of 59.5%. Twelve patients (11%) had thromboelastography performed and 58% of these patients had a hypercoagulable study. The calculated coagulation index was hypercoagulable in 50% of patients with thromboelastography.

<strong class="sub-title">Conclusions:</strong> These data show that coronavirus disease <em>2</em>019 results in a hypercoagulable state. Routine chemical venous thromboembolism prophylaxis may be inadequate in preventing venous thromboembolism in severe coronavirus disease <em>2</em>019.

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(<em>D</em>,L-lactic acid) and poly(<em>D</em>,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as <em>2</em> emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w(1)/o/w(<em>2</em>)) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from approximately 130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency>> 90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MAL<em>D</em>I-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A<em>2</em>1 desamido insulin and only traces of covalent insulin <em>dimer</em> were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MAL<em>D</em>I-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin.

BACKGROUND

Inflammation and hemostasis perturbation may be involved in vascular complications of HIV infection. We examined atherogenic biomarkers and subclinical atherosclerosis in HIV-infected adults before and after beginning highly active antiretroviral therapy (HAART).

METHODS

In the Women's Interagency HIV Study, 1<em>2</em>7 HIV-infected women studied pre and post HAART were matched to HIV-uninfected controls. Six semiannual measurements of soluble C<em>D</em>14, tumor necrosis factor (TNF) alfa, soluble interleukin (IL) <em>2</em> receptor, IL-6, IL-10, monocyte chemoattractant protein 1, <em>D</em>-<em>dimer</em>, and fibrinogen were obtained. Carotid artery intima-media thickness was measured by B-mode ultrasound.

RESULTS

Relative to HIV-uninfected controls, HAART-naive HIV-infected women had elevated levels of soluble C<em>D</em>14 (1945 vs 166<em>2</em> ng/mL, Wilcoxon signed rank P < 0.0001), TNF-α (6.3 vs 3.4 pg/mL, P < 0.0001), soluble IL-<em>2</em> receptor (1587 vs 949 pg/mL, P < 0.0001), IL-10 (3.3 vs 1.9 pg/mL, P < 0.0001), monocyte chemoattractant protein 1 (190 vs 163 pg/mL, P < 0.0001), and <em>D</em>-<em>dimer</em> (0.43 vs 0.31 μg/mL, P < 0.01). Elevated biomarker levels declined after HAART. Although most biomarkers normalized to HIV-uninfected levels, in women on effective HAART, TNF-α levels remained elevated compared with HIV-uninfected women (+0.8 pg/mL, P = 0.000<em>2</em>). Higher post-HAART levels of soluble IL-<em>2</em> receptor (P = 0.0<em>2</em>), IL-6 (P = 0.05), and <em>D</em>-<em>dimer</em> (P = 0.03) were associated with increased carotid artery intima-media thickness.

CONCLUSIONS

Untreated HIV infection is associated with abnormal hemostasis (eg, D-dimer), proatherogenic (eg, TNF-α), and antiatherogenic (eg, IL-10) inflammatory markers. HAART reduces most inflammatory mediators to HIV-uninfected levels. Increased inflammation and hemostasis are associated with subclinical atherosclerosis in recently treated women. These findings have potential implications for long-term risk of cardiovascular disease in HIV-infected patients, even with effective therapy.

Clinical diagnosis of acute mesenteric ischemia is difficult. The aim of this review is to provide current status on the search for an accurate plasma biomarker for acute mesenteric ischemia. A search using the medical subject heading terms marker and mesenteric ischemia or intestinal ischemia or superior mesenteric artery occlusion or mesenteric venous thrombosis in the Medline and Embase databases from 1980 to <em>2</em>011. Studies without a control group or a control group consisted of healthy individuals (human studies), or studies on intestinal reperfusion were excluded. Twenty animal and twelve human studies were identified. In human studies, the studied series of patients had a control group that had a need of laparotomy (n = <em>2</em>), suspected acute mesenteric ischemia (n = 7), acute abdomen (n = <em>2</em>) or systemic inflammatory response syndrome (n = 1). <em>D</em>: -<em>dimer</em> has been found to be the most consistent highly sensitive early marker, but specificity was low. The follow-up study on α-glutathione S-transferase yielded inferior sensitivity and accuracy than the preliminary study, clearly questioning the value of this marker. Intestinal fatty acid binding globulin (I-FABP) and <em>D</em>: -lactate are both interesting markers, but the results were conflicting. <em>D</em>ifferent cut-off levels have been used in the studies on I-FABP. The encouraging preliminary result of cobalt-albumin and urinary FABP as an accurate marker needs to be addressed in other study populations. The early clinical and laboratory diagnosis of intestinal ischemia remains a challenge. None of the proposed plasma-derived tests for acute mesenteric ischemia has as yet entered routine clinical practice. The proposed biomarkers need to be evaluated in a prospective clinical research project in patients with acute abdomen.

Among a wide range of noncovalent interactions, hydrogen (H) bonds are well known for their specific roles in various chemical and biological phenomena. When describing conventional hydrogen bonding, researchers use the notation AH···<em>D</em> (where A refers to the electron acceptor and <em>D</em> to the donor). However, the AH molecule engaged in a AH···<em>D</em> H-bond can also be pivoted around by roughly 180°, resulting in a HA···<em>D</em> arrangement. Even without the H atom in a bridging position, this arrangement can be attractive, as explained in this Account. The electron density donated by <em>D</em> transfers into a AH σ* antibonding orbital in either case: the lobe of the σ* orbital near the H atom in the H-bonding AH···<em>D</em> geometry, or the lobe proximate to the A atom in the HA···<em>D</em> case. A favorable electrostatic interaction energy between the two molecules supplements this charge transfer. When A belongs to the pnictide family of elements, which include phosphorus, arsenic, antimony, and bismuth, this type of interaction is called a pnicogen bond. This bonding interaction is somewhat analogous to the chalcogen and halogen bonds that arise when A is an element in group 16 or 17, respectively, of the periodic table. Electronegative substitutions, such as a F for a H atom opposite the electron donor atom, strengthen the pnicogen bond. For example, the binding energy in FH(<em>2</em>)P···NH(3) greatly exceeds that of the paradigmatic H-bonding water <em>dimer</em>. Surprisingly, di- or tri-halogenation does not produce any additional stabilization, in marked contrast to H-bonds. Chalcogen and halogen bonds show similar strength to the pnicogen bond for a given electron-withdrawing substituent. This insensitivity to the electron-acceptor atom distinguishes these interactions from H-bonds, in which energy depends strongly upon the identity of the proton-donor atom. As with H-bonds, pnicogen bonds can extract electron density from the lone pairs of atoms on the partner molecule, such as N, O, and S. The π systems of carbon chains can donate electron density in pnicogen bonds. Indeed, the strength of A···π pnicogen bonds exceeds that of H-bonds even when using strong proton donors such as water with the same π system. H-bonds typically have a high propensity for a linear AH···<em>D</em> arrangement, but pnicogen bonds show an even greater degree of anisotropy. <em>D</em>istortions of pnicogen bonds away from their preferred geometry cause a more rapid loss of stability than in H-bonds. Although often observed in <em>dimers</em> in the gas phase, pnicogen bonds also serve as the glue in larger aggregates, and researchers have found them in a number of diffraction studies of crystals.

We recently <em>d</em>evelope<em>d</em> a high-resolution genome-wi<em>d</em>e assay for mapping DNA excision repair name<em>d</em> eXcision Repair-sequencing (XR-seq) an<em>d</em> have now use<em>d</em> XR-seq to <em>d</em>etermine which regions of the genome are subject to repair very soon after UV exposure an<em>d</em> which regions are repaire<em>d</em> later. Over a time course, we measure<em>d</em> repair of the UV-in<em>d</em>uce<em>d</em> <em>d</em>amage of cyclobutane pyrimi<em>d</em>ine <em>dimers</em> (CPDs) (at 1, 4, 8, 16, <em>2</em>4, an<em>d</em> 48 h) an<em>d</em> (6-4)pyrimi<em>d</em>ine-pyrimi<em>d</em>one photopro<em>d</em>ucts [(6-4)PPs] (at 5 an<em>d</em> <em>2</em>0 min an<em>d</em> 1, <em>2</em>, an<em>d</em> 4 h) in normal human skin fibroblasts. Each type of <em>d</em>amage has <em>d</em>istinct repair kinetics. The (6-4)PPs are <em>d</em>etecte<em>d</em> as early as 5 min after UV treatment, with the bulk of repair complete<em>d</em> by 4 h. Repair of CPDs, which we previously showe<em>d</em> is intimately couple<em>d</em> to transcription, is slower an<em>d</em> in certain regions persists even <em>2</em> <em>d</em> after UV irra<em>d</em>iation. We compare<em>d</em> our results to the Encyclope<em>d</em>ia of DNA Elements <em>d</em>ata regar<em>d</em>ing histone mo<em>d</em>ifications, chromatin state, an<em>d</em> transcription. For both <em>d</em>amage types, an<em>d</em> for both transcription-couple<em>d</em> an<em>d</em> general excision repair, the earliest repair occurre<em>d</em> preferentially in active an<em>d</em> open chromatin states. Conversely, repair in regions classifie<em>d</em> as "heterochromatic" an<em>d</em> "represse<em>d</em>" was relatively low at early time points, with repair persisting into the late time points. Damage that remains <em>d</em>uring DNA replication increases the risk for mutagenesis. In<em>d</em>ee<em>d</em>, late-repaire<em>d</em> regions are associate<em>d</em> with a higher level of cancer-linke<em>d</em> mutations. In summary, we show that XR-seq is a powerful approach for stu<em>d</em>ying relationships among chromatin state, DNA repair, genome stability, mutagenesis, an<em>d</em> carcinogenesis.

Haemostatic reference intervals are generally based on samples from non-pregnant women. Thus, they may not be relevant to pregnant women, a problem that may hinder accurate diagnosis and treatment of haemostatic disorders during pregnancy. In this study, we establish gestational age-specific reference intervals for coagulation tests during normal pregnancy. Eight hundred one women with expected normal pregnancies were included in the study. Of these women, 391 had no complications during pregnancy, vaginal delivery, or postpartum period. Plasma samples were obtained at gestational weeks 13-<em>2</em>0, <em>2</em>1-<em>2</em>8, <em>2</em>9-34, 35-4<em>2</em>, at active labor, and on postpartum days 1 and <em>2</em>. Reference intervals for each gestational period using only the uncomplicated pregnancies were calculated in all 391 women for activated partial thromboplastin time (aPTT), fibrinogen, fibrin <em>D</em>-<em>dimer</em>, antithrombin, free protein S, and protein C and in a subgroup of 186 women in addition for prothrombin time (PT), Owren and Quick PT, protein S activity, and total protein S and coagulation factors II, V, VII, VIII, IX, X, XI, and XII. The level of coagulation factors II, V, X, XI, XII and antithrombin, protein C, aPTT, PT remained largely unchanged during pregnancy, delivery, and postpartum and were within non-pregnant reference intervals. However, levels of fibrinogen, <em>D</em>-<em>dimer</em>, and coagulation factors VII, VIII, and IX increased markedly. Protein S activity decreased substantially, while free protein S decreased slightly and total protein S was stable. Gestational age-specific reference values are essential for the accurate interpretation of a subset of haemostatic tests during pregnancy, delivery, and puerperium.