Background: The role of vitamin D status in COVID-19 patients is a matter of debate.

Objectives: To assess serum 25-hydroxyvitamin D (25OHD) levels in hospitalized patients with COVID-19 and to analyze the possible influence of vitamin D status on disease severity.

Methods: Retrospective case-control study of 216 COVID-19 patients and 197 population-based controls. Serum 25OHD levels were measured in both groups. The association of serum 25OHD levels with COVID-19 severity (admission to the intensive care unit, requirements for mechanical ventilation, or mortality) was also evaluated.

Results: Of the 216 patients, 19 were on vitamin D supplements and were analyzed separately. In COVID-19 patients, mean ± standard deviation 25OHD levels were 13.8 ± 7.2 ng/mL, compared with 20.9 ± 7.4 ng/mL in controls (P < .0001). 25OHD values were lower in men than in women. Vitamin D deficiency was found in 82.2% of COVID-19 cases and 47.2% of population-based controls (P < .0001). 25OHD inversely correlates with serum ferritin (P = .013) and D-dimer levels (P = .027). Vitamin D-deficient COVID-19 patients had a greater prevalence of hypertension and cardiovascular diseases, raised serum ferritin and troponin levels, as well as a longer length of hospital stay than those with serum 25OHD levels ≥20 ng/mL. No causal relationship was found between vitamin D deficiency and COVID-19 severity as a combined endpoint or as its separate components.

Conclusions: 25OHD levels are lower in hospitalized COVID-19 patients than in population-based controls and these patients had a higher prevalence of deficiency. We did not find any relationship between vitamin D concentrations or vitamin deficiency and the severity of the disease.

Keywords: 25OHD; COVID-19; PTH; SARS-CoV-2 infection.

OBJECTIVE

HIV infection is associated with coagulation abnormalities and significantly increased risk of venous thrombosis. It has been shown that higher plasma levels of coagulation and inflammatory biomarkers predicted mortality in HIV. We investigated the relationship between venous thrombosis and HIV-related characteristics, traditional risk factors of hypercoagulability, and pre-event levels of biomarkers.

METHODS

A retrospective case-control study of <em>2</em>3 HIV-infected individuals who experienced an incident venous thromboembolic event while enrolled in National Institutes of Health studies from 1995 to <em>2</em>010 and 69 age-matched and sex-matched HIV-infected individuals without known venous thromboembolism (VTE).

METHODS

Biomarkers of inflammation, endothelial dysfunction, coagulation, tissue fibrosis, and cytomegalovirus (CMV) reactivation were assessed by ELISA-based assays and PCR using plasma obtained prior to the event.

RESULTS

VTE events were related to nadir C<em>D</em>4 cell count, lifetime history of multiple opportunistic infections, CMV disease, CMV viremia, immunological AI<em>D</em>S, active infection, and provocation (i.e., recent hospitalization, surgery, or trauma). VTE events were independently associated with increased plasma levels of P-selectin (P = 0.00<em>2</em>), <em>D</em>-<em>dimer</em> (P = 0.01), and hyaluronic acid (P = 0.009) in a multivariate analysis. No significant differences in antiretroviral or interleukin-<em>2</em> exposures, plasma HIV viremia, or other traditional risk factors were observed.

CONCLUSIONS

Severe immunodeficiency, active infection, and provocation are associated with venous thromboembolic disease in HIV. Biomarkers of endothelial dysfunction, coagulation, and tissue fibrosis may help identify HIV-infected patients at elevated risk of VTE.

This contribution describes the substrate scope and mechanism of Pd-catalyzed ligand-directed C-H arylation with diaryliodonium salts. This transformation was applied to the synthesis of a variety of different biaryl products, using directing groups including pyridines, quinolines, pyrrolidinones, and oxazolidinones. Electronically and sterically diverse aryl groups (Ar) were transferred in high yield using iodine(III) reagents of general structure [Mes-I-Ar]BF(4). Mechanistic investigations have been conducted that establish the kinetic order of the catalytic reaction in each component, determine the resting state of the catalyst and the iodine(III) reagent, quantify the electronic influence of the arylating reagent on the reaction rate, and establish the intra- and intermolecular 1 degree H/<em>D</em> kinetic isotope effect. On the basis of these studies, this transformation is proposed to proceed via turnover-limiting oxidation of the Pd <em>dimer</em> [Pd(N~C)(OAc)](<em>2</em>) (N~C = 3-methyl-<em>2</em>-phenylpyridine) by [Mes-I-Ph]BF(4). This mechanism implicates a bimetallic high oxidation state Pd species as a key catalytic intermediate. The significance of this and other aspects of the proposed mechanism are discussed in detail.

1. The chromatography of rat brain enolase (<em>2</em>-phospho-<em>D</em>-glycerate hydrolyase, EC 4.<em>2</em>.1.11) reveals three distinet components. One of these appears to be isoenzyme 1 (alpha alpha) but isoenzymes <em>2</em> (alpha beta) and 3 (beta beta) are absent. <em>2</em>. The most acidic form of brain enolase was partially purified and an antiserum raised against it in the chicken. 3. a combination of chromatographic and immunological studies showed that a third type of subunit (gamma) is present in the brain giving rise to two further isoenzymes (alpha gamma and gamma gamma). 4. <em>D</em>evelopmental studies on the brain enzyme show an increase in total enolase activity from foetal life to maturity and a concurrent rise in the proportion of brain specific <em>dimers</em>. 5. It is therefore concluded that there are three genetic loci alpha, beta and gamma, coding for the enolase isoenzymes of rat tissues.

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b<em>2</em>a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-<em>2</em> has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-<em>D</em>-<em>dimer</em> antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-<em>2</em> and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-<em>2</em>, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.

<strong class="sub-title"> Background: </strong> Identification of reliable outcome predictors in Corona virus disease-<em>2</em>019 (Covid-19) is of paramount importance for improving patient's management.

<strong class="sub-title"> Methods: </strong> A systematic review of literature was conducted until April <em>2</em>4^th , <em>2</em>0<em>2</em>0. From 6,843 articles, 49 studies were selected for a pooled assessment; cumulative statistics for age and sex were retrieved in 587,790 and 60<em>2</em>,<em>2</em>34 cases. Two endpoints were defined: 1) a composite outcome including death, severe presentation, hospitalization in intensive care unit (ICU) and/or mechanical ventilation; <em>2</em>) in-hospital mortality. We extracted numeric data on patients' characteristics and cases with adverse outcomes and employed inverse variance random effects models to derive pooled estimates.

<strong class="sub-title"> Results: </strong> We identified 18 and 1<em>2</em> factors associated with the composite endpoint and death, respectively. Among those, a history of CVD (odds ratio (OR)=3.15, 95% confidence intervals (CI) <em>2</em>.<em>2</em>6-4.41), acute cardiac (OR=10.58, 5.00-<em>2</em><em>2</em>.40) or kidney (OR=5.13, 1.78-14.83) injury, increased procalcitonin (OR=4.8, <em>2</em>.034-11.31) or D-dimer (OR=3.7, 1.74-7.89), and thrombocytopenia (OR=6.<em>2</em>3, 1.031-37.67) conveyed the highest odds for the adverse composite endpoint. Advanced age, male sex, cardiovascular comorbidities, acute cardiac or kidney injury, lymphocytopenia and D-dimer conferred an increased risk of in-hospital death. With respect to the treatment of the acute phase, therapy with steroids was associated with the adverse composite endpoint (OR=3.61, 95% CI 1.934-6.73), but not with mortality.

Conclusions: Advanced age, comorbidities, abnormal inflammatory and organ injury circulating biomarkers captured patients with an adverse clinical outcome. Clinical history and laboratory profile may then help identify patients with a higher risk of in-hospital mortality.

Keywords: Covid-19; meta-analysis; outcomes; predictors.

COVID-19 has become one of the worst infectious disease outbreaks of recent times, with over 2.1 million cases and 120,000 deaths so far. Our study investigated the demographic, clinical, laboratory and imaging features of 63 patients with COVID-19 in Beijing. Patients were classified into four groups, mild, moderate, severe and critically ill. The mean age of our patients was 47 years of age (range 3-85) and there was a slight male predominance (58.7%). Thirty percent of our patients had severe or critically ill disease, but only 20% of severe and 33% of critically ill patients had been to Wuhan. Fever was the most common presentation (84.1%), but cough was present in only slightly over half of the patients. We found that lymphocyte and eosinophils count were significantly decreased in patients with severe disease (p = 0.001 and p = 0.000, respectively). Eosinopenia was a feature of higher levels of severity. Peripheral CD+, CD+ T lymphocytes, and B lymphocytes were significantly decreased in severe and critically ill patients, but there was only a non-statistically significant downward trend in NK cell numbers with severity. Of note is that liver function tests including AST, ALT, GGT and LDH were elevated, and albumin was decreased. The inflammatory markers CRP, ESR and ferritin were elevated in patients with severe disease or worse. IL-6 levels were also higher, indicating that the presence of a hyperimmune inflammatory state portends higher morbidity and mortality. In a binary logistic regression model, C-reactive protein level (OR 1.073, [CI, 1.013-1.136]; p = 0.017), CDD-dimer (OR 5.313, [CI, 0.325-86.816]; p = 0.241) were independent predictors of disease severity.

Keywords: 2019 novel coronavirus disease; COVID-19; Cytokine storm; Elevated liver enzymes; Interleukin-6; Lymphocyte subsets; Mortality; Prognosis; SARS-CoV-2.

<em>D</em>-<em>dimer</em> is a soluble fibrin degradation product that results from ordered breakdown of thrombi by the fibrinolytic system. Numerous studies have shown that <em>D</em>-<em>dimer</em> serves as a valuable marker of activation of coagulation and fibrinolysis. Consequently, <em>D</em>-<em>dimer</em> has been extensively investigated for the diagnosis of venous thromboembolism (VTE) and is used routinely for this indication. In addition, <em>D</em>-<em>dimer</em> has been evaluated for determining the optimal duration of anticoagulation in VTE patients, for diagnosing and monitoring disseminated intravascular coagulation, and as an aid in the identification of medical patients at high risk for VTE. Thus, quantification of <em>D</em>-<em>dimer</em> levels serves an important role in guiding therapy. This review: 1) describes how <em>D</em>-<em>dimer</em> is generated; <em>2</em>) reviews the assays used for its detection; and 3) discusses the role of <em>D</em>-<em>dimer</em> determination in these various conditions.

<strong class="sub-title">Background:</strong> The pandemic of new severe acute respiratory syndrome (SARS) due to coronavirus (CoV) <em>2</em> (SARS-CoV-<em>2</em>) has stressed the importance of effective diagnostic and prognostic biomarkers of clinical worsening and mortality. Epidemiological data showing a differential impact of SARS-CoV-<em>2</em> infection on women and men have suggested a potential role for testosterone (T) in determining gender disparity in the SARS-CoV-<em>2</em> clinical outcomes.

<strong class="sub-title">Objectives:</strong> To estimate the association between T level and SARS-CoV-<em>2</em> clinical outcomes (defined as conditions requiring transfer to higher or lower intensity of care or death) in a cohort of patients admitted in the respiratory intensive care unit (RICU).

<strong class="sub-title">Materials and methods:</strong> A consecutive series of 31 male patients affected by SARS-CoV-<em>2</em> pneumonia and recovered in the respiratory intensive care unit (RICU) of the "Carlo Poma" Hospital in Mantua were analyzed. Several biochemical risk factors (ie, blood count and leukocyte formula, C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), ferritin, D-dimer, fibrinogen, interleukin 6 (IL-6)) as well as total testosterone (TT), calculated free T (cFT), sex hormone-binding globulin (SHBG), and luteinizing hormone (LH) were determined.

Results: Lower TT and cFT were found in the transferred to ICU/deceased in RICU group vs groups of patients transferred to IM or maintained in the RICU in stable condition. Both TT and cFT showed a negative significant correlation with biochemical risk factors (ie, the neutrophil count, LDH, and PCT) but a positive association with the lymphocyte count. Likewise, TT was also negatively associated with CRP and ferritin levels. A steep increase in both ICU transfer and mortality risk was observed in men with TT < 5 nmol/L or cFT < 100 pmol/L.

<strong class="sub-title">Discussion and conclusion:</strong> Our study demonstrates for the first time that lower baseline levels of TT and cFT levels predict poor prognosis and mortality in SARS-CoV-<em>2</em>-infected men admitted to RICU.

Keywords: COVID-19; inflammatory markers; mortality; prognosis; sex hormones.

OBJECTIVE

Acute aortic syndrome (AAS), a potentially fatal pathologic process within the aortic wall, should be suspected in patients presenting with severe thoracic pain and hypertension. AAS, including aortic dissection (approximately 90% of cases) and intramural hematoma, may be complicated by poor perfusion, aneurysm, or uncontrollable pain and hypertension. AAS is uncommon (approximately 3.5-6.0 per 100,000 patient-years) but rapid diagnosis is imperative as an emergency surgical procedure is frequently necessary.

OBJECTIVE

To systematically review the current evidence on diagnosis and treatment of AAS.

METHODS

Searches of MEDLINE, EMBASE, and the Cochrane Register of Controlled Trials for articles on diagnosis and treatment of AAS from June 1994 to January <em>2</em>9, <em>2</em>016, were performed. Only clinical trials and prospective observational studies of 10 or more patients were included. Eighty-two studies (<em>2</em> randomized clinical trials and 80 observational) describing 57,311 patients were reviewed.

RESULTS

Chest or back pain was the most commonly reported presenting symptom of AAS (61.6%-84.8%). Patients were typically aged 60 to 70 years, male (50%-81%), and had hypertension (45%-100%). Sensitivities of computerized tomography and magnetic resonance imaging for diagnosis of AAS were 100% and 95% to 100%, respectively. Transesophageal echocardiography was 86% to 100% sensitive, whereas D-dimer was 51.7% to 100% sensitive and 3<em>2</em>.8% to 89.<em>2</em>% specific among 6 studies (n = 876). An immediate open surgical procedure is needed for dissection of the ascending aorta, given the high mortality (<em>2</em>6%-58%) and proximity to the aortic valve and great vessels (with potential for dissection complications such as tamponade). An RCT comparing endovascular surgical procedure to medical management for uncomplicated AAS in the descending aorta (n = 61) revealed no dissection-related deaths in either group. Endovascular surgical procedure was better than medical treatment (97% vs 43%, P < .001) for the primary end point of "favorable aortic remodeling" (false lumen thrombosis and no aortic dilation or rupture). The remaining evidence on therapies was observational, introducing significant selection bias.

CONCLUSIONS

Because of the high mortality rate, AAS should be considered and diagnosed promptly in patients presenting with acute chest or back pain and high blood pressure. Computerized tomography, magnetic resonance imaging, and transesophageal echocardiography are reliable tools for diagnosing AAS. Available data suggest that open surgical repair is optimal for treating type A (ascending aorta) AAS, whereas thoracic endovascular aortic repair may be optimal for treating type B (descending aorta) AAS. However, evidence is limited by the paucity of randomized trials.

OBJECTIVE

To evaluate the coagulative/fibrinolytic cascade and the circulating markers of the endothelial injury in systemic sclerosis (SSc).

METHODS

Plasma was obtained from <em>2</em>9 patients with SSc and tested for thrombin-antithrombin (TAT), fragments 1+<em>2</em> (F1+<em>2</em>), dermatansulphate (<em>D</em>S), thrombomodulin (TM), lipoprotein (a) [Lp(a)], von Willebrand factor (vWF), tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI), <em>D</em>-<em>dimers</em>, intercellular adhesion molecole-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and E-selectin. The data were correlated with lung (forced vital capacity, diffusing lung capacity for carbon monoxide, vital capacity) and skin (skin score) involvement.

RESULTS

Coagulation was significantly activated (increase in F1+<em>2</em>, P <.001; TAT, P <.01; and Lp(a), P <.05). TM was not significantly different from controls. vWF was significantly increased (P <.01), and its supranormal multimers increased in more than 50% of patients. <em>D</em>S was significantly increased in diffuse cutaneous SSc (P <.01). Fibrinolysis was impaired as shown by reduced <em>D</em>-<em>dimers</em> (P <.01) and decreased levels of PAI (P < 0.01). The markers of endothelial injury were also significantly elevated. <em>D</em>S correlated significantly with forced vital capacity (P <.01) and forced vital capacity ratio (P <.01).

CONCLUSIONS

Injury to the endothelium reduces endothelial function, as suggested by impairment of fibrinolysis and activation of the coagulative pathway. The loss of the balance between fibrinolysis and coagulation contributes to vessel engulfment with fibrin and breakdown of vessel patency. The increase of circulating DS suggests that this factor may be a new marker of endothelial injury.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of <em>dimer</em>ic regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII <em>dimer</em> interaction with the PKA substrate molecule microtubule-associated protein <em>2</em> (MAP<em>2</em>). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP<em>2</em> binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha <em>dimer</em>ization always prevented MAP<em>2</em> interaction because 1) RII <em>delta</em> 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP<em>2</em> or form <em>dimers</em>, and <em>2</em>) a modified RII alpha monomer including residues 1-14 did not bind MAP<em>2</em>. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed <em>dimers</em> but did not bind MAP<em>2</em>. This suggested other side-chains between residues 30-79 also participate in MAP<em>2</em> interaction. Peptide studies indicate additional contact with MAP<em>2</em> may occur through an acidic region (residues 68-8<em>2</em>) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP<em>2</em> as to allow its preferential phosphorylation upon kinase activation.

BACKGROUND

Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(<em>2</em>) conditions.

RESULTS

The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to <em>2</em>.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit.

CONCLUSIONS

The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(<em>2</em>) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(<em>2</em>) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (P<em>D</em>E6), is a catalytic hetero<em>dimer</em> (alphabeta) to which low molecular weight inhibitory gamma subunits bind to form the nonactivated P<em>D</em>E holoenzyme (alphabetagamma(<em>2</em>)). Although it is known that gamma binds tightly to alphabeta, the binding affinity for each gamma subunit to alphabeta, the domains on gamma that interact with alphabeta, and the allosteric interactions between gamma and the regulatory and catalytic regions on alphabeta are not well understood. We show here that the gamma subunit binds to two distinct sites on the catalytic alphabeta <em>dimer</em> (K(<em>D</em>)(1) < 1 pm, K(<em>D</em>)(<em>2</em>) = 3 pm) when the regulatory GAF domains of bovine rod P<em>D</em>E6 are occupied by cGMP. Binding heterogeneity of gamma to alphabeta is absent when cAMP occupies the noncatalytic sites. Two major domains on gamma can interact independently with alphabeta with the N-terminal half of gamma binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of gamma is responsible for the positive cooperativity between gamma and cGMP binding sites on alphabeta but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of gamma that bind to alphabeta, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of gamma interaction with alphabeta, and positive cooperativity of gamma with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of P<em>D</em>E6 during visual transduction in rod photoreceptors.

Maleylacetate reductase (EC 1.3.1.3<em>2</em>) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of <em>2</em>,4-dichlorophenoxyacetate (<em>2</em>,4-<em>D</em>)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a <em>dimer</em> of two identical subunits of 35 k<em>D</em>a. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by E<em>D</em>TA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and <em>2</em>-chloromaleylacetate were converted with similar efficiencies (with NA<em>D</em>H as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,<em>2</em>80/min, respectively). NA<em>D</em>H was preferred to NA<em>D</em>PH as the cosubstrate. Upon reduction of <em>2</em>-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NA<em>D</em>H. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the <em>2</em>,4-<em>D</em> degradation intermediate <em>2</em>-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH<em>2</em>-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in <em>2</em>,4-<em>D</em> degradation.

BACKGROUND

Patients experiencing massive haemorrhage are at high risk of developing coagulopathy through loss, consumption, and dilution of coagulation factors and platelets. It has been reported that plasma fibrinogen concentrations may reach a critical low level relatively early during bleeding, calling for replacement fibrinogen therapy. Cryoprecipitate has been widely used in the past, but more recently, a pasteurized fibrinogen concentrate has become available. We audited the effects of fibrinogen concentrate therapy on laboratory and clinical outcome in patients with massive haemorrhage.

METHODS

We identified 43 patients over the previous <em>2</em> yr to whom a fibrinogen concentrate had been administered as treatment for hypofibrinogenaemia during serious haemorrhage. Platelet count, P-fibrinogen, activated partial thromboplastin time (APTT), prothrombin time (PT), <em>D</em>-<em>dimer</em>, and volume of blood lost were obtained from medical and laboratory records. Numbers of units of red blood cells (RBC), fresh frozen plasma (FFP), and pooled platelet concentrates were recorded before and after fibrinogen substitution.

RESULTS

A significant increase in plasma fibrinogen concentration was observed after fibrinogen concentrate therapy. Platelet counts and fibrin D-dimer values remained unchanged, whereas the APTT and PT improved significantly. Requirements for RBC, FFP, and platelets were significantly reduced. Blood loss decreased significantly.

CONCLUSIONS

Off-label substitution therapy with a fibrinogen concentrate generally improved global laboratory coagulation results and as supplementary intervention, appeared to diminish the requirements for RBC, FFP, and platelet substitution in this patient cohort.

Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of CP. We demonstrate that the unusual His(4) motif (site <em>2</em>) formed at the S100A8/S100A9 <em>dimer</em> interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters <em>D</em> = <em>2</em>70 MHz and E/<em>D</em> = 0.30 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H<em>2</em>7 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The His(3)Asp motif (site 1), which is also formed at the S100A8/S100A9 <em>dimer</em> interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the His(4) site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the His(4) site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site <em>2</em>. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth.

The barrier-to-autointegration factor BAF bin<em>d</em>s to the LEM <em>d</em>omain (Em(LEM)) of the nuclear envelope protein emerin an<em>d</em> plays an essential role in the nuclear architecture of metazoan cells. In a<em>d</em><em>d</em>ition, the BAF(<em>2</em>) <em>dimer</em> bri<em>d</em>ges an<em>d</em> compacts <em>d</em>ouble-stran<em>d</em>e<em>d</em> DNA nonspecifically via two symmetry-relate<em>d</em> DNA bin<em>d</em>ing sites. In this article we present biophysical an<em>d</em> structural stu<em>d</em>ies on a complex of BAF(<em>2</em>) an<em>d</em> Em(LEM). Light scattering, analytical ultracentrifugation, an<em>d</em> NMR in<em>d</em>icate a stoichiometry of one molecule of Em(LEM) boun<em>d</em> per BAF(<em>2</em>) <em>dimer</em>. The equilibrium <em>d</em>issociation constant (K(<em>d</em>)) for the interaction of the BAF(<em>2</em>) <em>dimer</em> an<em>d</em> Em(LEM), <em>d</em>etermine<em>d</em> by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between correspon<em>d</em>ing cross-peaks of the magnetically non-equivalent subunits of the BAF(<em>2</em>) <em>dimer</em> in the complex yiel<em>d</em>s a <em>d</em>issociation rate constant of 78 +/- <em>2</em>s(-1). The solution NMR structure of the BAF(<em>2</em>)-Em(LEM) complex reveals that the LEM an<em>d</em> DNA bin<em>d</em>ing sites on BAF(<em>2</em>) are non-overlapping an<em>d</em> that both subunits of the BAF(<em>2</em>) <em>dimer</em> contribute approximately equally to the Em(LEM) bin<em>d</em>ing site. The relevance of the implications of the structural an<em>d</em> biophysical <em>d</em>ata on the complex in the context of the interaction between the BAF(<em>2</em>) <em>dimer</em> an<em>d</em> Em(LEM) at the nuclear envelope is <em>d</em>iscusse<em>d</em>.

We have examined the binding of two radioligands ([(3)H]spiperone and [(3)H]raclopride) to <em>D</em>(<em>2</em>) dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites, whereas in the absence of sodium ions [(3)H]raclopride labeled about half the number of sites labeled by [(3)H]spiperone. In competition experiments in the absence of sodium ions, however, raclopride was able to inhibit [(3)H]spiperone binding fully. In saturation analyses with [(3)H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a <em>dimer</em>, and in the absence of sodium ions, raclopride exerts negative cooperativity across the <em>dimer</em> both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here.

MUC5AC mucins secreted by HT-<em>2</em>9 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, <em>D</em>. J. (<em>2</em>000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and <em>dimer</em>, GalNAc-substituted <em>dimer</em>, fully glycosylated <em>dimer</em>, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately <em>2</em>-4 h). The results show that the mucin polypeptide undergoes <em>dimer</em>ization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin <em>dimer</em>, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology <em>2</em> (SH<em>2</em>) domain of APS. Here, we present the crystal structure of the APS SH<em>2</em> domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel <em>dimer</em>ic configuration of the APS SH<em>2</em> domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH<em>2</em> domains. The APS SH<em>2</em> <em>dimer</em> engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH<em>2</em> domain and a second phosphotyrosine, pTyr-116<em>2</em>, coordinated by two lysine residues in beta strand <em>D</em>. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its <em>dimer</em>ic receptor.

Advanced atherosclerosis is often associated with dystrophic calcification, which may contribute to plaque rupture and thrombosis. In this work, the localization and association of the noncollagenous bone matrix proteins osteonectin, osteopontin, and osteocalcin with calcification, lipoproteins, thrombus/hemorrhage (T/H), and matrix metalloproteinases (MMPs) in human carotid arteries from endarterectomy samples have been determined. According to the recent American Heart Association classification, 6 of the advanced lesions studied were type V (fibroatheroma) and 16 type VI (complicated). Osteonectin, osteocalcin, and osteopontin were identified by monoclonal antibodies IIIA(3)A(8), G1<em>2</em>, and MPIIIB10(1) and antiserum LF-1<em>2</em>3. Apolipoprotein (apo) AI, B, and E; lipoprotein(a); fibrinogen; fibrin; fragment <em>D</em>/<em>D</em>-<em>dimer</em>; MMP-<em>2</em> (gelatinase A); and MMP-3 (stromelysin-1) were identified with previously characterized antibodies. Calcium phosphate deposits (von Kossa's stain) were present in 8<em>2</em>% of samples (3 type V and 15 type VI). Osteonectin was localized in endothelial cells, SMCs, and macrophages and was associated with calcium deposits in 33% of type V and 88% of type VI lesions. Osteopontin was distributed similarly to osteonectin and was associated with calcium deposits in 50% of type V and 94% of type VI lesions. Osteocalcin was localized in large calcified areas only (in 17% of type V and 38% of type VI lesions). ApoB colocalized with cholesterol crystals and calcium deposits. Lipoprotein(a) was localized in the intima, subintima, and plaque shoulder. Fibrin (T/H) colocalized with bone matrix proteins in 33% of type V and 69% of type VI lesions. MMP-3 was cytoplasmic in most cells and colocalized with calcium and fibrin deposits. MMP-<em>2</em> was less often associated with calcification. The results of this study show that osteonectin, osteopontin, and osteocalcin colocalized with calcium deposits with apoB, fibrin, and MMP-3 in advanced, symptomatic carotid lesions. These data suggest that the occurrence of T/H might contribute to dystrophic arterial calcification in the progression and complications of atherosclerosis.

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-<em>D</em> sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA <em>dimers</em> and monomers in all-atom, explicit dilauroylphosphatidylcholine (<em>D</em>LPC), dimyristoylphosphatidylcholine (<em>D</em>MPC), dioleoylphosphatidylcholine (<em>D</em>OPC), and 1-palmitoyl-<em>2</em>-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4-5 Å from the channel, which was followed by an increase in <em>D</em>OPC and POPC or a further decrease in <em>D</em>LPC and <em>D</em>MPC bilayers. The bilayer thickness varied little in the monomer simulations-except one of three independent simulations for <em>D</em>MPC and all three <em>D</em>LPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA <em>dimers</em> due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA <em>dimers</em> are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.

OBJECTIVE

Pharmacological prophylaxis can reduce the risk of deep venous thrombosis (DVT), pulmonary embolism (PE), and death, and it is recommended 10–35 days after total hip arthroplasty (THA) and at least 10 days after total knee arthroplasty (TKA). However, early mobilization might also reduce the risk of DVT and thereby the need for prolonged prophylaxis, but this has not been considered in the previous literature. Here we report our results with short-duration pharmacological prophylaxis combined with early mobilization and reduced hospitalization.

METHODS

1,977 consecutive, unselected patients were operated with primary THA, TKA, or bilateral simultaneous TKA (BSTKA) in a well-described standardized fast-track set-up from 2004–2008. Patients received DVT prophylaxis with low-molecular-weight heparin starting 6–8 h after surgery until discharge. All re-admissions and deaths within 30 and 90 days were analyzed using the national health register, concentrating especially on clinical DVT (confirmed by ultrasound and elevated D-dimer), PE, or sudden death. Numbers were correlated to days of prophylaxis (LOS).

RESULTS

The mean LOS decreased from 7.3 days in 2004 to 3.1 days in 2008. 3 deaths (0.15%) were associated with clotting episodes and overall, 11 clinical DVTs (0.56%) and 6 PEs (0.30%) were found. The vast majority of events took place within 30 days; only 1 death and 2 DVTs occurred between 30 and 90 days. During the last 2 years (854 patients), when patients were mobilized within 4 h postoperatively and the duration of DVT prophylaxis was shortest (1–4 days), the mortality was 0% (95% CI: 0–0.5). Incident cases of DVT in TKA was 0.60% (CI: 0.2–2.2), in THA it was 0.51% (CI: 0.1–1.8), and in BSTKA it was 0% (CI: 0–2.9). Incident cases of PE in TKA was 0.30% (CI: 0.1–1.7), in THA it was 0% (CI: 0–1.0), and in BSTKA it was 0% (CI: 0–2.9).

CONCLUSIONS

The risk of clinical DVT, and of fatal and non-fatal PE after THA and TKA following a fast-track set-up with early mobilization, short hospitalization, and short duration of DVT prophylaxis compares favorably with published regimens with extended prophylaxis (up to 36 days) and hospitalization up to 11 days. This calls for a reconsideration of optimal duration of chemical thromboprophylaxis.