Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone N(<em>delta</em>)-(5-hydro-5-methyl-4-imidazolon-<em>2</em>-yl)-ornithine (9<em>2</em>% of total modification) with a minor formation of argpyrimidine, N(epsilon)-(1-carboxyethyl)lysine, and methylglyoxal lysine <em>dimer</em>. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-<em>2</em>18, and Arg-4<em>2</em>8. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pK(a) = 7.9 +/- 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pK(a) to 8.8 +/- 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of arginine-directed hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation.

Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system.

METHODS

Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody.

RESULTS

PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization.

CONCLUSIONS

(124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.

OBJECTIVE

Venous thromboembolism (VTE) is a well-recognized complication of cancer. Laboratory parameters might be useful to assess the VTE risk in patients with cancer. The aim of this study was to investigate <em>D</em>-<em>dimer</em> and prothrombin fragment 1 + <em>2</em> (F 1 + <em>2</em>), which reflect activation of blood coagulation and fibrinolysis, for prediction of cancer-associated VTE.

METHODS

In a prospective, observational, cohort study of 8<em>2</em>1 patients with newly diagnosed cancer or progression of disease who did not recently receive chemotherapy, radiotherapy, or surgery were enrolled and followed for a median of 501 days (interquartile range, <em>2</em>55 to 731 days). The malignancies in these patients were as follows: breast (n = 13<em>2</em>), lung (n = 119), stomach (n = 35), lower gastrointestinal tract (n = 106), pancreas (n = 46), kidney (n = <em>2</em><em>2</em>), and prostate (n = 101) cancers; high-grade glioma (n = 10<em>2</em>); malignant lymphoma (n = 94); multiple myeloma (n = 17); and other tumor types (n = 47). The study end point was occurrence of objectively confirmed symptomatic or fatal VTE.

RESULTS

VTE occurred in 6<em>2</em> patients (7.6%). The cutoff level for elevated <em>D</em>-<em>dimer</em> and elevated F 1 + <em>2</em> was set at the 75th percentile of the total study population. In multivariable analysis that included elevated <em>D</em>-<em>dimer</em>, elevated F 1 + <em>2</em>, age, sex, surgery, chemotherapy, and radiotherapy, the hazard ratios (HRs) of VTE in patients with elevated <em>D</em>-<em>dimer</em> (HR, 1.8; 95% CI, 1.0 to 3.<em>2</em>; P = .048) and elevated F 1 + <em>2</em> (HR, <em>2</em>.0; 95% CI, 1.<em>2</em> to 3.6; P = .015) were statistically significantly increased. The cumulative probability of developing VTE after 6 months was highest in patients with both elevated <em>D</em>-<em>dimer</em> and elevated F 1 + <em>2</em> (15.<em>2</em>%) compared with patients with nonelevated <em>D</em>-<em>dimer</em> and nonelevated F 1 + <em>2</em> (5.0%; P < .001).

CONCLUSIONS

High <em>D</em>-<em>dimer</em> and F 1 + <em>2</em> levels independently predict occurrence of VTE in patients with cancer.

<em>D</em>epression is a multicausal disorder and has been associated with the risk to develop cancer, dementia, diabetes, epilepsy and stroke. As a metabolic disorder depression has been associated with obesity, diabetes, insulin sensitivity, neuropeptide Y, glucose regulation, poor glycemic control, glucagone-like peptide-1, cholezystokinin, ghrelin, leptin, the endocannabinoid system, insulin-like growth factor and gastrin-releasing peptide. As a cardiovascular disease a close relationship exists between depression and blood pressure, heart rate, norepinephrine, sympathetic tone, vascular resistance, blood viscosity, plasma volume, intima thickness and atherosclerosis. Additionally blood coagulation, fibrinolysis, <em>D</em>-<em>dimers</em>, plasminogen activator inhibitor-1 protein, platelet activation, VEGF, plasma nitric oxide and its synthase are changed in depressed patients. As an endocrinological and stress disorder depression has been connected with the concentration of free T4, TSH, CRH, arginine vasopressin, corticotrophin, corticosteroid release and ACTH. <em>D</em>epression as an inflammatory disorder is mediated by pro-inflammatory cytokines, interleukin-1, interleukin-6, TNF-alpha, soluble interleukin-<em>2</em> receptors, interferon-alpha, interleukin 8, interleukin-10, hs-CRP, acute phase proteins, haptoglobin, toll like receptor 4, interleukin-1beta, mammalian target of rapamycin pathway, substance P, cyclooxygenase-<em>2</em>, prostaglandin-E<em>2</em>, lipid peroxidation levels and acid sphingomyelinase. Nutritional factors might influence depression risk, i.e. the consumption of folate, omega-3 fatty acids, monounsaturated fatty acids, olive oil, fish, fruits, vegetables, nuts, legumes, vitamin B6 and vitamin B1<em>2</em>. The neurodegenerative hypothesis of depression explains decreased hippocampal volumes in depressed patients and changes of neurotrophic support by B<em>D</em>NF, erythropoietin, G<em>D</em>NF, FGF-<em>2</em>, NT3, NGF and growth hormone. In this context, a fast neuroprotective and antidepressant effect has also been observed by ketamine, which acts via the glutamatergic system. Hence, GABA, AMPA, EAAT, NM<em>D</em>A- and metabotropic glutamate receptors (mGluR1 to mGluR8) have gained interest in depression recently. Alternative, causative or also easy available treatment strategies beyond serotonin and noradrenaline reuptake inhibition might be a major topic of future psychiatric care. In this review, an attempt is made to overview concepts of the disease and search for perspectives on antidepressant treatment strategies beyond approved medications.

OBJECTIVE

Among a subgroup of participants in the Strategies for Management of Antiretroviral Therapy (SMART) Trial that were naïve to antiretroviral therapy (ART) or off ART (6 months or longer) at study entry, risk of AIDS and serious non-AIDS events were increased for participants who deferred ART compared with those randomized to (re)initiate ART immediately. Our objective was to determine whether ART initiation in this group reduced markers of inflammation and coagulation that have been associated with increased mortality risk in SMART. Changes in these biomarkers have been described after stopping ART, but not after starting ART in SMART.

METHODS

Stored specimens for <em>2</em>54 participants (1<em>2</em>6 drug conservation [<em>D</em>C] and 1<em>2</em>8 viral suppression [VS]) who were naïve to ART or off ART (6 months or longer) were analyzed for interleukin-6, high sensitivity C-reactive protein, and <em>D</em>-<em>dimer</em> at baseline and Months <em>2</em> and 6.

RESULTS

At Month 6, 6<em>2</em>% of the VS group had HIV RNA less than 400 copies/mL and median C<em>D</em>4 count was 190 cells/mm3 higher than for the <em>D</em>C group (590 versus 400 cells/mm3). Compared with <em>D</em>C, the VS group had 3<em>2</em>% (95% confidence interval, 19%-43%) lower <em>D</em>-<em>dimer</em> levels at Month 6 (P < 0.001); differences were not significant for high sensitivity C-reactive protein or interleukin-6 levels.

CONCLUSIONS

In this randomized comparison of immediate versus delayed ART initiation, D-dimer, but not interleukin-6 and high sensitivity C-reactive protein, declined significantly after starting ART. Further studies are needed to determine whether improvements in D-dimer are associated with reduced risk of clinical disease and whether adjunct treatments used in combination with ART can reduce inflammation among individuals with HIV infection.

BACKGROUND

Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease.

METHODS

In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in <em>2</em>5 consecutive patients with sickle cell disease.

RESULTS

The majority of microparticles originated from platelets (GPIIIa,C<em>D</em>61) and erythrocytes (glycophorin A,C<em>D</em><em>2</em>35), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=-0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and <em>D</em>-<em>dimer</em> and prothrombin fragment F1+<em>2</em> (r=0.5<em>2</em>, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.0<em>2</em>3).

CONCLUSIONS

We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles.

BACKGROUND

The degree to which human immunodeficiency virus (HIV) continues to replicate during antiretroviral therapy (ART) is controversial. We conducted a randomized, double-blind, placebo-controlled study to assess whether raltegravir intensification reduces low-level viral replication, as defined by an increase in the level of <em>2</em>-long terminal repeat (<em>2</em>-LTR) circles.

METHODS

Thirty-one subjects with an ART-suppressed plasma HIV RNA level of <40 copies/mL and a CD4(+) T-cell count of ≥350 cells/mm(3) for ≥1 year were randomly assigned to receive raltegravir 400 mg twice daily or placebo for <em>2</em>4 weeks. <em>2</em>-LTR circles were analyzed by droplet digital polymerase chain reaction at weeks 0, 1, <em>2</em>, and 8.

RESULTS

The median duration of ART suppression was 3.8 years. The raltegravir group had a significant increase in the level of <em>2</em>-LTR circles, compared to the placebo group. The week 1 to 0 ratio was 8.8-fold higher (P = .00<em>2</em>5) and the week <em>2</em> to 0 ratio was 5.7-fold higher (P = .0<em>2</em>3) in the raltegravir vs. placebo group. Intensification also led to a statistically significant decrease in the D-dimer level, compared to placebo (P = .045).

CONCLUSIONS

Raltegravir intensification resulted in a rapid increase in the level of <em>2</em>-LTR circles in a proportion of subjects, indicating that low-level viral replication persists in some individuals even after long-term ART. Intensification also reduced the D-dimer level, a coagulation biomarker that is predictive of morbidity and mortality among patients receiving treatment for HIV infection.

Patients with hypertension, diabetes, coronary heart disease, cerebrovascular illness, COP<em>D</em>, and kidney dysfunction have worse clinical outcomes when infected with SARS-CoV-<em>2</em>, for unknown reasons. The purpose of this review is to summarize the evidence for the existence of elevated plasmin(ogen) in COVI<em>D</em>-19 patients with these comorbid conditions. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-<em>2</em>, extracellularly, which increases its infectivity and virulence. Hyper-fibrinolysis associated with plasmin leads to elevated <em>D</em>-<em>dimer</em> in severe patients. The plasmin(ogen) system may prove a promising therapeutic target for combating COVI<em>D</em>-19.

OBJECTIVE

To investigate the hemostatic status of critically ill, nonbleeding trauma patients. We hypothesized that a hypercoagulable state exists in patients early after severe injury and that the pattern of clotting and fibrinolysis are similar between burned and nonburn trauma patients.

METHODS

Patients admitted to the surgical or burn intensive care unit within 24 hours after injury were enrolled. Blood samples were drawn on days 0 through 7. Laboratory tests included prothrombin time (PT), activated partial thromboplastin time (aPTT), levels of activated factor XI, D-dimer, protein C percent activity, antithrombin III percent activity, and thromboelastography (TEG).

RESULTS

Study subjects were enrolled from April 1, 2004, to May 31, 2005, and included nonburn trauma patients (n = 33), burned patients (n = 25), and healthy (control) subjects (n = 20). Despite aggressive thromboprophylaxis, three subjects (2 burned and 1 nonburn trauma patients [6%]) had pulmonary embolism during hospitalization. Compared with controls, all patients had prolonged PT and aPTT (p < 0.05). The rate of clot formation (alpha angle) and maximal clot strength were higher for patients compared with those of controls (p < 0.05), indicating a hypercoagulable state. Injured patients also had lower protein C and antithrombin III percent activities and higher fibrinogen levels (p < 0.05 for all). Activated factor XI was elevated in 38% of patients (control subjects had undetectable levels).

CONCLUSIONS

Thromboelastography analysis of whole blood showed that patients were in a hypercoagulable state; this was not detected by plasma PT or aPTT. The high incidence of pulmonary embolism indicated that our current prophylaxis regimen could be improved.

The molecular mechanism un<em>d</em>erlying the promotion of woun<em>d</em> healing by TGF-beta 1 is incompletely un<em>d</em>erstoo<em>d</em>. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by mo<em>d</em>ulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratifie<em>d</em> culture<em>d</em> epi<em>d</em>ermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, an<em>d</em> the collagen receptor alpha <em>2</em> beta 1 by <em>d</em>ifferentially mo<em>d</em>ulating the synthesis of their alpha an<em>d</em> beta subunits; (b) <em>d</em>ownregulates the multifunctional alpha 3 beta 1 hetero<em>d</em>imer; (c) in<em>d</em>uces the <em>d</em>e novo expression an<em>d</em> surface exposure of the alpha v beta 6 fibronectin receptor; (<em>d</em>) stimulates keratinocyte migration towar<em>d</em> fibronectin an<em>d</em> vitronectin; (e) in<em>d</em>uces a marke<em>d</em> perturbation of the general mechanism of polarize<em>d</em> <em>d</em>omain sorting of both beta 1 an<em>d</em> beta 4 <em>dimers</em>; an<em>d</em> (f) causes a pericellular re<em>d</em>istribution of alpha v beta 5. These <em>d</em>ata suggest that alpha 5 beta 1, alpha v beta 6, an<em>d</em> alpha v beta 5, not routinely use<em>d</em> by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, an<em>d</em> uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human woun<em>d</em>s. In<em>d</em>ee<em>d</em>, TGF-beta 1 repro<em>d</em>uces the integrin expression pattern of keratinocytes locate<em>d</em> at the injury site, particularly of cells in the migrating epithelial tongue at the lea<em>d</em>ing e<em>d</em>ge of the woun<em>d</em>. Since these keratinocytes are inhibite<em>d</em> in their proliferative capacity, these <em>d</em>ata might account for the apparent para<em>d</em>ox of a TGF-beta 1-<em>d</em>epen<em>d</em>ent stimulation of epi<em>d</em>ermal woun<em>d</em> healing associate<em>d</em> with a growth inhibitory effect on epithelial cells.

A<em>D</em>P-ribosylation factors (ARFs) are essential and ubiquitous in eukaryotes, being involved in vesicular transport and functioning as an activator of phospholipase <em>D</em> (refs 1, <em>2</em>) and cholera toxin. The functions of ARF proteins in membrane traffic and organelle integrity are intimately tied to its reversible association with membranes and specific interactions with membrane phospholipids. One common feature of these functions is their regulation by the binding and hydrolysis of GTP. Here we report the three-dimensional structure of full-length human ARF1 (M(r) <em>2</em>1,000) in its G<em>D</em>P-bound non-myristoylated form. The presence of a unique amino-terminal alpha-helix and loop, together with differences in Mg<em>2</em>+ ligation and the existence of a non-crystallographic <em>dimer</em>, set this structure apart from other GTP-binding proteins. These features provide a structural basis for the GTP-dependent modulation of membrane affinity, the lack of intrinsic GTPase activity, and the nature of effector binding surfaces.

Background: Severe COVID-19 can manifest in rapid decompensation and respiratory failure with elevated inflammatory markers, consistent with cytokine release syndrome for which IL-6 blockade is approved treatment.

Methods: We assessed effectiveness and safety of IL-6 blockade with tocilizumab in a single-center cohort of patients with COVID-19 requiring mechanical ventilation. The primary endpoint was survival probability post-intubation; secondary analyses included an ordinal illness severity scale integrating superinfections. Outcomes in patients who received tocilizumab compared to tocilizumab-untreated controls were evaluated using multivariable Cox regression with propensity score inverse probability weighting (IPTW).

Results: 154 patients were included, of whom 78 received tocilizumab and 76 did not. Median follow-up was 47 days (range 28-67). Baseline characteristics were similar between groups, although tocilizumab-treated patients were younger (mean 55 vs. 60 years), less likely to have chronic pulmonary disease (10% vs. 28%), and had lower D-dimer values at time of intubation (median 2.4 vs. 6.5 mg/dL). In IPTW-adjusted models, tocilizumab was associated with a 45% reduction in hazard of death [hazard ratio 0.55 (95% CI 0.33, 0.90)] and improved status on the ordinal outcome scale [odds ratio per 1-level increase: 0.58 (0.36, 0.94)]. Though tocilizumab was associated with an increased proportion of patients with superinfections (54% vs. 26%; p<0.001), there was no difference in 28-day case fatality rate among tocilizumab-treated patients with versus without superinfection [22% vs. 15%; p=0.42]. Staphylococcus aureus accounted for ~50% of bacterial pneumonia.

Conclusions: In this cohort of mechanically ventilated COVID-19 patients, tocilizumab was associated with lower mortality despite higher superinfection occurrence.

Keywords: COVID-19; SARS-CoV-2; interleukin-6; tocilizumab.

This paper concerns the properties of herpes simplex virus 1 <em>D</em>NA replicating in HEp-<em>2</em> an<em>d</em> human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral <em>D</em>NA entere<em>d</em> the replicative pool. The bulk of the input viral <em>D</em>NA cose<em>d</em>imente<em>d</em> with marker viral <em>D</em>NA an<em>d</em> <em>d</em>i<em>d</em> not appear to be <em>d</em>egra<em>d</em>e<em>d</em> or <em>d</em>issociate<em>d</em> into L an<em>d</em> S components. (ii) Nascent <em>D</em>NA se<em>d</em>imente<em>d</em> faster an<em>d</em> ban<em>d</em>e<em>d</em> at a higher <em>d</em>ensity than that of mature viral <em>D</em>NA extracte<em>d</em> from virions. Pulse-chase experiments in<em>d</em>icate<em>d</em> that nascent <em>D</em>NA acquires the se<em>d</em>imentation rate an<em>d</em> buoyant <em>d</em>ensity of viral <em>D</em>NA within 30 to 40 min after its synthesis. (iii) Electron microscopic stu<em>d</em>ies in<em>d</em>icate<em>d</em> that the <em>D</em>NA extracte<em>d</em> from cells replicating viral <em>D</em>NA an<em>d</em> ban<em>d</em>ing at the <em>d</em>ensity of viral <em>D</em>NA containe<em>d</em>: (a) linear, full-size molecules with internal gaps an<em>d</em> single-stran<em>d</em>e<em>d</em> regions at termini; (b) molecules with lariats, consisting of a linear segment up to <em>2</em>x the size of mature <em>D</em>NA an<em>d</em> a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous an<em>d</em> <em>d</em>iscontinuous forks; (c) circular, <em>d</em>ouble-stran<em>d</em>e<em>d</em> molecules, both full-size an<em>d</em> multiples of 18 x 10(6) in molecular weight, but without forks or loops; (<em>d</em>) molecules showing "eye" an<em>d</em> "<em>D</em>" loops at or near one en<em>d</em> of the <em>D</em>NA; (e) large, tangle<em>d</em> masses of <em>D</em>NA, similar to those observe<em>d</em> for T4 an<em>d</em> pseu<em>d</em>orabies virus replicating <em>D</em>NAs, containing loops an<em>d</em> continuous an<em>d</em> <em>d</em>iscontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stran<em>d</em>e<em>d</em> en<em>d</em>s on the <em>D</em>NA anneal to form a hairpin, that the <em>D</em>NA synthesis is initiate<em>d</em> at or near that en<em>d</em> an<em>d</em> procee<em>d</em>s bi<em>d</em>irectionally to form a lariat, an<em>d</em> that resulting progeny <em>d</em>erive<em>d</em> by semiconservative replication are "hea<em>d</em>-to-hea<em>d</em>" an<em>d</em> "tail-to-tail" <em>dimers</em>.

For many ionic polysaccharides, the ability to form gels in the presence of divalent cations such as calcium is the key to biological functions and technological applications. This is particularly true for alginates and pectins, where the regular occurrence of respectively alpha-L-(1-4)-guluronate residues and alpha-<em>D</em>-galacturonate residues generates ordered templates for polymer chain associations that are involved in physical gels. The molecular basis responsible for the strength and the stereospecificity of calcium interactions for the two polysaccharides were investigated in a previous paper (Braccini; et al. Carbohydr. Res. 1999, 119). In the present work, a novel molecular modeling procedure has been developed; it involves a pairing procedure that evaluates all the possible associations of the ordered polyuronate chains with calcium ions to form <em>dimers</em>. Starting from the stable ordered forms of polygalacturonate and polyguluronate, all possible ways to form Ca(<em>2</em>+)-bridged <em>dimers</em> were computed; the parallel and antiparallel relative arrangements of the chains were also considered. <em>D</em>espite the structural analogy between polyguluronate and polygalacturonate chains, significant differences at the level of chain-chain associations are found. The popular "egg box model" can still be referred to in the case of polyguluronate. However, it cannot be used to describe a pectate junction zone as the unique feature of two consecutive chelation site per repeat, that provides a favorable entropic contribution to the interchain association is not reproduced by this pioneering model. The body of these results corroborates the two-stage process in the mechanism of calcium gelation, where the formation of strongly linked <em>dimer</em> associations is followed by the formation of weak inter-<em>dimer</em> associations mainly governed by electrostatic interactions.

Background: Patients critically ill with coronavirus disease-2019 (COVID-19) feature hyperinflammation, and the associated biomarkers may be beneficial for risk stratification. We aimed to investigate the association between several biomarkers, including serum C-reactive protein (CRP), procalcitonin (PCT), D-dimer, and serum ferritin, and COVID-19 severity.

Methods: We performed a comprehensive systematic literature search through electronic databases. The outcome of interest for this study was the composite poor outcome, which comprises mortality, acute respiratory distress syndrome, need for care in an intensive care unit, and severe COVID-19.

Results: A total of 5350 patients were pooled from 25 studies. Elevated CRP was associated with an increased composite poor outcome [risk ratio (RR) 1.84 (1.45, 2.33), p < 0.001; I²: 96%] and its severe COVID-19 (RR 1.41; I²: 93%) subgroup. A CRP ⩾10 mg/L has a 51% sensitivity, 88% specificity, likelihood ratio (LR) + of 4.1, LR- of 0.5, and an area under curve (AUC) of 0.84. An elevated PCT was associated with an increased composite poor outcome [RR 3.92 (2.42, 6.35), p < 0.001; I²: 85%] and its mortality (RR 6.26; I²: 96%) and severe COVID-19 (RR 3.93; I²: 63%) subgroups. A PCT ⩾0.5 ng/ml has an 88% sensitivity, 68% specificity, LR+ of 2.7, LR- of 0.2, and an AUC of 0.88. An elevated D-dimer was associated with an increased composite poor outcome [RR 2.93 (2.14, 4.01), p < 0.001; I²: 77%], including its mortality (RR 4.15; I²: 83%) and severe COVID-19 (RR 2.42; I²: 58%) subgroups. A D-dimer >0.5 mg/L has a 58% sensitivity, 69% specificity, LR+ of 1.8, LR- of 0.6, and an AUC of 0.69. Patients with a composite poor outcome had a higher serum ferritin with a standardized mean difference of 0.90 (0.64, 1.15), p < 0.0001; I²: 76%.

Conclusion: This meta-analysis showed that an elevated serum CRP, PCT, D-dimer, and ferritin were associated with a poor outcome in COVID-19. The reviews of this paper are available via the supplemental material section.

Keywords: COVID-19; SARS-CoV-2; biomarker; coronavirus; inflammatory.

BACKGROUND

Whether to continue oral anticoagulant therapy beyond 6 months after an "unprovoked" venous thromboembolism is controversial. We sought to determine clinical predictors to identify patients who are at low risk of recurrent venous thromboembolism who could safely discontinue oral anticoagulants.

METHODS

In a multicentre prospective cohort study, 646 participants with a first, unprovoked major venous thromboembolism were enrolled over a 4-year period. Of these, 600 participants completed a mean 18-month follow-up in September <em>2</em>006. We collected data for 69 potential predictors of recurrent venous thromboembolism while patients were taking oral anticoagulation therapy (5-7 months after initiation). <em>D</em>uring follow-up after discontinuing oral anticoagulation therapy, all episodes of suspected recurrent venous thromboembolism were independently adjudicated. We performed a multivariable analysis of predictor variables (p < 0.10) with high interobserver reliability to derive a clinical decision rule.

RESULTS

We identified 91 confirmed episodes of recurrent venous thromboembolism during follow-up after discontinuing oral anticoagulation therapy (annual risk 9.3%, 95% CI 7.7%-11.3%). Men had a 13.7% (95% CI 10.8%-17.0%) annual risk. There was no combination of clinical predictors that satisfied our criteria for identifying a low-risk subgroup of men. Fifty-two percent of women had 0 or 1 of the following characteristics: hyperpigmentation, edema or redness of either leg; <em>D</em>-<em>dimer</em>>> or = <em>2</em>50 microg/L while taking warfarin; body mass index>> or = 30 kg/m(<em>2</em>); or age>> or = 65 years. These women had an annual risk of 1.6% (95% CI 0.3%-4.6%). Women who had <em>2</em> or more of these findings had an annual risk of 14.1% (95% CI 10.9%-17.3%).

CONCLUSIONS

Women with 0 or 1 risk factor may safely discontinue oral anticoagulant therapy after 6 months of therapy following a first unprovoked venous thromboembolism. This criterion does not apply to men.

<em>D</em>eep vein thrombosis (<em>D</em>VT) is a common disease with an annual incidence of about 1 in 1000. Many risk factors have already been studied, both genetic and acquired. It is unclear whether obesity affects thrombotic risk in unselected patients. Obesity is common, with a prevalence of <em>2</em>0-<em>2</em>5% and may therefore have a considerable impact on the overall incidence of thrombosis. We evaluated the risk of thrombosis due to overweight and obesity using data from a large population based case-control study. Four hundred and fifty-four consecutive patients with a first episode of objectively diagnosed thrombosis from three Anticoagulation Clinics in the Netherlands were enrolled in a case-control study. Controls were matched on age and sex to patients and were introduced by the patients. All patients completed a standard questionnaire and interview, with weight and height measured under standard conditions. The associations of obesity with clotting factor levels were studied to investigate possible mechanisms. Obesity (BMI>>/=30 kg/m(<em>2</em>)) increased the risk of thrombosis twofold (CI95: 1.5 to 3.4), adjusted for age and sex. Obese individuals had higher levels of factor VIII and factor IX, but not of fibrinogen. The effect on risk of obesity was not changed after adjustment for coagulation factors levels (fibrinogen, F VIII, F IX, <em>D</em>-<em>dimer</em>). The relative risk estimates were similar in different age groups and in both sexes, indicating a larger absolute effect in older age groups. Evaluation of the combined effect of obesity and oral contraceptive pills among women aged 15-45 revealed that oral contraceptives further increased the effect of obesity on the risk of thrombosis, leading to 10-fold increased risk amongst women with a BMI greater than <em>2</em>5 kg/m(<em>2</em>) who used oral contraceptives. Obesity is a risk factor for deep vein thrombosis. Among women with a BMI greater than <em>2</em>5 kg/m(<em>2</em>) the synergistic effect with oral contraceptives should be considered when prescribing these.

The conformational stability of <em>dimer</em>ic globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many <em>dimer</em>ic proteins denature with a <em>2</em>-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native <em>dimer</em> to denatured monomer, the conformational stabilities, <em>delta</em> Gu (H<em>2</em>O), range from 10 to <em>2</em>7 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the <em>dimer</em> can be estimated by comparing <em>delta</em> Gu (H<em>2</em>O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of <em>dimers</em> is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of <em>dimers</em> by equilibrium denaturation methods are discussed.

Objective: To determine whether tocilizumab improves clinical outcomes for patients with severe or critical coronavirus disease 2019 (covid-19).

Design: Randomised, open label trial.

Setting: Nine hospitals in Brazil, 8 May to 17 July 2020.

Participants: Adults with confirmed covid-19 who were receiving supplemental oxygen or mechanical ventilation and had abnormal levels of at least two serum biomarkers (C reactive protein, D dimer, lactate dehydrogenase, or ferritin). The data monitoring committee recommended stopping the trial early, after 129 patients had been enrolled, because of an increased number of deaths at 15 days in the tocilizumab group.

Interventions: Tocilizumab (single intravenous infusion of 8 mg/kg) plus standard care (n=65) versus standard care alone (n=64).

Main outcome measure: The primary outcome, clinical status measured at 15 days using a seven level ordinal scale, was analysed as a composite of death or mechanical ventilation because the assumption of odds proportionality was not met.

Results: A total of 129 patients were enrolled (mean age 57 (SD 14) years; 68% men) and all completed follow-up. All patients in the tocilizumab group and two in the standard care group received tocilizumab. 18 of 65 (28%) patients in the tocilizumab group and 13 of 64 (20%) in the standard care group were receiving mechanical ventilation or died at day 15 (odds ratio 1.54, 95% confidence interval 0.66 to 3.66; P=0.32). Death at 15 days occurred in 11 (17%) patients in the tocilizumab group compared with 2 (3%) in the standard care group (odds ratio 6.42, 95% confidence interval 1.59 to 43.2). Adverse events were reported in 29 of 67 (43%) patients who received tocilizumab and 21 of 62 (34%) who did not receive tocilizumab.

Conclusions: In patients with severe or critical covid-19, tocilizumab plus standard care was not superior to standard care alone in improving clinical outcomes at 15 days, and it might increase mortality.

Trial registration: ClinicalTrials.gov NCT04403685.

In eukaryotes, 14-3-3 <em>dimers</em> regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 1<em>2</em> 14-3-3 isoforms expressed in Arabidopsis. The identification here of 1<em>2</em>1 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(<em>2</em>+) channels belonging to the glutamate receptor family (1.<em>2</em>, <em>2</em>.1, <em>2</em>.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase <em>D</em> (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

<em>D</em>uring the course of the coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) pandemic, reports of a new multisystem inflammatory syndrome in children (MIS-C) have been increasing in Europe and the United States (1-3). Clinical features in children have varied but predominantly include shock, cardiac dysfunction, abdominal pain, and elevated inflammatory markers, including C-reactive protein (CRP), ferritin, <em>D</em>-<em>dimer</em>, and interleukin-6 (1). Since June <em>2</em>0<em>2</em>0, several case reports have described a similar syndrome in adults; this review describes in detail nine patients reported to C<em>D</em>C, seven from published case reports, and summarizes the findings in 11 patients described in three case series in peer-reviewed journals (4-6). These <em>2</em>7 patients had cardiovascular, gastrointestinal, dermatologic, and neurologic symptoms without severe respiratory illness and concurrently received positive test results for SARS-CoV-<em>2</em>, the virus that causes COVI<em>D</em>-19, by polymerase chain reaction (PCR) or antibody assays indicating recent infection. Reports of these patients highlight the recognition of an illness referred to here as multisystem inflammatory syndrome in adults (MIS-A), the heterogeneity of clinical signs and symptoms, and the role for antibody testing in identifying similar cases among adults. Clinicians and health departments should consider MIS-A in adults with compatible signs and symptoms. These patients might not have positive SARS-CoV-<em>2</em> PCR or antigen test results, and antibody testing might be needed to confirm previous SARS-CoV-<em>2</em> infection. Because of the temporal association between MIS-A and SARS-CoV-<em>2</em> infections, interventions that prevent COVI<em>D</em>-19 might prevent MIS-A. Further research is needed to understand the pathogenesis and long-term effects of this newly described condition.

We characterize<em>d</em> the sequence an<em>d</em> protein interactions of cingulin, an M(r) 140-160-kD phosphoprotein localize<em>d</em> on the cytoplasmic surface of epithelial tight junctions (TJ). The <em>d</em>erive<em>d</em> amino aci<em>d</em> sequence of a full-length Xenopus laevis cingulin cDNA shows globular hea<em>d</em> (resi<em>d</em>ues 1-439) an<em>d</em> tail (1,3<em>2</em>6-1,368) <em>d</em>omains an<em>d</em> a central alpha-helical ro<em>d</em> <em>d</em>omain (440-1,3<em>2</em>5). Sequence analysis, electron microscopy, an<em>d</em> pull-<em>d</em>own assays in<em>d</em>icate that the cingulin ro<em>d</em> is responsible for the formation of coile<em>d</em>-coil parallel <em>dimers</em>, which can further aggregate through intermolecular interactions. Pull-<em>d</em>own assays from epithelial, insect cell, an<em>d</em> reticulocyte lysates show that an NH(<em>2</em>)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(<em>d</em>) approximately 5 nM), ZO-<em>2</em>, ZO-3, myosin, an<em>d</em> AF-6, but not with symplekin, an<em>d</em> a COOH-terminal fragment (377-1,368) interacts with myosin an<em>d</em> ZO-3. ZO-1 an<em>d</em> ZO-<em>2</em> immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH(<em>2</em>)-terminal an<em>d</em> COOH-terminal fragments, colocalizes with en<em>d</em>ogenous cingulin in transfecte<em>d</em> MDCK cells, in<em>d</em>icating that sequences within both hea<em>d</em> an<em>d</em> ro<em>d</em> <em>d</em>omains are require<em>d</em> for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque <em>d</em>omain of TJ to the actomyosin cytoskeleton.

BACKGROUND

Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD.

METHODS

This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD.

RESULTS

Patients with SC<em>D</em> (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+<em>2</em>, <em>D</em>-<em>dimer</em>) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SC<em>D</em> patients with PHT (n=<em>2</em>6) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation.

CONCLUSIONS

SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.

OBJECTIVE

The prevalence of depression increases with age, as does the prevalence of higher levels of the cytokine interleukin-6 (IL-6). This analysis was performed to determine the association between increased levels of this cytokine and depression in a population-based sample.

METHODS

Cross-sectional cohort study.

METHODS

Rural and urban counties in North Carolina.

METHODS

Community-dwelling older people.

METHODS

The association between IL-6 and other biologic variables with self-report depression was examined in 1686 persons aged 70 years and older in the third in-person survey wave (1991) of the Duke Established Population for Epidemiologic Studies of the Elderly (EPESE). Bivariate associations were established by the Spearman correlation, adjusted for age. A stepwise linear logistic regression model was used to derive a final model to assess multivariable effects on CES-D scores.

RESULTS

Depression was correlated with IL-6 (P = .011), D-Dimer (P = .017), alpha-1-globulin (P = .023), alpha-2-globulin (P = .002), and beta globulin (P = .012). After controlling for age, race, and gender, IL-6 levels remained the only biologic variable significantly associated with depression (P = .035).

CONCLUSIONS

These data suggest that the inflammatory marker, IL-6, is associated with depression in older people in this cross-sectional study. These results are compatible with the hypothesis of cytokine (IL-6) stimulation in geriatric depression as part of an overall immunoendocrine dysregulation.