Nitric oxi<em>d</em>e an<em>d</em> nitrovaso<em>d</em>ilators in<em>d</em>uce vascular smooth muscle cell relaxation in part by cGMP-<em>d</em>epen<em>d</em>ent protein kinase I (PKG-Ialpha)-me<em>d</em>iate<em>d</em> activation of myosin phosphatase (MLCP). Mechanistically it has been propose<em>d</em> that protein-protein interactions between the N-terminal leucine zipper (LZ) <em>d</em>omain of PKG-Ialpha ((PKG-Ialpha(1-59)) an<em>d</em> the LZ an<em>d</em>/or coile<em>d</em> coil (CC) <em>d</em>omain of the myosin bin<em>d</em>ing subunit (MBS) of MLCP are localize<em>d</em> in the C terminus of MBS. Although recent stu<em>d</em>ies have supporte<em>d</em> these interactions, the critical amino aci<em>d</em>s responsible for these interactions have not been i<em>d</em>entifie<em>d</em>. Here we present structural an<em>d</em> biophysical <em>d</em>ata i<em>d</em>entifying that the LZ <em>d</em>omain of PKG-Ialpha(1-59) interacts with a well <em>d</em>efine<em>d</em> 4<em>2</em>-resi<em>d</em>ue CC motif (MBS(CT4<em>2</em>)) within the C terminus of MBS. Using glutathione S-transferase pull<em>d</em>own experiments, chemical cross-linking, size exclusion chromatography, circular <em>d</em>ichroism, an<em>d</em> isothermal titration calorimetry we i<em>d</em>entifie<em>d</em> a weak <em>dimer</em>-<em>dimer</em> interaction between PKG-Ialpha(1-59) an<em>d</em> this C-terminal CC <em>d</em>omain of MBS. The K(<em>d</em>) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR <em>d</em>ata illustrate that this interaction is me<em>d</em>iate<em>d</em> by several PKG-Ialpha resi<em>d</em>ues that are on the a, <em>d</em>, e, an<em>d</em> g hy<em>d</em>rophobic an<em>d</em> electrostatic interface of the C-terminal hepta<em>d</em> layers <em>2</em>, 4, an<em>d</em> 5 of PKG-Ialpha. Taken together these <em>d</em>ata support a role for the LZ <em>d</em>omain of PKG-Ialpha an<em>d</em> the CC <em>d</em>omain of MBS in this requisite contractile complex.

We have use<em>d</em> a combination of simulate<em>d</em> annealing (SA), molecular <em>d</em>ynamics (MD) an<em>d</em> locally enhance<em>d</em> sampling (LES) metho<em>d</em>s in or<em>d</em>er to pre<em>d</em>ict the favourable topologies an<em>d</em> loop conformations of <em>dimer</em>ic DNA qua<em>d</em>ruplexes with T<em>2</em> or T3 loops. This follows on from our previous MD simulation stu<em>d</em>ies on the influence of loop lengths on the topology of intramolecular qua<em>d</em>ruplex structures [P. Hazel et al. (<em>2</em>004) J. Am. Chem. Soc., 1<em>2</em>6, 16 405-16 415], which provi<em>d</em>e<em>d</em> results consistent with biophysical <em>d</em>ata. The recent crystal structures of <em>d</em>(G4T3G4)<em>2</em> an<em>d</em> <em>d</em>(G4BrUT<em>2</em>G4) (P. Hazel et al. (<em>2</em>006) J. Am. Chem. Soc., in press) an<em>d</em> the NMR-<em>d</em>etermine<em>d</em> topology of <em>d</em>(TG4T<em>2</em>G4T)<em>2</em> [A.T. Phan et al. (<em>2</em>004) J. Mol. Biol., 338, 93-10<em>2</em>] have been use<em>d</em> in the present stu<em>d</em>y for comparison with simulation results. These together with MM-PBSA free-energy calculations in<em>d</em>icate that lateral T3 loops are favoure<em>d</em> over <em>d</em>iagonal loops, in accor<em>d</em>ance with the experimental structures; however, <em>d</em>istinct loop conformations have been pre<em>d</em>icte<em>d</em> to be favoure<em>d</em> compare<em>d</em> to those foun<em>d</em> experimentally. Several lateral an<em>d</em> <em>d</em>iagonal loop conformations have been foun<em>d</em> to be similar in energy. The simulations suggest an explanation for the <em>d</em>istinct patterns of observe<em>d</em> <em>dimer</em> topology for sequences with T3 an<em>d</em> T<em>2</em> loops, which <em>d</em>epen<em>d</em> on the loop lengths, rather than only on G-quartet stability.

To help identify the etiological agents for amyloid-related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H(<em>2</em>)O- and <em>D</em>(<em>2</em>)O-based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three <em>dimers</em> or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end-on-end association of <em>dimers</em> to form three-<em>dimer</em> nuclei, and then exhibits broadening or side-on-side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics.

BACKGROUND

Pulmonary embolism causes significant morbidity in hospitalized patients, yet few studies have explored the impact of spiral computed tomography (CT) scanning on diagnosis and clinical outcome.

METHODS

Incidence rates of pulmonary embolism, chest and spiral CT rates, D-dimer assay, anticoagulation, and in-hospital mortality were assessed on statewide pulmonary embolism discharge data (1997-2001) from the Pennsylvania Health Care Cost Containment Council.

RESULTS

The incidence of pulmonary embolism increased from 47 to 63 per 100,000 patients from 1997 to 2001 (mean of 0.004% per year, P < .001). Mean pulmonary embolism incidence rates were higher for African American patients (0.031% per year higher than for white patients), patients aged 70 years or more (0.007% higher than for patients aged<70 years), and female patients (0.013% higher than for male patients) (all P < .001). Concomitantly, the proportion undergoing CT (including spiral) scans increased from 23.23% to 45.18% (odds ratio=1.30; P<.001), controlling for age, gender, race, and cancer, whereas rates for other procedures remained unchanged. By comparing 1999 and before with 2000 and after, there was a significant decrease in the 2 highest Atlas Severity of Illness categories (49.4%-37.7%) and a significant increase in the 3 lowest categories (50.6%-62.3%; P < .001). The risk of in-hospital deaths among patients with pulmonary embolism decreased in this period from 12.8% to 11.1% (P < .001).

CONCLUSIONS

The incidence of pulmonary embolism is increasing with the increasing use of spiral CT scans, with a lower severity of illness and lower mortality, suggesting the increase is due to earlier diagnosis.

Cyclic <em>d</em>iguanosine monophosphate (c-<em>d</em>i-GMP) is a key signalling molecule involve<em>d</em> in regulating many important biological functions in bacteria. The synthesis of c-<em>d</em>i-GMP is catalyze<em>d</em> by the GGDEF-<em>d</em>omain-containing <em>d</em>iguanylate cyclase (DGC), the activity of which is regulate<em>d</em> by the bin<em>d</em>ing of pro<em>d</em>uct at the allosteric inhibitory (I) site. However, a significant number of GGDEF <em>d</em>omains lack the RxxD motif characteristic of the allosteric I site. Here, the structure of XCC4471(GGDEF), the GGDEF <em>d</em>omain of a DGC from Xanthomonas campestris, in complex with c-<em>d</em>i-GMP has been solve<em>d</em>. Unexpecte<em>d</em>ly, the structure of the complex reveale<em>d</em> a GGDEF-<em>d</em>omain <em>dimer</em> cross-linke<em>d</em> by two molecules of c-<em>d</em>i-GMP at the strongly conserve<em>d</em> active sites. In the complex (c-<em>d</em>i-GMP)(<em>2</em>) a<em>d</em>opts a novel partially intercalate<em>d</em> form, with the peripheral guanine bases boun<em>d</em> to the guanine-bin<em>d</em>ing pockets an<em>d</em> the two central bases stacke<em>d</em> upon each other. Alteration of the resi<em>d</em>ues involve<em>d</em> in specific bin<em>d</em>ing to c-<em>d</em>i-GMP le<em>d</em> to <em>d</em>ramatically re<em>d</em>uce<em>d</em> K(<em>d</em>) values between XCC4471(GGDEF) an<em>d</em> c-<em>d</em>i-GMP. In a<em>d</em><em>d</em>ition, these key resi<em>d</em>ues are strongly conserve<em>d</em> among the many thousan<em>d</em>s of GGDEF-<em>d</em>omain sequences i<em>d</em>entifie<em>d</em> to <em>d</em>ate. These results in<em>d</em>icate a new pro<em>d</em>uct-boun<em>d</em> form for GGDEF-<em>d</em>omain-containing proteins obtaine<em>d</em> via (c-<em>d</em>i-GMP)(<em>2</em>) bin<em>d</em>ing at the active site. This novel XCC4471(GGDEF)-c-<em>d</em>i-GMP complex structure may serve as a general mo<em>d</em>el for the <em>d</em>esign of lea<em>d</em> compoun<em>d</em>s to block the DGC activity of GGDEF-<em>d</em>omain-containing proteins in X. campestris or other microorganisms that contain multiple GGDEF-<em>d</em>omain proteins.

OBJECTIVE

The purpose of this study was to examine the association between hemostatic activation and stroke severity, and to provide data on hemostatic variables in acute ischemic stroke.

METHODS

The patient material comprised 76 consecutive patients with acute ischemic stroke (median 16 h, interquartile range 3-48). Levels of hemostatic variables were determined in blood samples collected on the day of hospitalization. Stroke severity was assessed on admission by the Oxfordshire Community Stroke Project (OCSP) classification, and on discharge (median 9 days, interquartile range 6-14) by Barthel Index (BI, scores 0-50, 55-90, or 95-100) and modified Rankin Scale (mRS, scores 0-1 or <em>2</em>-6). Associations were assessed by multiple linear regression analyses.

RESULTS

Levels of the fibrin degradation product D-Dimer and the activation peptide prothrombin fragment 1 + <em>2</em> (F1 + <em>2</em>) were linearly related to stroke severity, whether assessed on admission (P = .001 and.03, respectively, for the OCSP classification), or on discharge (P = .009 and.43, respectively, for BI; and.001 and.05, respectively, for mRS). High levels of D-Dimer and F(1 + <em>2</em>), as well as low levels of antithrombin and protein C were also present in patients with a presumed embolic source, and low antithrombin or protein C was borderline significantly associated with atrial fibrillation (P = .07<em>2</em> and.058, respectively). Low levels of protein C or protein S, and the presence of antiphospholipid antibodies, including lupus anticoagulant (LA), was detected in 13/73 (18%) and 15/70 (<em>2</em>1%) of the patients, respectively.

CONCLUSIONS

Activation of the hemostatic system is independently related to acute stroke severity and short-term outcome. Low levels of coagulation inhibitors or presence of antiphospholipid antibodies is a relatively frequent finding in unselected patients with acute ischemic stroke, but a causative role cannot be inferred from our study.

The mechanism of tight junction (TJ) assembly an<em>d</em> the structure of clau<em>d</em>ins (Cl<em>d</em>n) that form the TJ stran<em>d</em>s are unclear. This limits the molecular un<em>d</em>erstan<em>d</em>ing of paracellular barriers an<em>d</em> strategies for <em>d</em>rug <em>d</em>elivery across tissue barriers. Cl<em>d</em>n3 an<em>d</em> Cl<em>d</em>n5 are both common in the bloo<em>d</em>-brain barrier but form TJ stran<em>d</em>s with <em>d</em>ifferent ultrastructures. To i<em>d</em>entify the molecular <em>d</em>eterminants of fol<em>d</em>ing an<em>d</em> assembly of these classic clau<em>d</em>ins, Cl<em>d</em>n3/Cl<em>d</em>n5 chimeric mutants were generate<em>d</em> an<em>d</em> analyze<em>d</em> by cellular reconstitution of TJ stran<em>d</em>s, live cell confocal imaging, an<em>d</em> freeze-fracture electron microscopy. A comprehensive screening was performe<em>d</em> on the basis of the rescue of mutants <em>d</em>eficient for stran<em>d</em> formation. Cl<em>d</em>n3/Cl<em>d</em>n5 resi<em>d</em>ues in transmembrane segment 3, TM3 (Ala-1<em>2</em>7/Cys-1<em>2</em>8, Ser-136/Cys-137, Ser-138/Phe-139), an<em>d</em> the transition of TM3 to extracellular loop <em>2</em>, ECL<em>2</em> (Thr-141/Ile-14<em>2</em>) an<em>d</em> ECL<em>2</em> (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were i<em>d</em>entifie<em>d</em> to be involve<em>d</em> in clau<em>d</em>in fol<em>d</em>ing an<em>d</em>/or assembly. Blue native PAGE an<em>d</em> FRET assays reveale<em>d</em> 1% n-<em>d</em>o<em>d</em>ecyl β-<em>d</em>-maltosi<em>d</em>e-resistant cis-<em>dimer</em>ization for Cl<em>d</em>n5 but not for Cl<em>d</em>n3. This homophilic interaction was foun<em>d</em> to be stabilize<em>d</em> by resi<em>d</em>ues in TM3. The resulting subtype-specific cis-<em>dimer</em> is suggeste<em>d</em> to be a subunit of polymeric TJ stran<em>d</em>s an<em>d</em> contributes to the specific ultrastructure of the TJ <em>d</em>etecte<em>d</em> by freeze-fracture electron microscopy. In particular, the Cl<em>d</em>n5-like exoplasmic face-associate<em>d</em> an<em>d</em> particle-type stran<em>d</em>s were foun<em>d</em> to be relate<em>d</em> to cis-<em>dimer</em>ization. These results provi<em>d</em>e new insight into the mechanisms of paracellular barrier formation by <em>d</em>emonstrating that <em>d</em>efine<em>d</em> non-conserve<em>d</em> resi<em>d</em>ues in TM3 an<em>d</em> ECL<em>2</em> of classic clau<em>d</em>ins contribute to the formation of TJ stran<em>d</em>s with <em>d</em>iffering ultrastructures.

Technetium-99m ethyl cysteinate <em>dimer</em> ([99mTc]EC<em>D</em>) is a neutral, lipophilic complex which rapidly crosses the blood-brain barrier. Brain retention and tissue metabolism of [99mTc]EC<em>D</em> is dependent upon the stereochemical configuration of the complex. While both L,L and <em>D</em>,<em>D</em> enantiomers are extracted by the brain, only the L,L but not the <em>D</em>,<em>D</em> form, is metabolized and retained in the monkey brain (4.7% injected dose initially, T 1/<em>2</em> greater than <em>2</em>4 hr). <em>D</em>ynamic single photon emission computed tomography imaging studies in one monkey indicates 99mTc-L,L-EC<em>D</em> to be distributed in a pattern consistent with regional cerebral blood flow for up to 16 hr postinjection. <em>D</em>ual-labeled 99mTc-L,L-EC<em>D</em> and [14C]iodoantipyrine autoradiography studies performed 1 hr after administration show cortical gray to white matter ratios of both isotopes to be equivalent (approximately 4-5:1). These data suggest that 99mTc-L,L-EC<em>D</em> will be useful for the scintigraphic assessment of cerebral perfusion in humans.

OBJECTIVE

To investigate platelet and leukocyte activation and interaction in patients with rheumatoid arthritis (RA) and the effect of methotrexate (MTX) or anti-tumor necrosis factor-a (TNF-a) treatment on these variables.

METHODS

Four-color flow cytometry analysis was performed for quantitative measurement of platelet (P-selectin, PAC-1) and leukocyte (CDD, D-dimer, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin 6 (IL-6), and TNF-a and tender and swollen joint counts were carried out.

RESULTS

Before therapy, PAC-1 binding, expression of C<em>D</em>11b and C<em>D</em>64 on monocytes and neutrophils, circulating levels of monocyte (C<em>D</em>11b+ or C<em>D</em>64+)-platelet complexes, monocyte-PAC-1+ platelet complexes, CRP, ESR, IL-6, TNF-a, fibrinogen, <em>D</em>-<em>dimer</em> and sP-selectin were significantly higher in RA patients compared to controls. The anti-TNF-a therapy significantly reduced levels of monocyte-PAC-1+ platelet complexes, sP-selectin, CRP, ESR, IL-6, TNF-a, fibrinogen, and <em>D</em>-<em>dimer</em> and tender and swollen joint counts. C<em>D</em>64 expression on monocytes was significantly decreased by MTX therapy. PAC-1 binding was not inhibited by MTX or anti-TNF-a.

CONCLUSIONS

Increased platelet and leukocyte activation and increased formation of leukocyte-platelet complexes in patients with RA suggest a status of simultaneous activation of the immune and hemostatic systems.

This study sought to describe and evaluate any relationship between <em>D</em>-<em>dimer</em> values and progressive hemorrhagic injury (PHI) after traumatic brain injury (TBI). In patients with TBI, plasma <em>D</em>-<em>dimer</em> was measured while a computed tomography (CT) scan was conducted as soon as the patient was admitted to the emergency department. A series of other clinical and laboratory parameters were also measured and recorded. A logistic multiple regression analysis was used to identify risk factors for PHI. A cohort of 194 patients with TBI was evaluated in this clinical study. Eighty-one (41.8%) patients suffered PHI as determined by a second CT scan. The plasma <em>D</em>-<em>dimer</em> level was higher in patients who demonstrated PHI compared with those who did not (P < 0.001. Using a receiver-operator characteristic curve to predict the possibility by measuring the <em>D</em>-<em>dimer</em> level, a value of 5.00 mg/L was considered the cutoff point, with a sensitivity of 7<em>2</em>.8% and a specificity of 78.8%. Eight-four patients had <em>D</em>-<em>dimer</em> levels higher than the cut point value (5.0 mg/L); PHI was seen in 71.4% of these patients and in 19.1% of the other patients (P < 0.01). Factors with P < 0.<em>2</em> on bivariate analysis were included in a stepwise logistic regression analysis to identify independent risk factors for TBI coagulopathy. Logistic regression analysis showed that the <em>D</em>-<em>dimer</em> value was a predictor of PHI, and the odds ratio (OR) was 1.341 with per milligram per liter (P = 0.0<em>2</em>0). The stepwise logistic regression also identified that time from injury to the first CT shorter than <em>2</em> h (OR = <em>2</em>.118, P = 0.047), PLT counts lesser than 100 x 109/L (OR = 7.853, P = 0.018), and Fg lower than <em>2</em>.0 g/L (OR = 3.001, P = 0.01<em>2</em>) were risk factors for the development of PHI. When <em>D</em>-<em>dimer</em> values were dichotomized at 5 mg/L, time from injury to the first CT scan was no longer a risk factor statistically while the OR value of <em>D</em>-<em>dimer</em> to the occurrence of PHI elevated to 11.850(P < 0.001). The level of plasma <em>D</em>-<em>dimer</em> after TBI can be a useful prognostic factor for PHI and should be considered in the clinical management of patients in combination with neuroimaging and other data.

The new antigen receptor (IgNAR) family has been detected in all elasmobranch species so far studied and has several intriguing structural and functional features. IgNAR protein, found in both transmembrane and secretory forms, is a <em>dimer</em> of heavy chains with no associated light chains, with each chain of the <em>dimer</em> having a single free and flexible V region. Four rearrangement events (among 1V, 3<em>D</em>, and 1J germline genes) generate an expressed NAR V gene, resulting in long and diverse C<em>D</em>R3 regions that contain cysteine residues. IgNAR mutation frequency is very high and "selected" mutations are found only in genes encoding the secreted form, suggesting that the primary repertoire is entirely C<em>D</em>R3-based. Here we further analyzed the two IgNAR types, "type 1" having one cysteine in C<em>D</em>R3 and "type <em>2</em>" with an even number (two or four) of C<em>D</em>R3 cysteines, and discovered that placement of the disulfide bridges in the IgNAR V domain differentially influences the selection of mutations in C<em>D</em>R1 and C<em>D</em>R<em>2</em>. Ontogenetic analyses showed that IgNAR sequences from young animals were infrequently mutated, consistent with the paradigm that the shark immune system must become mature before high levels of mutation accompanied with selection can occur. Nevertheless, also in agreement with the idea that the IgNAR repertoire is entirely C<em>D</em>R3-based, but unlike studies in most other vertebrates, N-region diversity is present in expressed IgNAR clones at birth. <em>D</em>uring the investigation of this early IgNAR repertoire we serendipitously detected a third type of IgNAR gene that is expressed in all neonatal tissues; later in life its expression is perpetuated only in the epigonal organ, a tissue recently shown to be a (the?) primary lymphoid tissue in elasmobranchs. This "type 3" IgNAR gene still undergoes three rearrangement events (two <em>D</em> regions are "germline-joined"), yet C<em>D</em>R3 sequences were exactly of the same length and very similar sequence, suggesting that "type 3" C<em>D</em>R3s are selected early in ontogeny, perhaps by a self-ligand.

Antennae of the tobacco hornworm moths Man<em>d</em>uca sexta contain an al<em>d</em>ehy<em>d</em>e oxi<em>d</em>ase (AOX) that oxi<em>d</em>izes al<em>d</em>ehy<em>d</em>es to carboxylic aci<em>d</em>s. The enzyme, which is <em>d</em>istinguishable from al<em>d</em>ehy<em>d</em>e-oxi<em>d</em>izing activities in other tissues, is secrete<em>d</em> into the receptor lymph that bathes the primary olfactory <em>d</em>en<em>d</em>rites. First <em>d</em>etectable about 3 <em>d</em> before eclosion, AOX levels increase through the first <em>d</em>ay after eclosion. This parallels the <em>d</em>evelopment of the antennal responsiveness to bombykal (a male attractant al<em>d</em>ehy<em>d</em>ic pheromone pro<em>d</em>uce<em>d</em> by female M. sexta) an<em>d</em> trans-<em>2</em>-hexenal (an al<em>d</em>ehy<em>d</em>e commonly foun<em>d</em> in leaves). The AOX is about 60% more abun<em>d</em>ant in antennae of males than in antennae of females. The antennal AOX is a <em>dimer</em> with Mr of <em>2</em>95 kDa an<em>d</em> is capable of oxi<em>d</em>izing a variety of al<em>d</em>ehy<em>d</em>es. Of all al<em>d</em>ehy<em>d</em>es examine<em>d</em>, the pheromone bombykal was the best substrate with an apparent Km of 5 microM, whereas the next best substrate, benzal<em>d</em>ehy<em>d</em>e, ha<em>d</em> an apparent Km of <em>2</em>55 microM. Using kinetic parameters estimate<em>d</em> in vitro an<em>d</em> the assumption of first-or<em>d</em>er kinetics, the half-life of bombykal in sensilla was estimate<em>d</em> to be about 0.6 msec. The affinity of the antennal AOX for bombykal, its location in the receptor lymph, an<em>d</em> its pattern of <em>d</em>evelopmental expression all suggest that it plays a role in mo<em>d</em>ulating the sensitivity of a<em>d</em>ult M. sexta to al<em>d</em>ehy<em>d</em>e o<em>d</em>ors an<em>d</em>, in particular, the sensitivity of males to the pheromone bombykal.

BACKGROUND

Lower activated partial thromboplastin times are associated with higher levels of some coagulation factors and may represent a procoagulant tendency.

METHODS

In the Atherosclerosis Risk in Communities study, we studied the 13-year risk of venous thromboembolism in relation to baseline activated partial thromboplastin time in 13,880 individuals. We also studied <em>2</em>58 venous thromboembolism cases and 589 matched controls with measurements of additional coagulation factors.

RESULTS

After adjustment for demographics and procoagulant factors reflected in the activated partial thromboplastin time (fibrinogen, factors VIII, IX, and XI, and von Willebrand factor), participants in the lowest <em>2</em> quartiles of activated partial thromboplastin time compared with the fourth quartile had <em>2</em>.4-fold (95% confidence interval [CI], 1.4-4.<em>2</em>) and 1.9-fold (95% CI, 1.1-3.<em>2</em>) higher risks of venous thromboembolism. The risk associated with activated partial thromboplastin times below the median was higher for idiopathic (odds ratio 5.5; 95% CI, <em>2</em>.0-15.5) than secondary venous thromboembolism (odds ratio 1.74; 95% CI, 0.88-3.43). Subjects with both activated partial thromboplastin times below the median and factor V Leiden were 1<em>2</em>.6-fold (95% CI, 5.7-<em>2</em>8.0) more likely to develop venous thromboembolism compared with those with neither risk factor (P interaction<.01). A lower activated partial thromboplastin time also added to the thrombosis risk associated with obesity and elevated <em>D</em>-<em>dimer</em>.

CONCLUSIONS

A single determination of the activated partial thromboplastin time below the median increased the risk of future venous thromboembolism. Findings were independent of coagulation factor levels, and a low activated partial thromboplastin time added to the risk associated with other risk factors.

Postinjury fibrinolysis can manifest as three distinguishable phenotypes: 1) hyperfibrinolysis, <em>2</em>) physiologic, and 3) hypofibrinolysis (shutdown). Hyperfibrinolysis is associated with uncontrolled bleeding due to clot dissolution; whereas, fibrinolysis shutdown is associated with organ dysfunction due to microvascular occlusion. The incidence of fibrinolysis phenotypes at hospital arrival in severely injured patients is: 1) hyperfibrinolysis 18%, physiologic 18%, and shutdown 64%. The mechanisms responsible for dysregulated fibrinolysis following injury remain uncertain. Animal work suggests hypoperfusion promotes fibrinolysis, while tissue injury inhibits fibrinolysis. Clinical experience is consistent with these observations. The predominant mediator of postinjury hyperfibrinolysis appears to be tissue plasminogen activator (tPA) released from ischemic endothelium. The effects of tPA are accentuated by impaired hepatic clearance. Fibrinolysis shutdown, on the other hand, may occur from inhibition of circulating tPA, enhanced clot strength impairing the binding of tPA and plasminogen to fibrin, or the inhibition of plasmin. Plasminogen activator inhibitor -1 (PAI-1) binding of circulating tPA appears to be a major mechanism for postinjury shutdown. The sources of PAI-1 include endothelium, platelets, and organ parenchyma. The laboratory identification of fibrinolysis phenotype, at this moment, is best determined with viscoelastic hemostatic assays (TEG, ROTEM). While <em>D</em>-<em>dimer</em> and plasmin antiplasmin (PAP) levels corroborate fibrinolysis, they do not provide real-time assessment of the circulating blood capacity. Our clinical studies indicate that fibrinolysis is a very dynamic process and our experimental work suggests plasma first resuscitation reverses hyperfibrinolysis. Collectively, we believe recent clinical and experimental work suggest antifibrinolytic therapy should be employed selectively in the acutely injured patient, and optimally guided by TEG or ROTEM.

<strong class="sub-title"> Introduction and objectives: </strong> Common laboratory parameters are crucial in aiding coronavirus disease <em>2</em>019 (COVID-19) case detection. This study aimed to determine the differences between laboratory parameters in (1) COVID-19 versus non-COVID-19 pneumonia, and (<em>2</em>) severe versus non-severe COVID-19 cases.

<strong class="sub-title"> Methods: </strong> Studies were collected until March <em>2</em>0<em>2</em>0, and retrieved parameters include leukocyte, neutrophil, thrombocyte, and lymphocyte counts in addition to C-reactive protein (CRP), procalcitonin (PCT) and D-dimer levels. In the presence of heterogeneity, the random-effect model (REM) was used instead of the fixed-effect model (FEM).

<strong class="sub-title"> Results: </strong> Seven studies in the first analysis showed significantly lower leukocyte, neutrophil and platelet counts in COVID-19 pneumonia (SMD=-0.4<em>2</em>, 95%CI -0.60 to -0.<em>2</em>5, p<0.00001, SMD=-0.<em>2</em>3, 95%CI -0.41 to -0.06, p=0.01, SMD=-0.54, 95%CI -0.91 to -0.16, p=0.0005) compared to non-COVID-19 pneumonia. Twenty-six studies in the second analysis showed significantly lower lymphocyte and thrombocyte counts (SMD=-0.56, 95%CI -0.71 to -0.40, p<0.0001, SMD=-0.3<em>2</em>, 95%CI -0.49 to -0.15, p=0.000<em>2</em>) and significantly higher leukocyte, neutrophil, D-dimer, and CRP (SMD=0.31, 95%CI 0.07-0.56, p=0.01; SMD=0.44, 95%CI 0.<em>2</em>4-0.64, p<0.0001; SMD=0.53, 95%CI 0.31-0.75, p<0.00001; SMD=0.97, 95%CI 0.70-1.<em>2</em>4, p<0.00001) in severe COVID-19 compared to non-severe COVID-19.

Conclusions: In conclusion, thrombocyte count is key in both diagnosis and prognosis. Low leukocyte and neutrophil counts are markers of COVID-19 infection, but contrastingly higher counts indicate progressive COVID-19. And although lymphocyte, D-dimer and CRP levels did not demonstrate diagnostic value, all indicate severity of COVID-19. Confirmation of these findings should be performed in future studies.

<strong class="sub-title"> Introducción y objetivos: </strong> Los parámetros comunes de laboratorio son cruciales para ayudar a la detección de casos de enfermedad por coronavirus <em>2</em>019 (COVID-19). Este estudio tuvo como objetivo determinar las diferencias entre los parámetros de laboratorio en: 1) COVID-19 versus neumonía no COVID-19, y <em>2</em>) Casos severos versus no severos de COVID-19.

<strong class="sub-title"> Métodos: </strong> Los estudios se recolectaron hasta marzo de <em>2</em>0<em>2</em>0, y los parámetros recuperados incluyen recuentos de leucocitos, neutrófilos, trombocitos y linfocitos además de los niveles de proteína C reactiva (PCR), procalcitonina (PCT) y dímero-D. En presencia de heterogeneidad, se utilizó el modelo de efectos aleatorios en lugar del modelo de efectos fijos.

<strong class="sub-title"> Resultados: </strong> Siete estudios en el primer análisis mostraron recuentos de leucocitos, neutrófilos y plaquetas significativamente más bajos en la neumonía por COVID-19 (SMD = −0,4<em>2</em>; IC 95%: −0,60 a −0,<em>2</em>5; p < 0,00001; SMD = −0,<em>2</em>3; IC 95%: −0,41 a −0,06; p = 0,01; SMD = −0,54; IC 95%: −0,91 a −0,16; p = 0,0005) en comparación con la neumonía no COVID-19. Veintiséis estudios en el segundo análisis mostraron recuentos de linfocitos y trombocitos significativamente más bajos (SMD = −0,56; IC 95%: −0,71 a −0,40; p < 0,0001; SMD = −0,3<em>2</em>; IC 95%: −0,49 a −0,15; p = 0,000<em>2</em>) y leucocitos, neutrófilos, dímero D y PCR significativamente más altos (SMD = 0,31; IC 95%: 0,07-0,56; p = 0,01; SMD = 0,44; IC 95%: 0,<em>2</em>4-0,64; p < 0,0001; SMD = 0,53; IC 95%: 0,31-0,75; p < 0,00001; SMD = 0,97; IC 95%: 0,70-1,<em>2</em>4; p < 0,00001) en COVID-19 severo en comparación con COVID-19 no severo.

Conclusiones: En conclusión, el recuento de trombocitos es clave tanto en el diagnóstico como en el pronóstico. Los recuentos bajos de leucocitos y neutrófilos son marcadores de infección por COVID-19, pero los recuentos contrastantemente más altos indican COVID-19 progresivo. Y aunque los niveles de linfocitos, dímero D y PCR no mostraron valor diagnóstico, todos indican la gravedad de COVID-19. La confirmación de estos hallazgos debe realizarse en futuros estudios.

<strong class="sub-title"> Keywords: </strong> COVID-19; Diagnosis; Diagnóstico; Laboratory parameters; Parámetros de laboratorio; SARS-CoV-<em>2</em>.

The incidence of venous thromboembolism (VTE) is well recognized to increase with aging. Concurrently, the plasma concentrations of many coagulation factors (e.g., fibrinogen, factor [F] V, FVII, FVIII, and FIX) increase with aging, as does von Willebrand factor (VWF), thrombin generation, and platelet activation. <em>D</em>ata are conflicting regarding age-related changes in the natural anticoagulants, including protein C, protein S, and antithrombin. Changes are also observed with components of the fibrinolytic pathway. All in all, aging is associated with a variety of hemostasis changes that on balance reflects a heightened procoagulant status compared with earlier age. It has to be recognized that as this occurs in the otherwise normal general population, this can also be considered a normal phenomenon of progressive life. An element of this heightened procoagulant status may reflect ongoing inflammatory processes, given some markers, notably FVIII and fibrinogen, are acute phase reactants. A variety of acquired prothrombotic risk factors (e.g., cancer, autoimmune disorders, and diabetes) also gradually develop with aging, some of which may induce profound abnormalities of hemostasis, and confound the age-related changes in hemostasis, as well as their influence on thrombotic risk. In this article, we review the changes in hemostasis markers measurable within many hemostasis laboratories, and consider many of the important implications for clinical and laboratory practice. Apart from representing an increased thrombotic risk, additional considerations entail the potential need (1) to utilize age-adjusted normal ranges (e.g., for <em>D</em>-<em>dimer</em>), (<em>2</em>) to consider the consequence on previous diagnoses (e.g., "mild type 1" von Willebrand disease [VW<em>D</em>], where VWF test results may "normalize" with aging), and (3) to consider the effect of these changes of risk factors on the (perceived) therapeutic efficacy of antithrombotic medications such as aspirin.

CD<em>2</em>3/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD<em>2</em>3 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD<em>2</em>3, corresponding to the soluble lectin domain of CD<em>2</em>3, and Fc epsilon3-4, a <em>dimer</em> of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD<em>2</em>3 and Fc epsilon3-4 indicate that IgE has two binding sites for CD<em>2</em>3, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (<em>delta</em> H(degree)To = -<em>2</em>.1 +/- 3.3 kcal mol(-1)) and heat capacity (<em>delta</em> Cp(degree) = -3<em>2</em>0 +/- 3<em>2</em>0 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (<em>delta</em> H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (<em>delta</em> Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD<em>2</em>3, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD<em>2</em>3 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the <em>2</em>:1 complex between CD<em>2</em>3 and IgE is consistent with the thermodynamic data presented.

Prostaglandin (PG) H(<em>2</em>) (PGH(<em>2</em>)), formed from arachidonic acid, is an unstable intermediate and is converted efficiently into more stable arachidonate metabolites (PG<em>D</em>(<em>2</em>), PGE(<em>2</em>), and PGF(<em>2</em>)) by the action of three groups of enzymes. Prostaglandin E synthase catalyzes an isomerization reaction, PGH(<em>2</em>) to PGE(<em>2</em>). Microsomal prostaglandin E synthase type-<em>2</em> (mPGES-<em>2</em>) has been crystallized with an anti-inflammatory drug indomethacin (IMN), and the complex structure has been determined at <em>2</em>.6A resolution. mPGES-<em>2</em> forms a <em>dimer</em> and is attached to lipid membrane by anchoring the N-terminal section. Two hydrophobic pockets connected to form a V shape are located in the bottom of a large cavity. IMN binds deeply in the cavity by placing the OMe-indole and chlorophenyl moieties into the V-shaped pockets, respectively, and the carboxyl group interacts with S(gamma) of C110 by forming a H-bond. A characteristic H-bond chain formation (N-H...S(gamma)-H...S(gamma)...H-N) is seen through Y107-C113-C110-F11<em>2</em>, which apparently decreases the pK(a) of S(gamma) of C110. The geometry suggests that the S(gamma) of C110 is most likely the catalytic site of mPGES-<em>2</em>. A search of the RCSB Protein <em>D</em>ata Bank suggests that IMN can fit into the PGH(<em>2</em>) binding site in various proteins. On the basis of the crystal structure and mutation data, a PGH(<em>2</em>)-bound model structure was built. PGH(<em>2</em>) fits well into the IMN binding site by placing the alpha and omega-chains in the V-shaped pockets, and the endoperoxide moiety interacts with S(gamma) of C110. A possible catalytic mechanism is proposed on the basis of the crystal and model structures, and an alternative catalytic mechanism is described. The fold of mPGES-<em>2</em> is quite similar to those of GSH-dependent hematopoietic prostaglandin <em>D</em> synthase, except for the two large loop sections.

BACKGROUND

Recent investigations suggest that activation of coagulation and fibrinolysis occurs in patients with ulcerative colitis (UC). However, the role of the hypercoagulable state in UC has not been determined. On the other hand, there are no reports dealing with coagulation in ischemic colitis (IC), in which acute bowel inflammation and reversible vascular occlusion affect the colon. Thus, our aim was to evaluate the hyper states of coagulation and fibrinolysis in UC by comparing activations of coagulation and fibrinolysis in patients with active UC and in those with IC.

METHODS

Twenty-four patients with active UC and 1<em>2</em> patients with IC were studied, with 18 patients with inactive UC serving as controls. We investigated the activation of the coagulation system, including platelet counts, activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), serum concentrations of von Willebrand factor (vWF), activated factors XII, XI, X, IX, VIII, VII, V, II, fibrinogen, prothrombin fragments 1+<em>2</em> (F1+<em>2</em>), thrombin-antithrombin complexes (TAT), protein S, protein C, plasminogen, alpha-<em>2</em> plasminogen inhibitor (alpha-<em>2</em>PI) and <em>D</em>-<em>dimer</em> (<em>D</em>-<em>D</em>).

RESULTS

Median serum vWF concentrations, F1+<em>2</em>, TAT, fibrinogen, activated factor XI, IX, VIII and V were significantly elevated in patients with active UC and IC compared to those in patients with inactive UC. There was no significant difference between active UC and IC patients in the mean values of any of the factors that were measured.

CONCLUSIONS

The results of the present study indicate that the coagulation-fibrinolysis system is activated in patients with active bowel inflammation such as active UC and IC, and that the hyper states of coagulation and fibrinolysis in patients with active UC are secondary to bowel inflammation.

<AbstractText><em>D</em>ifferent skin manifestations of COVI<em>D</em>-19 are being reported. Acral lesions on the hands and feet, closely resembling chilblains, have been recognized during the peak incidence of the COVI<em>D</em>-19 pandemic MATERIAL AN<em>D</em> METHO<em>D</em>S: A retrospective review of <em>2</em><em>2</em> children and adolescents with chilblain-like lesions seen over a short period of time in the Emergency <em>D</em>epartment of a children's hospital during the peak incidence of COVI<em>D</em>-19 in Madrid, Spain.</AbstractText><AbstractText>All patients had lesions clinically consistent with chilblains of the toes or feet, with 3 also having lesions of the fingers. Pruritus and mild pain were the only skin symptoms elicited, and only 10 had mild respiratory and/or GI symptoms. None had fever. Coagulation tests, hemogram, serum chemistry and lupus anticoagulant were normal in all patients tested. One out of 16 tested cases had elevated <em>D</em>-<em>dimer</em> results, but without systemic symptoms or other lab anomalies. SARS-CoV-<em>2</em> PCR tested in 19 cases was positive in just 1 case. Skin biopsies obtained in 6 patients were consistent with chilblains. On follow-up, all cases showed spontaneous marked improvement or complete healing.</AbstractText><AbstractText>Acute chilblains were observed during COVI<em>D</em>-19 pandemic in children and teenagers. It is a mildly symptomatic condition with an excellent prognosis, usually requiring no therapy. Etiopathogenesis remains unknown.</AbstractText>

The effects of <em>d</em>ietary n-3 fatty aci<em>d</em>s (n-3FAs) on the frequency of pain episo<em>d</em>es an<em>d</em> ex vivo bloo<em>d</em> tests of thrombosis have been evaluate<em>d</em> in patients with sickle cell <em>d</em>isease (SC<em>D</em>) utilizing a <em>d</em>ouble-blin<em>d</em>, olive oil-controlle<em>d</em> clinical trial. <em>D</em>ietary n-3FA therapy (0.1 g/kg/<em>d</em>) was provi<em>d</em>e<em>d</em> as menha<em>d</em>en fish oil (0.<em>2</em>5 g/kg/<em>d</em>) containing 1<em>2</em>% eicosapentaenoic aci<em>d</em> (EPA), an<em>d</em> 18% <em>d</em>ocosahexaenoic aci<em>d</em> (<em>D</em>HA). Within 1 month <em>d</em>ietary n-3FAs exchange<em>d</em> with n-6FAs in plasma an<em>d</em> erythrocyte membrane phospholipi<em>d</em>s (p <0.01 in all cases). Treatment with <em>d</em>ietary n-3FAs for 1 year re<em>d</em>uce<em>d</em> the frequency of pain episo<em>d</em>es requiring presentation to the hospital from 7.8 events <em>d</em>uring the prece<em>d</em>ing year to 3.8 events/year (p <0.01; n = 5). By contrast, subjects receiving control <em>d</em>ietary olive oil (n = 5) experience<em>d</em> 7.1 pain events/year, compare<em>d</em> to 7.6 <em>d</em>uring the previous year (p >0.4). The re<em>d</em>uction in episo<em>d</em>es in n-3FA-treate<em>d</em> subjects was also significant when compare<em>d</em> to control subjects (p <0.01). <em>D</em>ietary n-3FA therapy was not associate<em>d</em> with hemorrhagic, gastrointestinal or other a<em>d</em>verse effects. Compare<em>d</em> to 10 asymptomatic African-American controls, sickle cell subjects <em>d</em>emonstrate<em>d</em> significantly increase<em>d</em> pretreatment: 1) flow cytometric expression of platelet membrane P-selectin (C<em>D</em>6<em>2</em>p; p <0.01) an<em>d</em> annexin V bin<em>d</em>ing sites (p = 0.0<em>2</em>); <em>2</em>) plasma levels of platelet-specific secretory proteins platelet factor 4 (PF4) an<em>d</em> beta-thromboglobulin (betaTG) (p <0.01 in both cases); 3) plasma pro<em>d</em>ucts of thrombin generation, prothrombin fragment 1.<em>2</em> (F1.<em>2</em>) an<em>d</em> thrombin:antithrombin (TAT) complex (p <0.01 in both cases); an<em>d</em> 4) plasma levels of thrombolytic pro<em>d</em>ucts, <em>D</em>-<em>dimer</em> an<em>d</em> plasmin:antiplasmin (PAP) complex (p <0.01 in both cases). Treatment with <em>d</em>ietary n-3FAs concurrently <em>d</em>ecrease<em>d</em> plasma levels of F1.<em>2</em>, <em>D</em>-<em>dimer</em>, an<em>d</em> PAP (p <0.05, compare<em>d</em> to olive oil controls), implying that the re<em>d</em>uction in pain events was relate<em>d</em> to n-3FA-<em>d</em>epen<em>d</em>ent inhibition of thrombosis. We conclu<em>d</em>e that <em>d</em>ietary n-3FAs re<em>d</em>uce the frequency of pain episo<em>d</em>es perhaps by re<em>d</em>ucing prothrombotic activity in sickle cell <em>d</em>isease.

Biotin synthase and lipoate synthase are homo<em>dimer</em>s that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (<em>2</em>Fe-<em>2</em>S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per <em>dimer</em> was obtained, suggesting that two (<em>2</em>Fe-<em>2</em>S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report Mössbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per <em>dimer</em>. The (4Fe-4S) cluster is quantitatively converted into a (<em>2</em>Fe-<em>2</em>S)(<em>2</em>+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the <em>2</em>+ and 1+ oxidation states. The Mössbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and <em>delta</em> = 0.44 mm/s for the diamagnetic (4Fe-4S)(<em>2</em>+) and DeltaE(q) = 0.51 mm/s, <em>delta</em> = 0.85 mm/s, g(par) = <em>2</em>.035, and g(perp) = 1.93 for the S = (1)/(<em>2</em>) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per <em>dimer</em>, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.<em>2</em>0 mm/s and <em>delta</em> = 0.44 mm/s for the diamagnetic (4Fe-4S)(<em>2</em>+) and g(par) = <em>2</em>.039 and g(perp) = 1.93 for the S = (1)/(<em>2</em>) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (<em>2</em>Fe-<em>2</em>S)(<em>2</em>+) center.

Some species of puffer fish have been reporte<em>d</em> to possess both of tetro<em>d</em>otoxin an<em>d</em> saxitoxin, which share one bin<em>d</em>ing site on so<em>d</em>ium channels. We purifie<em>d</em> a novel soluble glycoprotein that bin<em>d</em>s to these toxins from plasma of the puffer fish, Fugu par<em>d</em>alis, an<em>d</em> name<em>d</em> puffer fish saxitoxin an<em>d</em> tetro<em>d</em>otoxin bin<em>d</em>ing protein (PSTBP). PSTBP possesse<em>d</em> a bin<em>d</em>ing capacity of 10.6 +/- 0.97 nmol x mg(-1) protein an<em>d</em> a K(<em>d</em>) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium bin<em>d</em>ing assays. [(3)H]Saxitoxin (10 nm) bin<em>d</em>ing to PSTBPs was half-inhibite<em>d</em> by the presence of tetro<em>d</em>otoxin an<em>d</em> saxitoxin at 1<em>2</em> microm an<em>d</em> 8.5 nm, respectively. From the results of gel filtration chromatography (<em>2</em>00 kDa) an<em>d</em> SDS/PAGE (104 kDa), PSTBP was suggeste<em>d</em> to consist of noncovalently linke<em>d</em> <em>dimers</em> of a single subunit. PSTBP was completely <em>d</em>eglycosylate<em>d</em> by glycopepti<em>d</em>ase F, pro<em>d</em>ucing a single ban<em>d</em> at 4<em>2</em> kDa. Two highly homologous cDNAs to each other co<em>d</em>ing PSTBP (PSTBP1 an<em>d</em> PSTBP<em>2</em>, the pre<em>d</em>icte<em>d</em> amino-aci<em>d</em> i<em>d</em>entity 93%), were obtaine<em>d</em> from a cDNA library of F. par<em>d</em>alis liver. These proteins consiste<em>d</em> to two tan<em>d</em>emly repeate<em>d</em> homologous <em>d</em>omains. The pre<em>d</em>icte<em>d</em> amino-aci<em>d</em> sequences of PSTBP1 an<em>d</em> <em>2</em> were not homologous to that of saxiphilin, a reporte<em>d</em> saxitoxin bin<em>d</em>ing protein, or so<em>d</em>ium channels, but their N-terminus sequences were homologous to that of the reporte<em>d</em> tetro<em>d</em>otoxin bin<em>d</em>ing protein from plasma of Fugu niphobles, which has not been fully characterize<em>d</em>. The partially homologous cDNA sequences to PSTBP1 an<em>d</em> <em>2</em> were also foun<em>d</em> in expresse<em>d</em> sequence tag clones of nontoxic floun<em>d</em>ers liver. Presumably, PSTBP is involve<em>d</em> in accumulation an<em>d</em>/or excretion of toxins in puffer fish.

Studies on the interactions between SARS-CoV-<em>2</em> and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVI<em>D</em>-19 pneumonia. COVI<em>D</em>-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM⁺ and IgM^- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, <em>D</em>-<em>dimer</em>, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov<em>2</em>, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.

<strong class="sub-title"> Keywords: </strong> B cells; COVI<em>D</em>‐19; Coronavirus; SARS‐CoV‐<em>2</em>; Uniform Manifold Approximation and Projection (UMAP); carboxyfluorescein succinimidyl ester CFSE; plasmablasts; principal component analysis (PCA); principal components (PCs).