The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; <em>2</em> mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in <em>D</em>-<em>dimer</em> release from thrombi, and prevented the occlusion of <em>2</em>-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (3<em>2</em> mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

Over 100 mutations in Cu/Zn-superoxi<em>d</em>e <em>d</em>ismutase (SOD1) result in familial amyotrophic lateral sclerosis. <em>Dimer</em> <em>d</em>issociation is the first step in SOD1 aggregation, an<em>d</em> stu<em>d</em>ies suggest nearly every amino aci<em>d</em> resi<em>d</em>ue in SOD1 is <em>d</em>ynamically connecte<em>d</em> to the <em>dimer</em> interface. Post-translational mo<em>d</em>ifications of SOD1 resi<em>d</em>ues might be expecte<em>d</em> to have similar effects to mutations, but few mo<em>d</em>ifications have been i<em>d</em>entifie<em>d</em>. Here we show, using SOD1 isolate<em>d</em> from human erythrocytes, that human SOD1 is phosphorylate<em>d</em> at threonine <em>2</em> an<em>d</em> glutathionylate<em>d</em> at cysteine 111. A secon<em>d</em> SOD1 phosphorylation was observe<em>d</em> an<em>d</em> mappe<em>d</em> to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a <em>2</em>-fol<em>d</em> increase in the K(<em>d</em>). This change in the <em>dimer</em> stability is expecte<em>d</em> to result in a 67% increase in monomer concentration, 315 nm rather than <em>2</em>1<em>2</em> nm at physiological SOD1 concentrations. Because protein glutathionylation is associate<em>d</em> with re<em>d</em>ox regulation, our fin<em>d</em>ing that glutathionylation promotes SOD1 monomer formation supports a mo<em>d</em>el in which increase<em>d</em> oxi<em>d</em>ative stress promotes SOD1 aggregation.

The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of <em>2</em>0 different human insulin mutants at both low pH (conditions favoring monomer/<em>dimer</em>) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in <em>2</em>0% acetic acid, 0.1 M NaCl, pH <em>2</em>. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation [Brange, J., Whittingham, J., Edwards, <em>D</em>., Youshang, Z., Wollmer, A., Brandenburg, <em>D</em>., <em>D</em>odson, G., and Finch, J. (1997) Curr. Sci. 7<em>2</em>, 470-476]. Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B<em>2</em>5. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation. A model for insulin fibril formation is proposed in which the formation of a partially folded intermediate is the precursor for associated species on the pathway to fibril formation.

Background: COVID-19-associated coagulopathy (CAC) characterized by the elevated D-dimer without remarkable changes of other global coagulation markers is associated with various thrombotic complications and disease severity. The purpose of this review is to elucidate the pathophysiology of this unique coagulopathy.

Methods: The authors performed online search of published medical literature through PubMed using the MeSH (Medical Subject Headings) term "COVID-19," "SARS-CoV-2," "coronavirus," "coagulopathy," and "thrombus." Then, selected 51 articles that closely relevant to coagulopathy in COVID-19.

Results: The primary targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the pneumocytes, immune cells, and vascular endothelial cells. The alveolar damage and the pulmonary microvascular thrombosis are the major causes of acute lung injury in COVID-19. The endotheliopathy that occurs is due to direct SARS-CoV-2 infection and activation of other pathways that include the immune system and thromboinflammatory responses leading to what is termed CAC. As a result, both microvascular and macrovascular thrombotic events occur in arterial, capillary, venule, and large vein vascular beds to produce multiorgan dysfunction and thrombotic complications. In addition to the endothelial damage, SARS-CoV-2 also can cause vasculitis and presents as a systemic inflammatory vascular disease. Clinical management of COVID-19 includes anticoagulation but novel therapies for endotheliopathy, hypercoagulability, and vasculitis are needed.

Conclusion: The endotheliopathy due to direct endothelial infection with SARS-COV-2 and the indirect damage caused by inflammation play the predominant role in the development of CAC. The intensive control of thromboinflammation is necessary to improve the outcome of this highly detrimental contagious disease.

Keywords: COVID-19; Coagulopathy; Endotheliopathy; Thromboembolism; Vasculitis.

<strong class="sub-title"> Background: </strong> In April, <em>2</em>0<em>2</em>0, clinicians in the UK observed a cluster of children with unexplained inflammation requiring admission to paediatric intensive care units (PICUs). We aimed to describe the clinical characteristics, course, management, and outcomes of patients admitted to PICUs with this condition, which is now known as paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-<em>2</em> (PIMS-TS).

<strong class="sub-title"> Methods: </strong> We did a multicentre observational study of children (aged <18 years), admitted to PICUs in the UK between April 1 and May 10, <em>2</em>0<em>2</em>0, fulfilling the case definition of PIMS-TS published by the Royal College of Paediatrics and Child Health. We analysed routinely collected, de-identified data, including demographic details, presenting clinical features, underlying comorbidities, laboratory markers, echocardiographic findings, interventions, treatments, and outcomes; serology information was collected if available. PICU admission rates of PIMS-TS were compared with historical trends of PICU admissions for four similar inflammatory conditions (Kawasaki disease, toxic shock syndrome, haemophagocytic lymphohistiocytosis, and macrophage activation syndrome).

<strong class="sub-title"> Findings: </strong> 78 cases of PIMS-TS were reported by <em>2</em>1 of <em>2</em>3 PICUs in the UK. Historical data for similar inflammatory conditions showed a mean of one (95% CI 0·85-1·<em>2</em><em>2</em>) admission per week, compared to an average of 14 admissions per week for PIMS-TS and a peak of 3<em>2</em> admissions per week during the study period. The median age of patients was 11 years (IQR 8-14). Male patients (5<em>2</em> [67%] of 78) and those from ethnic minority backgrounds (61 [78%] of 78) were over-represented. Fever (78 [100%] patients), shock (68 [87%]), abdominal pain (48 [6<em>2</em>%]), vomiting (49 [63%]), and diarrhoea (50 [64%]) were common presenting features. Longitudinal data over the first 4 days of admission showed a serial reduction in C-reactive protein (from a median of <em>2</em>64 mg/L on day 1 to 96 mg/L on day 4), D-dimer (4030 μg/L to 1659 μg/L), and ferritin (104<em>2</em> μg/L to 757 μg/L), whereas the lymphocyte count increased to more than 1·0 × 10⁹ cells per L by day 3 and troponin increased over the 4 days (from a median of 157 ng/mL to 358 ng/mL). 36 (46%) of 78 patients were invasively ventilated and 65 (83%) needed vasoactive infusions; 57 (73%) received steroids, 59 (76%) received intravenous immunoglobulin, and 17 (<em>2</em><em>2</em>%) received biologic therapies. <em>2</em>8 (36%) had evidence of coronary artery abnormalities (18 aneurysms and ten echogenicity). Three children needed extracorporeal membrane oxygenation, and two children died.

Interpretation: During the study period, the rate of PICU admissions for PIMS-TS was at least 11-fold higher than historical trends for similar inflammatory conditions. Clinical presentations and treatments varied. Coronary artery aneurysms appear to be an important complication. Although immediate survival is high, the long-term outcomes of children with PIMS-TS are unknown.

Funding: None.

Vascular endothelial growth factor (VEGF) C and VEGF-<em>D</em> stimulate lymphangiogenesis and angiogenesis in tissues and tumors by activating the endothelial cell surface receptor tyrosine kinases VEGF receptor (VEGFR) <em>2</em> and VEGFR-3. These growth factors are secreted as full-length inactive forms consisting of NH<em>2</em>- and COOH-terminal propeptides and a central VEGF homology domain (VH<em>D</em>) containing receptor binding sites. Proteolytic cleavage removes the propeptides to generate mature forms, consisting of <em>dimers</em> of the VEGF homology domain, that bind receptors with much greater affinity than the full-length forms. Therefore, proteolytic processing activates VEGF-C and VEGF-<em>D</em>, although the proteases involved were unknown. Here, we report that the serine protease plasmin cleaved both propeptides from the VEGF homology domain of human VEGF-<em>D</em> and thereby generated a mature form exhibiting greatly enhanced binding and cross-linking of VEGFR-<em>2</em> and VEGFR-3 in comparison to full-length material. Plasmin also activated VEGF-C. As lymphangiogenic growth factors promote the metastatic spread of cancer via the lymphatics, the proteolytic activation of these molecules represents a potential target for antimetastatic agents. Identification of an enzyme that activates the lymphangiogenic growth factors will facilitate development of inhibitors of metastasis.

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(<em>2</em>) (Kojima, S., and Blair, <em>D</em>. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(<em>2</em>) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical <em>dimer</em> of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. <em>D</em>isulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB hetero<em>dimer</em> upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked hetero<em>dimer</em>, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma <em>2</em>-CH gamma 3-15aa linker-CH epsilon <em>2</em>-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa <em>D</em> <em>dimer</em> that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.

The type II restriction endonuclease EcoRV was crystallized as a complex with the substrate <em>D</em>NA undecamer AAAGATATCTT (recognition sequence underlined). These crystals diffract to much better resolution (<em>2</em> A) than was the case for the previously reported complex with the decamer GGGATATCCC [Winkler, F. K., Banner, <em>D</em>. W., Oefner, C., Tsernoglou, <em>D</em>., Brown, R. S., Heathman, S. P., Bryan, R. K., Martin, P. <em>D</em>., Petratos, K., & Wilson, K. S. (1993) EMBO J. 1<em>2</em>, 1781-1795]. The crystal structure contains one <em>dimer</em> complex in the asymmetric unit and was solved by molecular replacement. The same kinked <em>D</em>NA conformation characteristic for enzyme-bound cognate <em>D</em>NA is observed. Crystals, soaked with Mg<em>2</em>+, show the essential cofactor bound at only one active site of the <em>dimer</em>, and the <em>D</em>NA is not cleaved. The Mg<em>2</em>+ has one oxygen from the scissile phosphodiester group and two carboxylate oxygens, one form Asp74 and one from Asp90, in its octahedral ligand sphere. The scissile phosphodiester group is pulled by 1 A toward the Mg<em>2</em>+. After substrate cleavage in solution, isomorphous crystals containing the enzyme--product--Mg<em>2</em>+ complex were obtained. In this structure, each of the 5'-phosphate groups is bound to two Mg<em>2</em>+. The kinked <em>D</em>NA conformation is essentially maintained, but the two central adenines, 3' to the cleavage sites, form an unusual cross-strand base stacking. The structures have been refined to R factors of 0.16 at <em>2</em>.1-<em>2</em>.0 A resolution maintaining very good stereochemistry. On the basis of these structures and inspired by recent kinetic data [Vipond, I. B., & Halford, S. E. (1994) Biochemistry (second paper of three in this issue)], we have constructed a transition state model with two metals bound to the scissile phosphorane group.

<b>Rationale:</b> In addition to the overwhelming lung inflammation that prevails in COVI<em>D</em>-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVI<em>D</em>-19. <b>Objective:</b> To evaluate the contribution of platelets to inflammation and thrombosis in COVI<em>D</em>-19 patients. <b>Methods and Results:</b> Blood was collected from 115 consecutive COVI<em>D</em>-19 patients presenting non-severe (n=71) and severe (n=44) respiratory symptoms. We document the presence of SARS-CoV-<em>2</em> RNA associated with platelets of COVI<em>D</em>-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both non-severe and severe COVI<em>D</em>-19 patients, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both non-severe and severe forms of COVI<em>D</em>-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in non-severe, but not in severe cases of COVI<em>D</em>-19. Levels of <em>D</em>-<em>dimers</em>, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVI<em>D</em>-19 subjects presenting non-severe and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. <b>Conclusions:</b> Taken together, the data suggest that platelets are at the frontline of COVI<em>D</em>-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVI<em>D</em>-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVI<em>D</em>-19.

<strong class="sub-title"> Keywords: </strong> SARS-CoV-<em>2</em>.

Thirty-three subjects with sickle cell disease (SC<em>D</em>), 11 during episodes of pain and <em>2</em><em>2</em> during periods without pain, were evaluated for in vivo thrombogenic activities as compared with 10 normal black control subjects. Measurements were performed for (1) platelet surface activation, assessing flow cytometric expression of activated integrin alpha(IIb)beta(3) receptor (GPIIb/IIIa, C<em>D</em>41a) and P-selectin (C<em>D</em>6<em>2</em>p); (<em>2</em>) platelet and erythrocyte surface procoagulant activities, measuring flow cytometric binding of activated factor (FVa) and annexin V; (3) plasma levels of platelet-specific secreted proteins platelet factor 4 (PF4) and beta-thromboglobulin (betaTG); (4) plasma markers of thrombin generation, prothrombin activation fragment (F(1.<em>2</em>)), and thrombin: antithrombin complex (TAT); and (5) plasma markers of fibrinolysis, <em>D</em> -<em>dimer</em>, and plasmin:antiplasmin complex (PAP). As compared with control subjects, asymptomatic subjects with SC<em>D</em> demonstrated significantly increased platelet activation (P <.01 for P-selectin and annexin V binding), elevated plasma levels of PF4 and betaTG (P <.01 and P <.03, respectively), and increased plasma concentrations of F(1.<em>2</em>), TAT, PAP, and <em>D</em> -<em>dimer</em> (P <.05 in all cases). <em>D</em>uring episodes of SC<em>D</em> pain, platelet activation was increased as compared with periods without pain (P <.01 for expression of activated integrin alpha(IIb)beta(3) receptor and P-selectin and binding of FVa and annexin V), erythrocytes expressed procoagulant activities (P <.01 for FVa and annexin V binding), and platelet microparticles appeared in the circulation (3% to 30%; P <.001). SC<em>D</em> pain episodes were associated with elevated plasma levels of F(1.<em>2</em>), TAT, PAP, and <em>D</em> -<em>dimer</em> (P <.05 as compared with asymptomatic intervals). The frequency of pain episodes correlated with enhanced platelet procoagulant activity (r = 0.61, P <.05) and elevated plasma fibrinolytic activity (r = 0.74, P <.01) measured during periods without pain. Plasma fibrinolytic activity was inversely correlated with time to the next pain episode (r = -0.50, P <.05). Thus, asymptomatic subjects with SC<em>D</em> exhibit ongoing platelet activation, thrombin generation, and fibrinolysis that increases during episodes of pain. These changes are predictive of frequency of pain and interval to next pain episode, thereby implicating thrombogenic activity in the development of SC<em>D</em> pain episodes.

In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homo<em>dimer</em> and binds with two alpha-helix-turn-alpha-helix motifs (HTH) to two tandemly orientated <em>D</em>NA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to <em>D</em>NA. TetR of class <em>D</em>, TEtR<em>D</em>, was cocrystallized with tetracycline (7HTc) and Mg<em>2</em>+ in space group I4(1)<em>2</em><em>2</em> and studied by X-ray diffraction. One TetR<em>D</em> monomer occupies the crystal asymmetric unit, and the <em>dimer</em> is formed by a crystallographic <em>2</em>-fold rotation. The crystal structure was determined by multiple isomorphous replacement at <em>2</em>.5 A resolution, and on this basis the structure of the nearly isomorphous complex with 7-chlorotetracycline, TetR<em>D</em>/(Mg 7CITc)+, has been refined to an R-factor of 18.3% using all reflections to <em>2</em>.1 A resolution. TetR<em>D</em> folds into ten alpha-helices with connecting turns and loops. The N-terminal three alpha-helices of the repressor form the <em>D</em>NA-binding domain, including the HTH with an inverse orientation compared with HTH in other <em>D</em>NA-binding proteins. The distance of 39 A between the two recognition helices explains the inability of the induced TetR to bind to B-form <em>D</em>NA. The core of the protein is formed by helices alpha 5 to alpha 10. It is responsible for <em>dimer</em>ization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation. The structure of the TetR<em>D</em>/(Mg 7CITc)+ complex reveals the octahedral coordination of Mg<em>2</em>+ by Tc (chelating O-11, and O-1<em>2</em>), His100 N epsilon and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.

<strong class="sub-title">Importance:</strong> Descriptions of the coronavirus disease <em>2</em>019 (COVID-19) experience in pediatrics will help inform clinical practices and infection prevention and control for pediatric facilities.

Objective: To describe the epidemiology, clinical, and laboratory features of patients with COVID-19 hospitalized at a children's hospital and to compare these parameters between patients hospitalized with and without severe disease.

<strong class="sub-title">Design, setting, and participants:</strong> This retrospective review of electronic medical records from a tertiary care academically affiliated children's hospital in New York City, New York, included hospitalized children and adolescents (≤<em>2</em>1 years) who were tested based on suspicion for COVID-19 between March 1 to April 15, <em>2</em>0<em>2</em>0, and had positive results for severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>).

<strong class="sub-title">Exposures:</strong> Detection of SARS-CoV-<em>2</em> from a nasopharyngeal specimen using a reverse transcription-polymerase chain reaction assay.

Main outcomes and measures: Severe disease as defined by the requirement for mechanical ventilation.

<strong class="sub-title">Results:</strong> Among 50 patients, <em>2</em>7 (54%) were boys and <em>2</em>5 (50%) were Hispanic. The median days from onset of symptoms to admission was <em>2</em> days (interquartile range, 1-5 days). Most patients (40 [80%]) had fever or respiratory symptoms (3<em>2</em> [64%]), but 3 patients (6%) with only gastrointestinal tract presentations were identified. Obesity (11 [<em>2</em><em>2</em>%]) was the most prevalent comorbidity. Respiratory support was required for 16 patients (3<em>2</em>%), including 9 patients (18%) who required mechanical ventilation. One patient (<em>2</em>%) died. None of 14 infants and 1 of 8 immunocompromised patients had severe disease. Obesity was significantly associated with mechanical ventilation in children <em>2</em> years or older (6 of 9 [67%] vs 5 of <em>2</em>5 [<em>2</em>0%]; P = .03). Lymphopenia was commonly observed at admission (36 [7<em>2</em>%]) but did not differ significantly between those with and without severe disease. Those with severe disease had significantly higher C-reactive protein (median, 8.978 mg/dL [to convert to milligrams per liter, multiply by 10] vs 0.64 mg/dL) and procalcitonin levels (median, 0.31 ng/mL vs 0.17 ng/mL) at admission (P < .001), as well as elevated peak interleukin 6, ferritin, and D-dimer levels during hospitalization. Hydroxychloroquine was administered to 15 patients (30%) but could not be completed for 3. Prolonged test positivity (maximum of <em>2</em>7 days) was observed in 4 patients (8%).

Conclusions and relevance: In this case series study of children and adolescents hospitalized with COVID-19, the disease had diverse manifestations. Infants and immunocompromised patients were not at increased risk of severe disease. Obesity was significantly associated with disease severity. Elevated inflammatory markers were seen in those with severe disease.

<em>D</em>isseminated intravascular coagulation, characterized by circulating fibrin(ogen) degradation products (F<em>D</em>P), is associated with both acute and chronic inflammatory conditions. Since the association of F<em>D</em>P with monocytes could influence the release of cytokines and other regulatory proteins with significant clinical ramifications, we have studied cytokine synthesis and release following the interaction of <em>D</em>-<em>dimer</em> (<em>D</em><em>D</em>), a terminal degradation product of fibrin, with human monocytes in vitro. Adherent peripheral blood monocytes were incubated with purified <em>D</em><em>D</em> for <em>2</em>4 and 48 h and secreted or cell-associated IL-1 beta and IL-6 antigen levels and activity determined. <em>D</em><em>D</em> (50 micrograms/ml) boosted the secretion of IL-1 beta antigen from median control levels of 659 pg/ml to <em>2</em>704 pg/ml and that of IL-6 antigen from 806 pg/ml to>> 3000 pg/ml at 48 h (P < 0.05). Similar increases in extracellular biologically active IL-1 and IL-6 were observed. Although <em>D</em><em>D</em> increased cell associated IL-1 beta antigen levels from median values of 188 to 1600 pg/106 cells and IL-6 antigen from 660 to <em>2</em><em>2</em>15 pg/106 cells (P < 0.05), cell-associated IL-1 functional activity decreased from control levels of 98 inhibitor units/ml to 65 units/ml for cells exposed to <em>D</em><em>D</em>. Secreted plasminogen activator inhibitor (PAI) bioactivity and PAI type <em>2</em> antigen levels were significantly increased following exposure of monocytes to <em>D</em><em>D</em>. This may explain the decreased cell associated IL-1 activity observed in our study as PAI are known to inhibit biologically active membrane bound IL-1. Our finding that <em>D</em><em>D</em> enhances monocyte release of biologically active cytokines suggests the presence of positive feedback pathways for fibrinogen synthesis by hepatocytes. Furthermore, the association of monocytes with <em>D</em><em>D</em> may potentiate localized coagulation processes by subsequent alterations in pericellular proteolysis.

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activate<em>d</em> upon bin<em>d</em>ing <em>d</em>ouble-stran<em>d</em>e<em>d</em> (<em>d</em>s) RNA to un<em>d</em>ergo autophosphorylation. Although PKR is known to <em>dimer</em>ize, the relationship between <em>dimer</em>ization an<em>d</em> activation remains unclear. Here, we <em>d</em>irectly characterize <em>dimer</em>ization of PKR in free solution using analytical ultracentrifugation an<em>d</em> correlate self-association with autophosphorylation activity. Latent, unphosphorylate<em>d</em> PKR exists pre<em>d</em>ominantly as a monomer at protein concentrations below <em>2</em> mg/ml. A monomer se<em>d</em>imentation coefficient of s(<em>2</em>0,w)(0)=3.58 S an<em>d</em> a frictional ratio of f/f(0)=1.6<em>2</em> in<em>d</em>icate an asymmetric shape. Se<em>d</em>imentation equilibrium measurements in<em>d</em>icate that PKR un<em>d</em>ergoes a weak, reversible monomer-<em>dimer</em> equilibrium with K(<em>d</em>)=450 microM. This <em>dimer</em>ization reaction serves to initiate a previously unrecognize<em>d</em> <em>d</em>sRNA-in<em>d</em>epen<em>d</em>ent autophosphorylation reaction. The resulting activate<em>d</em> enzyme is phosphorylate<em>d</em> on the two critical threonine resi<em>d</em>ues present in the activation loop an<em>d</em> is competent to phosphorylate the physiological substrate, eIF<em>2</em>alpha. <em>Dimer</em> stability is enhance<em>d</em> by approximately 500-fol<em>d</em> upon autophosphorylation. We propose a chain reaction mo<em>d</em>el for PKR <em>d</em>sRNA-in<em>d</em>epen<em>d</em>ent activation where <em>dimer</em>ization of latent enzyme followe<em>d</em> by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activate<em>d</em> enzyme within high-affinity hetero<em>dimer</em>s. Our results support a mo<em>d</em>el whereby <em>d</em>sRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the <em>dimer</em>ization of free PKR.

Fibrin deposition is a prominent finding in the synovium of patients with rheumatoid arthritis (RA). Macrophages are found in increased numbers in RA synovium, and these cells are known to produce a variety of procoagulant and anticoagulant molecules. Using immunohistologic techniques, the content and distribution of several important components of the coagulation system in the synovium of patients with RA, osteoarthritis (OA), or traumatic joint abnormalities requiring surgery were investigated. Samples from 3 patients from each category were examined in detail. RA synovium (compared with that of patients with OA or joint trauma) had increased numbers of macrophages and increased expression/content of fibrinogen, tissue factor, factor XIII, tissue transglutaminase, cross-linked fibrin (fibrin <em>D</em> <em>dimer</em>), urokinase-type plasminogen activator, and alpha <em>2</em>-plasmin inhibitor. Macrophage content in RA synovium was increased in both the lining cell areas and the interstitial cell areas. Fibrinogen was distributed throughout the tissue in all samples and was greater in RA synovium. In trauma and OA synovia, tissue factor was seen only in association with vessels (endothelial cells), but in RA synovium, it was markedly increased throughout the tissues. While fibrin <em>D</em> <em>dimer</em> was seen in small amounts in synovial lining cell areas of trauma and OA synovia, it was present in increased amounts in the lining cell and interstitial cell areas of RA synovium. Factor XIII and tissue transglutaminase were present in scant amounts in trauma and OA synovia, but there were increased amounts of both (especially tissue transglutaminase) in RA synovium in the vessel, lining cell, and interstitial cell areas. Urokinase and alpha <em>2</em>-plasmin inhibitor were also markedly increased in RA synovium. These results suggest that in inflamed synovium, there is ongoing extravascular tissue fibrin formation and dissolution that correlates with the degree of inflammation and macrophage content. Extravascular coagulation/fibrinolysis in RA represents a potential target for therapeutic intervention in this disease.

Malic enzyme is a tetrameric protein with double <em>dimer</em> structure in which the <em>dimer</em> interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and <em>dimer</em> interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and <em>D</em>. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-11<em>2</em> and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(<em>2</em>+) or Mg(<em>2</em>+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.

<strong class="sub-title"> Background: </strong> Pediatric SARS-CoV-<em>2</em> infection can be complicated by a dangerous hyperinflammatory condition termed multisystem inflammatory syndrome in children (MIS-C). The clinical and immunologic spectrum of MIS-C and its relationship to other inflammatory conditions of childhood have not been studied in detail.

<strong class="sub-title"> Methods: </strong> We retrospectively studied confirmed cases of MIS-C at our institution from March to June <em>2</em>0<em>2</em>0. The clinical characteristics, laboratory studies and treatment response were collected. Data were compared with historic cohorts of Kawasaki disease (KD) and macrophage activation syndrome (MAS).

<strong class="sub-title"> Results: </strong> Twenty-eight patients fulfilled the case definition of MIS-C. Median age at presentation was 9 years (range 1 month to 17 years); 50% of patients had pre-existing conditions. All patients had laboratory confirmation of SARS-CoV-<em>2</em> infection. Seventeen patients (61%) required intensive care, including 7 patients (<em>2</em>5%) requiring inotrope support. Seven patients (<em>2</em>5%) met criteria for complete or incomplete KD and coronary abnormalities were found in 6 cases. Lymphopenia, thrombocytopenia, and elevation in inflammatory markers, D-dimer, B-type natriuretic peptide, IL-6 and IL-10 levels were common but not ubiquitous. Cytopenias distinguished MIS-C from KD and the degree of hyperferritinemia and pattern of cytokine production differed between MIS-C and MAS. Immunomodulatory therapy given to MIS-C patients included IVIG (71%), corticosteroids (61%) and anakinra (18%). Clinical and laboratory improvement were observed in all cases, including 6 cases that did not require immunomodulatory therapy. No mortality was recorded in this cohort.

Conclusion: MIS-C encompasses a broad phenotypic spectrum with clinical and laboratory features distinct from Kawasaki disease and macrophage activation syndrome.

<strong class="sub-title"> Funding: </strong> This work was supported by the National Institute of Health / National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) K08-AR07456<em>2</em> (PYL), K08-AR AR073339 (LAH), R01-AR065538, R01-AR073<em>2</em>01 and P30-AR070<em>2</em>53 (PAN); National Institute of Allergy and Infectious Diseases 5T3<em>2</em>AI00751<em>2</em>-34 (JL, JR, TB, AAN and RWN); Rheumatology Research Foundation Investigator Awards (PYL and LAH) and Medical Education Award (JSH); Boston Children's Hospital Faculty Career Development Awards (PYL and LAH), the McCance Family Foundation (JWN), and the Samara Jan Turkel Center (JC, RPS, MBS).

Keywords: COVID-19; Clinical practice; Immunology.

Lack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype <em>2</em> (strain 16 681) at 1 x 10(7) PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of <em>D</em>-<em>dimer</em> was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. <em>D</em>engue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.

Triosephosphate isomerase (TIM) is a perfectly evolved enzyme which very fast interconverts dihydroxyacetone phosphate and <em>D</em>: -glyceraldehyde-3-phosphate. Its catalytic site is at the <em>dimer</em> interface, but the four catalytic residues, Asn11, Lys13, His95 and Glu167, are from the same subunit. Glu167 is the catalytic base. An important feature of the TIM active site is the concerted closure of loop-6 and loop-7 on ligand binding, shielding the catalytic site from bulk solvent. The buried active site stabilises the enediolate intermediate. The catalytic residue Glu167 is at the beginning of loop-6. On closure of loop-6, the Glu167 carboxylate moiety moves approximately <em>2</em> Å to the substrate. The dynamic properties of the Glu167 side chain in the enzyme substrate complex are a key feature of the proton shuttling mechanism. Two proton shuttling mechanisms, the classical and the criss-cross mechanism, are responsible for the interconversion of the substrates of this enolising enzyme.

We found previously that overexpression of an F-box protein betaTrCP1 and the structurally related betaTrCP<em>2</em> augments ubiquitination of phosphorylated IkappaBalpha (pIkappaBalpha) induced by tumor necrosis factor-alpha (TNF-alpha), but the relationship of the two homologous betaTrCP proteins remains unknown. Herein we reveal that deletion mutants of betaTrCP1 and betaTrCP<em>2</em> lacking the F-box domain suppressed ubiquitination and destruction of pIkappaBalpha as well as transcriptional activation of NF-kappaB. The ectopically expressed betaTrCP1 and betaTrCP<em>2</em> formed both homo<em>dimer</em> and hetero<em>dimer</em> complexes without displaying the trimer complex. <em>D</em>imerization of betaTrCP1 and/or betaTrCP<em>2</em> takes place at their conserved NH(<em>2</em>)-terminal regions, termed a "<em>D</em>-domain" (for <em>dimer</em>ization domain), located upstream of the F-box domain. The <em>D</em>-domain was necessary and sufficient for the <em>dimer</em> formation. Intriguingly, the betaTrCP homo<em>dimer</em>, but not the hetero<em>dimer</em>, was selectively recruited to pIkappaBalpha induced by TNF-alpha. These results indicate that not only betaTrCP1 but also betaTrCP<em>2</em> participates in the ubiquitination-dependent destruction of IkappaBalpha by forming SCF(betaTrCP1-betaTrCP1) and SCF(betaTrCP<em>2</em>-betaTrCP<em>2</em>) ubiquitin-ligase complexes.

<strong class="sub-title"> Objective: </strong> To assess clinical characteristics and outcomes of SARS-CoV-<em>2</em> associated multisystem inflammatory syndrome in children (MIS-C).

<strong class="sub-title"> Study design: </strong> Children with MIS-C admitted to pediatric intensive care units (PICU) in New York City between April <em>2</em>3 and May <em>2</em>3, <em>2</em>0<em>2</em>0 were included. Demographic and clinical data were collected.

<strong class="sub-title"> Results: </strong> Of 33 children with MIS-C, the median age was 10 years; 61% were male; 45% were Hispanic/Latino; 39% were black. Comorbidities were present in 45%. Fever (93%) and vomiting (69%) were the most common presenting symptoms. Depressed left ventricular ejection fraction (LVEF) was found in 63% of patients with median EF of 46.6% (IQR 39.5, 5<em>2</em>.8). C-reactive protein, procalcitonin, D-dimer, and pro-B-type natriuretic peptide levels were elevated in all patients. For treatment, intravenous immunoglobulin was used in 18 (54%), corticosteroids in 17 (51%), tocilizumab in 1<em>2</em> (36%), remdesivir in 7 (<em>2</em>1%), vasopressors in 17 (51%), mechanical ventilation in 5 (15%), extracorporeal membrane oxygenation (ECMO) in 1 (3%), and intra-aortic balloon pump in 1 (3%). The LVEF normalized in 95% of those with depressed EF. All patients were discharged home with median duration of PICU stay of 4.7 (4, 8) days and hospital stay of 7.8 (6, 10.1) days. One (3%) patient died after withdrawal of care secondary to stroke while on ECMO.

Conclusions: Critically ill children with COVID-19 associated MIS-C have a spectrum of severity broader than described previously but still require careful supportive intensive care. Rapid, complete clinical and myocardial recovery was almost universal.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (p<em>D</em><em>D</em>-<em>2</em>) containing only 165 bp of AAV sequence: two copies of the <em>D</em> element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. p<em>D</em><em>D</em>-<em>2</em> replication intermediates were characteristic of the AAV replication scheme in which linear monomer, <em>dimer</em>, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV <em>D</em>NA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

BACKGROUND

Widespread screening of patients with venous thromboembolism (VTE) for thrombophilic risk factors has become common clinical practice. Because of the increasing number of risk factors, assessing the risk of recurrence in an individual patient is intricate; therefore, a laboratory method that measures multifactorial thrombophilia is required.

OBJECTIVE

To prospectively study the relationship between the risk of recurrent VTE and D-dimer, a global marker of coagulation activation and fibrinolysis.

METHODS

Prospective cohort study of 610 patients older than 18 years who were treated with oral anticoagulants for at least 3 months with a first spontaneous VTE, in whom D-dimer levels were measured shortly after discontinuation of oral anticoagulation. The study was conducted at the Department of Internal Medicine I, University Hospital, Vienna, Austria. Patients entered the study at time of discontinuation of oral anticoagulants and were observed at 3-month intervals during the first year and every 6 months thereafter from July 1992 to October 2002.

METHODS

Objectively documented symptomatic recurrent VTE.

RESULTS

A total of 79 (13%) of 610 patients had recurrent VTE with a mean observation time of 38 months. Patients with recurrence had significantly higher D-dimer levels compared with those without recurrence (553 ng/mL vs 427 ng/mL, P =.01). Compared with patients with D-dimer levels of 750 ng/mL or higher, the relative risk (RR) of recurrence was 0.6 (95% confidence interval [CI], 0.3-1.4), 0.6 (95% CI, 0.3-1.2), and 0.3 (95% CI, 0.1-0.6) in patients with D-dimer levels of 500 to 749 ng/mL, 250 to 499 ng/mL, and less than 250 ng/mL, respectively. The cumulative probability of recurrent VTE at 2 years was 3.7% (95% CI, 0.9%-6.5%) among patients with D-dimer levels of less than 250 ng/mL compared with 11.5% (95% CI, 8.0%-15.0%) among patients with higher levels (P =.001). Patients with D-dimer levels of less than 250 ng/mL had a 60% lower RR of recurrence compared with patients with higher levels (RR, 0.4; 95% CI, 0.2-0.8).

CONCLUSIONS

Patients with a first spontaneous VTE and a D-dimer level of less than 250 ng/mL after withdrawal of oral anticoagulation have a low risk of VTE recurrence.