A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

Experiences of breast cancer in Arab countries. A thematic synthesis.

BACKGROUND: Breast cancer is the most common cancer in women globally. Its negative effects on a woman's quality of life are related to the individual and socio-cultural factors. This review aimed to identify and synthesise the reported experiences and quality of life of women with breast cancer in Arab countries. METHODS: PubMed, Embase, Web of Science, SCOPUS, PsychInfo, CINAHL, Allied and Complementary Medicine Database, and Index Medicus for the Eastern Mediterranean Region were searched for articles published from start to March 2019 using PRISMA guidelines. These searches were complimented by citation tracking and  hand searching of relevant journals. A thematic synthesis was carried out on the 'findings/results' sections from the identified papers. RESULTS: Of 5228 records identified, 19 were included in the review which represented 401 women from 11 Arab countries. All used qualitative methods of data collection to produce rich descriptions of experiences. Thematic synthesis of the extracted data identified three major themes, Perceptions and reactions, Coping or enduring and Changing roles. CONCLUSIONS: This review provides a rich description of the reported quality of life and experiences of women with breast cancer in Arab countries. These are influenced by the women's and society's views of cancer, the women's role in society and family, religious faith and the healthcare context and access to treatment choices and information.

Does pre-hospital telephone communication with a clinician result in more appropriate medication administration by parents during childhood asthma exacerbations?

BACKGROUND: The National Heart, Lung and Blood Institute asthma guidelines recommend that parents communicate with a clinician during childhood asthma exacerbations when symptoms worsen or do not improve with initial therapy. This study tested the hypothesis that communication by parents with a clinician before an Emergency Department visit was associated with more appropriate medication administration for children with asthma exacerbations. METHODS: This was a retrospective cohort study using data gathered from parents of children presenting with an asthma exacerbation to the emergency department. The communicating cohort included parents who communicated by telephone with a clinician during the exacerbation and the non-communicating cohort included parents who did not. Multivariate logistic regression models were used to test three hypotheses; communication with a clinician is associated with (1) administration of short-acting beta-agonists (SABAs), (2) increased dosing frequency of SABAs, and (3) administration of an oral corticosteroid. RESULTS: A total of 199 subjects were enrolled, with 104 (52.3%) in the communicating and 95 (47.7%) in the non-communicating cohort. There was an association between communication and provider practice type, with children who received routine care from a private practice provider more likely to communicate with the clinician than children in hospital-based clinics or community health centers (Adjusted OR 1.9, 95% CI 1.0-3.7). Impoverished children and children insured by Medicaid were less likely to communicate with a clinician (controlling for provider type). Parents who communicated with a clinician were more likely to administer a SABA (adjusted OR 3.6, 95% CI 1.3-9.4) and an oral corticosteroid (adjusted OR 3.3, 95% CI 1.3-8.4) but were not more likely to administer a SABA with increased dosing frequency (adjusted OR 0.9, 95% CI 0.5-1.6). CONCLUSIONS: Parents of children with asthma exacerbations who communicated with clinicians were more likely to administer SABAs and an oral corticosteroid before bringing their child to an emergency department. Frequency of SABA dosing was not associated with communication. Clinicians providing telephone advice to parents need to provide explicit instructions about medication administration, emphasizing the frequency with which SABAs should be administered.

IgM and stromal cell-associated heparan sulfate/heparin as complement-independent ligands for CD19.

The more severe phenotype of mice lacking CD19 as compared to CD21 suggests that a complement-independent ligand for the CD19/CD21 complex exists. We sought ligands for CD19 by examining binding reactions with fusion proteins comprised of the extracellular region of CD19 and the Fc region of IgG1. A fusion protein containing the third extracellular domain (D3-Fc) bound to WEHI-231 cells, and this was competed by soluble IgM. This function of IgM was confirmed by the binding of D3-Fc to beads coated with IgM. A second ligand for D3-Fc was found on stromal cells, and was shown to be heparin/heparan sulfate. These two ligands would be considered to reside on follicular dendritic cells, and may account for the observed ability of D3-Fc to bind to sites in germinal centers containing these cells.

Arrested differentiation, the self-renewing memory lymphocyte, and vaccination.

Vaccination for persistent viral or bacterial infections must program the immune system for a lifelong need to generate antigen-specific effector lymphocytes. How the immune system does this is not known, but recent studies have shown that a subset of B lymphocytes, the germinal center B cell, is capable of self-renewal because it expresses a transcriptional repressor, BCL6, that blocks terminal differentiation. If a similar mechanism for arresting differentiation exists for long-lived, antigen-selected lymphocytes, a stem cell-like capacity for self-renewal could be the basis for the continual generation of effector lymphocytes from the memory pool. Understanding how to regulate the terminal differentiation of lymphocytes will improve immunotherapeutic approaches for chronic infectious diseases and cancer.

Suppression of signal transducer and activator of transcription 3-dependent B lymphocyte terminal differentiation by BCL-6.

Lymphocytes usually differentiate into effector cells within days after antigen exposure, except in germinal centers where terminal differentiation is delayed while somatic hypermutation creates high-affinity antibody mutants. Here we investigate whether arrest of terminal differentiation can be mediated by BCL-6, a transcriptional repressor that is expressed by germinal center B cells and is required for this phase of B cell development. We find that BCL-6 suppresses the differentiation of transformed and primary B cells to plasma cells by inhibiting the signal transducer and activator of transcription 3-dependent expression of the major regulator of plasma cell development, the B lymphocyte-induced maturation protein (Blimp-1). This function of BCL-6 as a repressor of B lymphocyte differentiation may also underlie the association between chromosomal translocations of its gene and B cell lymphomas.

Enhanced immunogenicity of aldehyde-bearing antigens: a possible link between innate and adaptive immunity.

Innate immunity directs the adaptive immune response by identifying antigens that are associated with infectious agents. Although some microbial antigens can be recognized by innate immune receptors, most cannot, and these require identification by some other means. The introduction of aldehydes into antigens by glycolaldehyde, which can be produced by activated neutrophils reacting with serine, or by the oxidation of an N-linked oligosaccharide with NaIO4, enhances by several orders of magnitude their immunogenicity in mice. The augmented immunogenicity requires the presence of an aldehyde on the antigen, and is not dependent on protein aggregation. An in vitro correlate of augmented immunogenicity is the enhanced presentation of glycolaldehyde-modified antigen to T cells by macrophages and bone marrow-derived dendritic cells. The potential clinical importance of this form of antigen modification is twofold: glycolaldehyde renders a model self antigen immunogenic, and it converts a relatively non-immunogenic malaria antigen, merozoite surface protein-1, into an effective immunogen. Thus, the tagging of antigens by the addition of aldehydes, which may be an innate immune mechanism to facilitate their recognition by the adaptive immune system, may have a role in the genesis of autoimmunity and the development of vaccines.

Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex.

The membrane protein complex CD19/CD21 couples the innate immune recognition of microbial antigens by the complement system to the activation of B cells. CD21 binds the C3d fragment of activated C3 that becomes covalently attached to targets of complement activation, and CD19 co-stimulates signaling through the antigen receptor, membrane immunoglobulin. CD21 is also expressed by follicular dendritic cells and mediates the long-term retention of antigen that is required for the maintenance of memory B cells. Understanding of the biology of this receptor complex has been enriched by analyses of genetically modified mice; these analyses have uncovered roles not only in positive responses to foreign antigens, but also in the development of tolerance to self-antigens. Studies of signal transduction have begun to determine the basis for the coreceptor activities of CD19. The integration of innate and adaptive immune recognition at this molecular site on the B cell guides the appropriate selection of antigen by adaptive immunity and emphasizes the importance of this coreceptor complex.

The complement system and adaptive immunity.

The complement system covalently attaches C3d to microbial antigens which binds to CR2 on B lymphocytes, leading to a markedly enhanced adaptive immune response to that antigen. The enhancement is mediated by the cross-linking of the CR2-CD19 complex to mIg which augments the activation of several intracellular signalling pathways. Two additional receptors of the B lymphocyte, FcgammaRIIB and CD22, have opposing effects when cross-linked to mIg, the former suppressing signalling by recruiting the inositol phosphatase, SHIP, and the latter by activating the phosphotyrosine phosphatase, SHP-1. Two principles emerge from these studies: innate immunity guides the adaptive immune response, and activation of the B lymphocyte is determined by co-receptors which evaluate the biological characteristics of antigen.

Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors.

Despite the recent success of mAb in the treatment of certain malignancies, there is still considerable uncertainty about the mechanism of action of anti-cancer Abs. Here, a panel of rat anti-mouse B cell mAb, including Ab directed at surface IgM Id, CD19, CD22, CD40, CD74, and MHC class II, has been investigated in the treatment of two syngeneic mouse B cell lymphomas, BCL1 and A31. Only three mAb were therapeutically active in vivo, anti-Id, anti-CD19, and anti-CD40. mAb to the other Ags showed little or no therapeutic activity in either model despite giving good levels of surface binding and activity in Ag-dependent cellular cytotoxicity and complement assays, and in some cases inhibiting cell growth in vitro. We conclude that the activity of mAb in vitro does not predict therapeutic performance in vivo. Furthermore, in vivo tracking experiments using fluorescently tagged cells showed that anti-Id and anti-CD40 mAb probably operate via different mechanisms: the anti-Id mAb cause growth arrest that is almost immediate and does not eliminate cells over a period of 5 or 6 days, and the anti-CD40 mAb have a delayed effect that allows tumor to grow normally for 3 days, but then abruptly eradicates lymphoma cells. This work supports the belief that mAb specificity is critical to therapeutic success in lymphoma and that, in addition to any effector-recruiting activity they may possess, in vivo mAb operate via mechanisms that involve cross-linking and signaling of key cellular receptors.

CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav.

CD19 is a coreceptor that amplifies signaling by membrane immunoglobulin (mIg) to promote responses of the B lymphocyte to T-dependent antigens. Vav is a guanine nucleotide exchange factor for the Rho, Rac, Cdc42 family of small GTPases. We found that coligating mIg and CD19 causes a synergistic increase in the tyrosine phosphorylation of CD19. Phosphorylated tyrosine-391 of CD19 binds Vav to mediate a sustained increase in intracellular Ca2+ concentration. This response correlates with activation by the CD19-Vav complex of phosphatidylinositol 4-phosphate 5-kinase for the synthesis of phosphatidylinositol 4,5-bisphosphate. Interaction of CD19 with Vav also mediates the synergistic activation of the mitogen-activated protein kinase JNK. Therefore, CD19 is a membrane adaptor protein that recruits Vav for the activation of lipid and protein kinases.

Inhibition of the B cell by CD22: a requirement for Lyn.

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca2+] is lost. Therefore, Lyn is required for the SHP-1-mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.

Downregulated expression of SHP-1 in Burkitt lymphomas and germinal center B lymphocytes.

We wish to identify developmental changes in germinal center B cells that may contribute to their rapid growth. SHP-1 is an SH2 domain-containing phosphotyrosine phosphatase that negatively regulates activation of B cells and other cells of hematopoietic lineages. We have found that in all 13 EBV-negative and 11 EBV-positive Burkitt lymphomas with a nonlymphoblastoid phenotype, the mean concentration of SHP-1 was reduced to 5% of that of normal B and T cells. The possibility that this diminished expression of SHP-1 was related to the germinal center phenotype of Burkitt lymphomas was supported by the low to absent immunofluorescent staining for SHP-1 in germinal centers, and by the inverse relationship between the concentration of SHP-1 and the expression of the germinal center marker CD38 on purified tonsillar B cells. In CD38-high B cells, SHP-1 concentration was 20% of that of mantle zone B cells from the same donor. This reduction in SHP-1 is comparable to that of cells from motheaten viable mev/mev mice in which there is dysregulated, spontaneous signaling by cytokine and antigen receptors. Therefore, germinal center B cells may have a developmentally regulated, low threshold for cellular activation.