PEGylated Domain I of Beta-2-Glycoprotein I Inhibits Thrombosis in a Chronic Mouse Model of the Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

\ua9 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-l5(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine

Improving the normalization of complex interventions: measure development based on normalization process theory (NoMAD): study protocol

<b>Background</b> Understanding implementation processes is key to ensuring that complex interventions in healthcare are taken up in practice and thus maximize intended benefits for service provision and (ultimately) care to patients. Normalization Process Theory (NPT) provides a framework for understanding how a new intervention becomes part of normal practice. This study aims to develop and validate simple generic tools derived from NPT, to be used to improve the implementation of complex healthcare interventions.<p></p> <b>Objectives</b> The objectives of this study are to: develop a set of NPT-based measures and formatively evaluate their use for identifying implementation problems and monitoring progress; conduct preliminary evaluation of these measures across a range of interventions and contexts, and identify factors that affect this process; explore the utility of these measures for predicting outcomes; and develop an online users’ manual for the measures.<p></p> <b>Methods</b> A combination of qualitative (workshops, item development, user feedback, cognitive interviews) and quantitative (survey) methods will be used to develop NPT measures, and test the utility of the measures in six healthcare intervention settings.<p></p> <b>Discussion</b> The measures developed in the study will be available for use by those involved in planning, implementing, and evaluating complex interventions in healthcare and have the potential to enhance the chances of their implementation, leading to sustained changes in working practices

Targeted Disruption of the Low-Affinity Leukemia Inhibitory Factor-Receptor Gene Causes Placental, Skeletal, Neural and Metabolic Defects and Results in Perinatal Death

The low-affinity receptor for leukemia inhibitory factor (LIFR)* interacts with gp130 to induce an intracellular signal cascade, The LIFR-gp130 heterodimer is implicated in the function of diverse systems, Normal placentation is disrupted in LIFR mutant animals, which leads to poor intrauterine nutrition but allows fetuses to continue to term. Fetal bone volume is reduced greater than three-fold and the number of osteoclasts is increased six-fold, resulting in severe osteopenia of perinatal bone. Astrocyte numbers are reduced in the spinal cord and brain stem. Late gestation fetal livers contain relatively high stores of glycogen, indicating a metabolic disorder. Hematologic and primordial germ cell compartments appear normal. Pleiotropic defects in the mutant animals preclude survival beyond the day of birth

Bim and Bmf synergize to induce apoptosis in Neisseria gonorrhoeae infection

Abstract: Bcl-2 family proteins including the pro-apoptotic BH3-only proteins are central regulators of apoptotic cell death. Here we show by a focused siRNA miniscreen that the synergistic action of the BH3-only proteins Bim and Bmf is required for apoptosis induced by infection with Neisseria gonorrhoeae (Ngo). While Bim and Bmf were associated with the cytoskeleton of healthy cells, they both were released upon Ngo infection. Loss of Bim and Bmf from the cytoskeleton fraction required the activation of Jun-N-terminal kinase-1 (JNK-1), which in turn depended on Rac-1. Depletion and inhibition of Rac-1, JNK-1, Bim, or Bmf prevented the activation of Bak and Bax and the subsequent activation of caspases. Apoptosis could be reconstituted in Bim-depleted and Bmf-depleted cells by additional silencing of antiapoptotic Mcl-1 and Bcl-XL, respectively. Our data indicate a synergistic role for both cytoskeletal-associated BH3-only proteins, Bim, and Bmf, in an apoptotic pathway leading to the clearance of Ngo-infected cells. Author Summary: A variety of physiological death signals, as well as pathological insults, trigger apoptosis, a genetically programmed form of cell death. Pathogens often induce host cell apoptosis to establish a successful infection. Neisseria gonorrhoeae (Ngo), the etiological agent of the sexually transmitted disease gonorrhoea, is a highly adapted obligate human-specific pathogen and has been shown to induce apoptosis in infected cells. Here we unveil the molecular mechanisms leading to apoptosis of infected cells. We show that Ngo-mediated apoptosis requires a special subset of proapoptotic proteins from the group of BH3-only proteins. BH3-only proteins act as stress sensors to translate toxic environmental signals to the initiation of apoptosis. In a siRNA-based miniscreen, we found Bim and Bmf, BH3-only proteins associated with the cytoskeleton, necessary to induce host cell apoptosis upon infection. Bim and Bmf inactivated different inhibitors of apoptosis and thereby induced cell death in response to infection. Our data unveil a novel pathway of infection-induced apoptosis that enhances our understanding of the mechanism by which BH3-only proteins control apoptotic cell death

Acceleration of generalized hypergeometric functions through precise remainder asymptotics

We express the asymptotics of the remainders of the partial sums {s_n} of the generalized hypergeometric function q+1_F_q through an inverse power series z^n n^l \sum_k c_k/n^k, where the exponent l and the asymptotic coefficients {c_k} may be recursively computed to any desired order from the hypergeometric parameters and argument. From this we derive a new series acceleration technique that can be applied to any such function, even with complex parameters and at the branch point z=1. For moderate parameters (up to approximately ten) a C implementation at fixed precision is very effective at computing these functions; for larger parameters an implementation in higher than machine precision would be needed. Even for larger parameters, however, our C implementation is able to correctly determine whether or not it has converged; and when it converges, its estimate of its error is accurate.Comment: 36 pages, 6 figures, LaTeX2e. Fixed sign error in Eq. (2.28), added several references, added comparison to other methods, and added discussion of recursion stabilit

Cervico-vaginal immunoglobulin g levels increase post-ovulation independently of neutrophils

The prevalence of sexually transmitted infections (STIs) is often higher in females than in males. Although the reproductive cycle profoundly modulates local immunity in the female reproductive tract (FRT) system, significant gaps in our knowledge of the immunobiology of the FRT still exist. An intriguing and frequently observed characteristic of the FRT is the predominant presence of immunoglobulin (Ig) G in cervico-vaginal secretions. We show here that in the mouse, IgG accumulation was enhanced approximately 5-fold post-ovulation, and was accompanied by an influx of neutrophils into the FRT. To determine whether these two events were causally related, we performed short-term neutrophil depletion experiments at individual stages throughout the estrous cycle. Our results demonstrate that neutrophils were not necessary for cycle-dependent tissue remodeling and cycle progression and that cycle-dependent IgG accumulation occurred independent of neutrophils. We thus conclude that neutrophil influx and IgG accumulation are independent events that occur in the FRT during the reproductive cycle

The Impact of Global Warming and Anoxia on Marine Benthic Community Dynamics: an Example from the Toarcian (Early Jurassic)

The Pliensbachian-Toarcian (Early Jurassic) fossil record is an archive of natural data of benthic community response to global warming and marine long-term hypoxia and anoxia. In the early Toarcian mean temperatures increased by the same order of magnitude as that predicted for the near future; laminated, organic-rich, black shales were deposited in many shallow water epicontinental basins; and a biotic crisis occurred in the marine realm, with the extinction of approximately 5% of families and 26% of genera. High-resolution quantitative abundance data of benthic invertebrates were collected from the Cleveland Basin (North Yorkshire, UK), and analysed with multivariate statistical methods to detect how the fauna responded to environmental changes during the early Toarcian. Twelve biofacies were identified. Their changes through time closely resemble the pattern of faunal degradation and recovery observed in modern habitats affected by anoxia. All four successional stages of community structure recorded in modern studies are recognised in the fossil data (i.e. Stage III: climax; II: transitional; I: pioneer; 0: highly disturbed). Two main faunal turnover events occurred: (i) at the onset of anoxia, with the extinction of most benthic species and the survival of a few adapted to thrive in low-oxygen conditions (Stages I to 0) and (ii) in the recovery, when newly evolved species colonized the re-oxygenated soft sediments and the path of recovery did not retrace of pattern of ecological degradation (Stages I to II). The ordination of samples coupled with sedimentological and palaeotemperature proxy data indicate that the onset of anoxia and the extinction horizon coincide with both a rise in temperature and sea level. Our study of how faunal associations co-vary with long and short term sea level and temperature changes has implications for predicting the long-term effects of “dead zones” in modern oceans