Role of Gonococcal Neisserial Surface Protein A (NspA) in Serum Resistance and Comparison of Its Factor H Binding Properties with Those of Its Meningococcal Counterpart

Neisseria gonorrhoeae, the causative agent of gonorrhea, has evolved several mechanisms to subvert complement, including binding of the complement inhibitor factor H (FH). We previously reported FH binding to N. gonorrhoeae independently of lipooligosaccharide (LOS) sialylation. Here we report that factor H-like protein 1 (FHL-1), which contains FH domains 1 through 7 and possesses complement-inhibitory activity, also binds to N. gonorrhoeae The ligand for both FH and FHL-1 was identified as neisserial surface protein A (NspA), which has previously been identified as a ligand for these molecules on Neisseria meningitidis As with N. meningitidis NspA (Nm-NspA), N. gonorrhoeae NspA (Ng-NspA) bound FH/FHL-1 through FH domains 6 and 7. Binding of FH/FHL-1 to NspA was human specific; the histidine (H) at position 337 of domain 6 contributed to human-specific FH binding to both Ng- and Nm-NspA. FH/FHL-1 bound Nm-NspA better than Ng-NspA; introducing Q at position 73 (loop 2, present in Ng-NspA) or replacing V and D at positions 112 and 113 in Nm-NspA loop 3 with A and H (Ng-NspA), respectively, reduced FH/FHL-1 binding. The converse Ng-NspA to Nm-NspA mutations increased FH/FHL-1 binding. Binding of FH/FHL-1 through domains 6 and 7 to N. gonorrhoeae increased with truncation of the heptose I (HepI) chain of LOS and decreased with LOS sialylation. Loss of NspA significantly decreased serum resistance of N. gonorrhoeae with either wild-type or truncated LOS. This report highlights the role for NspA in enabling N. gonorrhoeae to subvert complement despite LOS phase variation. Knowledge of FH-NspA interactions will inform the design of vaccines and immunotherapies against the global threat of multidrug-resistant gonorrhea

Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA.

BACKGROUND: Trypanosoma cruzi, causative agent of Chagas disease in humans and dogs, is a vector-borne zoonotic protozoan parasite that can cause fatal cardiac disease. While recognized as the most economically important parasitic infection in Latin America, the incidence of Chagas disease in the United States of America (US) may be underreported and even increasing. The extensive genetic diversity of T. cruzi in Latin America is well-documented and likely influences disease progression, severity and treatment efficacy; however, little is known regarding T. cruzi strains endemic to the US. It is therefore important to expand our knowledge on US T. cruzi strains, to improve upon the recognition of and response to locally acquired infections. METHODOLOGY/PRINCIPLE FINDINGS: We conducted a study of T. cruzi molecular diversity in California, augmenting sparse genetic data from southern California and for the first time investigating genetic sequences from northern California. The vector Triatoma protracta was collected from southern (Escondido and Los Angeles) and northern (Vallecito) California regions. Samples were initially screened via sensitive nuclear repetitive DNA and kinetoplast minicircle DNA PCR assays, yielding an overall prevalence of approximately 28% and 55% for southern and northern California regions, respectively. Positive samples were further processed to identify discrete typing units (DTUs), revealing both TcI and TcIV lineages in southern California, but only TcI in northern California. Phylogenetic analyses (targeting COII-ND1, TR and RB19 genes) were performed on a subset of positive samples to compare Californian T. cruzi samples to strains from other US regions and Latin America. Results indicated that within the TcI DTU, California sequences were similar to those from the southeastern US, as well as to several isolates from Latin America responsible for causing Chagas disease in humans. CONCLUSIONS/SIGNIFICANCE: Triatoma protracta populations in California are frequently infected with T. cruzi. Our data extend the northern limits of the range of TcI and identify a novel genetic exchange event between TcI and TcIV. High similarity between sequences from California and specific Latin American strains indicates US strains may be equally capable of causing human disease. Additional genetic characterization of Californian and other US T. cruzi strains is recommended

Characterization of a monothiol glutaredoxin encoded by Chlorella virus PBCV-1

Annotation of the 330-kb Chlorella virus PBCV-1 genome identified a 237 nucleotide gene (a438l) that codes for a protein with ~35% amino acid identity to glutaredoxins (Grx) found in other organisms. The PBCV-1 protein resembles classical Grxs in both size (9 kDa) and location of the active site (N-terminus). However, the PBCV-1 Grx is unusual because it contains a monothiol active site (CPYS) rather than the typical dithiol active site (CPYC). To examine this unique active site, four sitespecific mutants (CPYC, CPYA, SPYC, and SPYS) were constructed to determine if the N-terminal cysteine is necessary for enzyme activity. Wild type and both mutants containing N-terminal cysteines catalyzed the reduction of disulfides in a coupled system with GSH, NADPH, and glutathione reductase. However, both mutants with an altered N-terminal cysteine were inactive. The grx gene is common in the Chlorella viruses. Molecular phylogenetic analyses of the PBCV-1 enzyme support its relatedness to those from other Chlorella viruses and yet demonstrate the divergence of the Grx molecule

Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis

The identification of factor H binding protein (fHbp)-null invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines. IMPORTANCE The widespread use of antimeningococcal vaccines based on factor H (fH) binding protein (fHbp) is imminent. Meningococci that lack fHbp were recently isolated from persons with invasive disease, and these fHbp-null strains could spawn vaccine failure. Our report provides a molecular basis for an explanation of how fHbp-null strains may evade the host immune system. Meningococci possess several mechanisms to subvert killing by the alternative pathway (AP) of complement, including production of the fHbp and NspA fH binding proteins. Here we show that a meningococcal protein called porin B2 (PorB2) contributes to inhibition of the AP on the bacterial surface. A majority of the fHbp-null isolates identified, as well as all members of a hypervirulent lineage (called ST-11), express PorB2. Our findings highlight the potential for the emergence of fHbp-negative strains that are able to regulate the AP and may be associated with fHbp vaccine failure

The Menninger Clinic

The authors propose that clinicians endeavor to differentiate between reversible and irreversible memory failures in patients with dissociative symptoms who report "memory gaps" and "lost time," The classic dissociative disorders, such as dissociative amnesia and dissociative identity disorder, entail reversible memory failures associated with encoding experience in altered states. The authors propose another realm of memory failures associated with severe dissociative detachment that may preclude the level of encoding of ongoing experience needed to support durable autobiographical memories. They describe how dissociative detachment may be intertwined with neurobiological factors that impair memory, and they spell out the significance of distinguishing reversible and irreversible memory impairment for diagnosis, patient education, psychotherapy, and research. Copyright © 1999 by W.B. Saunders Company M ANY PATIENTS with a severe childhood trauma history and dissociative disorders complain of gaps in their memory. These gaps include an inability to remember specific traumatic events in the past and a lack of memory for substantial periods of childhood. Many such patients also report ongoing memory lapses, for example, not remembering what they have said, what they have done, or where they have been. They complain that they have "lost time"--they cannot account for blocks of their day. These ongoing memory disturbances and the associated loss of continuity in experience profoundly disrupt their functioning. Clinicians treating these patients often conclude that the memory for the events transpiring during the "lost time" exists but is not currently accessible, and/or that complex activity during the time interval for which the patient is amnestic is evidence of dissociative identity disorder. Recent research on memory and the neurophysiology of trauma suggest that neither of these conclusions is invariably warranted. In the context of conducting diagnostic evaluation and treatment of patients with severe and chronic trauma-related disorders, we have endeavored to understand the basis of memory gaps and lost time. We propose that it is useful in principle, although not easy in practice, to distinguish between potentially reversible and relatively irreversible memory gaps. The diagnostic classification of dissociation includes three disorders that entail potentially reversible memory impairment: dissociative amnesia, fugue, and dissociative identity disorder. However, on the basis of routine clinical experience, we believe that another reason for irreversible memory impairment in patients with posttraumatic disorders is a failure to encode ongoing experience associated with severe dissociative detachment. To set the stage for our argument that encoding failures associated with dissociative detachment may contribute to memory impairment, we begin by delineating crucial distinctions among the types and stages of memory. After briefly reviewing the three dissociative disorders associated with reversible amnesia, we describe different degrees of dissociative detachment and their potential relation to irreversible memory impairment. We also review evidence suggesting that memory impairment associated with dissociative detachment may be intertwined with neurophysiological concomitants of stress. We conclude by defining the implications for clinical practice and research. To preview our argument, we propose that evidence of complex actions in dissociative states is not necessarily diagnostic of dissociative identity disorder, and that memory gaps are not necessarily reversible by various memory retrieval techniques. We do not advocate that clinicians aspire to answer definitively the question, "Reversible amnesia or encoding failure?" Instead, we propose that they ask the question. TYPES AND STAGES OF MEMORY Declarative Versus Nondeclarative Memory The distinction between nondeclarative and declarative memory is one of the most robust in memory research. 1-2 Nondeclarative (implicit) memory is a kind of procedural or "habit memory,&quot

Asymmetric cyclopropanation of conjugated cyanosulfones using a novel cupreine organocatalyst: rapid access to d3-amino acids

An organocatalytic asymmetric synthesis of a novel, highly functionalised cyclopropane system furnished with versatile substituents and containing a quaternary centre is described. The process utilises a new bifunctional catalyst based on the cinchona alkaloid framework and the products made using this catalyst were obtained as single diastereoisomers, with very high enantioselectivities (up to 96% ee). We have also demonstrated that these resulting cyclopropanes are very useful synthetic intermediates to interesting products, such as the difficult to access d3-amino acids

Editor's Choice - Bypass versus Angioplasty for Severe Ischaemia of the Leg (BASIL) Prospective Cohort Study and the Generalisability of the BASIL-2 Randomised Controlled Trial

OBJECTIVE: The Bypass versus Angioplasty in Severe Ischaemia of the Leg-2 (BASIL-2) randomised controlled trial has shown that, for patients with chronic limb threatening ischaemia (CLTI) who require an infrapopliteal (IP) revascularisation a vein bypass (VB) first revascularisation strategy led to a 35% increased risk of major amputation or death when compared with a best endovascular treatment (BET) first revascularisation strategy. The study aims are to place the BASIL-2 trial within the context of the CLTI patient population as a whole and to investigate the generalisability of the BASIL-2 outcome data.METHODS: This was an observational, single centre prospective cohort study. Between 24 June 2014 and 31 July 2018, the BASIL Prospective Cohort Study (PCS) was performed which used BASIL-2 trial case record forms to document the characteristics, initial and subsequent management, and outcomes of 471 consecutive CLTI patients admitted to an academic vascular centre. Ethical approval was obtained, and all patients provided fully informed written consent. Follow up data were censored on 14 December 2022.RESULTS: Of the 238 patients who required an infrainguinal revascularisation, 75 (32%) had either IP bypass (39 patients) or IP BET (36 patients) outside BASIL-2. Seventeen patients were initially randomised to BASIL-2. A further three patients who did not have an IP revascularisation as their initial management were later randomised in BASIL-2. Therefore, 95/471 (20%) of patients had IP revascularisation (16% outside, 4% inside BASIL-2). Differences in amputation free survival, overall survival, and limb salvage between IP bypass and IP BET performed outside BASIL-2 were not subject to hypothesis testing due to the small sample size. Reasons for non-randomisation into the trial were numerous, but often due to anatomical and technical considerations.CONCLUSION: CLTI patients who required an IP revascularisation procedure and were subsequently randomised into BASIL-2 accounted for a small subset of the CLTI population as a whole. For a wide range of patient, limb, anatomical and operational reasons, most patients in this cohort were deemed unsuitable for randomisation in BASIL-2. The results of BASIL-2 should be interpreted in this context.</p

Understanding Cytotoxicity and Cytostaticity in a High-Throughput Screening Collection.

While mechanisms of cytotoxicity and cytostaticity have been studied extensively from the biological side, relatively little is currently understood regarding areas of chemical space leading to cytotoxicity and cytostasis in large compound collections. Predicting and rationalizing potential adverse mechanism-of-actions (MoAs) of small molecules is however crucial for screening library design, given the link of even low level cytotoxicity and adverse events observed in man. In this study, we analyzed results from a cell-based cytotoxicity screening cascade, comprising 296 970 nontoxic, 5784 cytotoxic and cytostatic, and 2327 cytostatic-only compounds evaluated on the THP-1 cell-line. We employed an in silico MoA analysis protocol, utilizing 9.5 million active and 602 million inactive bioactivity points to generate target predictions, annotate predicted targets with pathways, and calculate enrichment metrics to highlight targets and pathways. Predictions identify known mechanisms for the top ranking targets and pathways for both phenotypes after review and indicate that while processes involved in cytotoxicity versus cytostaticity seem to overlap, differences between both phenotypes seem to exist to some extent. Cytotoxic predictions highlight many kinases, including the potentially novel cytotoxicity-related target STK32C, while cytostatic predictions outline targets linked with response to DNA damage, metabolism, and cytoskeletal machinery. Fragment analysis was also employed to generate a library of toxicophores to improve general understanding of the chemical features driving toxicity. We highlight substructures with potential kinase-dependent and kinase-independent mechanisms of toxicity. We also trained a cytotoxic classification model on proprietary and public compound readouts, and prospectively validated these on 988 novel compounds comprising difficult and trivial testing instances, to establish the applicability domain of models. The proprietary model performed with precision and recall scores of 77.9% and 83.8%, respectively. The MoA results and top ranking substructures with accompanying MoA predictions are available as a platform to assess screening collections.Biotechnology and Biological Sciences Research Council, AstraZenec

Identity-by-descent estimation with population- and pedigree-based imputation in admixed family data

Background: In the past few years, imputation approaches have been mainly used in population-based designs of genome-wide association studies, although both family- and population-based imputation methods have been proposed. With the recent surge of family-based designs, family-based imputation has become more important. Imputation methods for both designs are based on identity-by-descent (IBD) information. Apart from imputation, the use of IBD information is also common for several types of genetic analysis, including pedigree-based linkage analysis. Methods: We compared the performance of several family- and population-based imputation methods in large pedigrees provided by Genetic Analysis Workshop 19 (GAW19). We also evaluated the performance of a new IBD mapping approach that we propose, which combines IBD information from known pedigrees with information from unrelated individuals. Results: Different combinations of the imputation methods have varied imputation accuracies. Moreover, we showed gains from the use of both known pedigrees and unrelated individuals with our IBD mapping approach over the use of known pedigrees only. Conclusions: Our results represent accuracies of different combinations of imputation methods that may be useful for data sets similar to the GAW19 pedigree data. Our IBD mapping approach, which uses both known pedigree and unrelated individuals, performed better than classical linkage analysis