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Cynthia R Collins School of Nursing, College of Social Sciences, Loyola University, New Orleans, LA, USA Abstract: Communication and decision making in the health care workplace often involve finding solutions to ill-structured problems in uncertain, dynamic environments influenced by the competing interests of multiple stakeholders. In this environment, doctoral-prepared nurses who practice as administrators, policy makers, or advanced practice practitioners are often compelled to make important decisions based upon evaluating the merit of colleagues’ proposals against some desired organizational or population outcome. Of equal importance is the nurse leader’s own capacity to construct a compelling argument or proposal that will drive the organization forward to meet the evolving needs for quality health care. Where do we learn the skills necessary to foster this kind of critical thinking in our professional communications? The author suggests that one teaching–learning approach can be found through the thoughtful application of the work of British philosopher Steven Toulmin. Toulmin defined a model for both the analysis and derivation of logical arguments or proposals that can be readily learned and applied for use in health care systems. This model posits that a substantive argument or claim can be evaluated based on the assumptions it presumes (warrants) and the strength of the evidence base (backing). Several of the social science professions have adapted Toulmin’s model to generate analysis and creative solutions to complex or emergent problems. The author proposes that an application of this model be included in the pedagogy of doctoral level Philosophy of Science or Nursing Theory courses. The Toulmin process often provides the doctoral student or novice researcher with their first real learning experience in defining the scope and inherent challenges of framing a clinical issue to be the focus of their scholarly translational projects. Several illustrations, which were eventually researched and further developed for capstone scholarly projects, are presented as exemplars of this process. Keywords: Toulmin, argument, research process, hypothesis formation, nursing education

Cynthia R Collins Loyola University New Orleans, College of Social Sciences/School of Nursing, New Orleans, LA, USA Abstract: Emerging practice research on filial sources of health care support has indicated that there is a growing trend for sons to assume some responsibility for the health care needs of their aging parents. The purpose of this work is to propose that outcomes observed through a secondary analysis of data from a previous mixed methods research project, conducted with a sample of 60 elderly women residing in independent living centers, supports this concept in elder care. The present study is a retrospective interpretation utilizing the original database to examine the new question, “What specific roles do sons play in caregiving of their elderly mothers?” While daughters presently continue to emerge in existing health care studies as the primary care provider, there is a significant pattern in these data for older patients to depend upon sons for a variety of instrumental activities of daily living. As the baby-boomers age, there is more of cohort trend for their families to be smaller, adult daughters to be employed, and for adult children to be more geographically mobile. These factors may combine to make health care support networks more limited for the current aging population, challenging the elderly and their health care providers to revisit the cultural gender norms that are used to identify caregivers. Keywords: sons as caregivers, male caregivers, aging parents, filial support, informal caregivers

Malaria pathogenesis and parasite multiplication depend on the ability of Plasmodium merozoites to invade human erythrocytes. Invasion is a complex multi-step process involving multiple parasite proteins which can differ between species and has been most extensively studied in P. falciparum. However, dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed P. falciparum merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified strains including some generated specifically for this work, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment interactions. Conditional deletion of the major P. falciparum merozoite surface protein PfMSP1, long thought to play a central role in initial attachment, had no impact on the force needed to pull merozoites and erythrocytes apart, whereas interventions that disrupted the function of several members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families did have a significant negative impact on attachment. Deletion of individual PfEBA and PfRH ligands reinforced the known redundancy within these families, with the deletion of some ligands impacting detachment force while others did not. By comparing over 4000 individual merozoite-erythrocyte interactions in a range of conditions and strains, we establish that the PfEBA/PfRH families play a central role in P. falciparum merozoite attachment, not the major merozoite surface protein PfMSP1., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kals et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P 3 amino acid side chains as well as N -capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P 1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Unrecognized central venous catheter (CVC) infiltration is an uncommon but potentially life-threatening complication. For instance, a malpositioned subclavian line can infuse into the mediastinum, pleural cavity, or interstitial space of the neck. We present the case of a 30-year-old male with gunshot wounds to the right chest, resuscitated with an initially functional left subclavian CVC, which later infiltrated into the neck causing compression of the carotid sinus and consequent bradycardic arrest. Return of spontaneous circulation (ROSC) was achieved following intravenous epinephrine, cardiac massage, and emergency neck exploration and cervical fasciotomy. Our case highlights the importance of frequent reassessment of lines, especially those placed during fast-paced, high-intensity clinical situations. We recommend being mindful when using rapid transfusion devices as an interstitial catheter may not mount enough back pressure to trigger the system's alarm before significant tissue damage or compartment syndrome occurs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

Generations of scholars and activists have argued that racial inequities emerge not only because of racist ideologies but also from a hierarchical system of racial oppression. This theoretical tradition has highlighted numerous ways in which systemic racism manifests itself, from racist policies to differential access to material conditions and power. However, given that by definition systemic racism is focused on systems, theories of systemic racism would be more comprehensive and actionable by drawing on scholarship related to systems thinking. Systems thinking is a conceptual orientation that aims to understand how different types of systems function over time. This paper builds on the work of previous scholars to propose a systems thinking approach to understand and strategically disrupt racist systems. We provide a typology of system characteristics (organized into the categories of paradigms, structures, elements, and feedback loops) that together can be used to help understand the operation of systemic racism in different system contexts. The paper also provides an approach to identify and strategically target multiple system leverage points to simultaneously disrupt the status quo of racial inequity and promote the emergence of conditions enabling racial equity. This systems thinking approach can be used to guide learning and action within an ongoing process of antiracist praxis., (© 2022 Society for Community Research and Action.)

As White activists are growing the racial justice movement, their antiracism frequently disrupts the racial hierarchy, which features whiteness in a dominant role, especially in interpersonal relationships. We investigate how White antiracists disrupt whiteness in interpersonal relationships. We interviewed 16 White antiracists who had experienced significant relationship distance due to their antiracism. We conducted thematic analyses to understand the conflicts that emerged between antiracists and their White counterparts as activists challenged White racial dominance. Antiracists disrupted whiteness by exercising social power to punish racist offenders. In response, their White counterparts resisted these challenges by exerting their instruments of power to sanction antiracists. The conflict with White people led antiracists to build greater personal and social capacity for antiracist activism. This study illustrates how conflicts can emerge during social change efforts even at the microlevel as parties exercise power to contest or support the status quo., (© 2022 Wiley Periodicals LLC.)

Background: As healthcare costs rise, there is an increasing emphasis on alternative payment models to improve care efficiency. The bundled payment represents an alternative reimbursement model gaining popularity within the surgical sphere. We aimed to assess where the largest opportunities for care improvement lay and how best to identify patients at high risk of suffering costly complications., Methods: We utilized itemized CMS claims data for a retrospective cohort of patients between 2014 and 2016 who met inclusion criteria for the Major Bowel Bundled Payment Program and performed a cost analysis to identify opportunities for improved care efficiency. Based on the results of this cost analysis, we identified readmissions as a target for improvement. We then assessed whether the American College of Surgeons' National Surgical Quality Improvement Program surgical risk calculator (ACS NSQIP SRC) could accurately identify patients within our bundled payment population who were at high risk of readmission using a logistic regression model., Results: Our study cohort included 252 patients. Readmissions accounted for 12.8% of the average total care episode cost with a coefficient of variation of 2.72, thereby representing the most substantial, non-fixed cost for our bundled payment patients. Patients readmitted within their 90-day care episode were 2.53 times more likely to be high-cost (>$60,000) than patients not readmitted. However, the ACS NSQIP SRC did not accurately predict patients at high risk of readmission within the first 30 days with an AUROC of 0.58., Conclusions: Our study highlights the importance of reducing readmissions as a central component of improving care for bowel surgery bundled payment patients. Preventing such readmissions requires accurate identification of patients at high risk of readmission; however, current risk prediction models lack the adaptability necessary for this task., (© 2022. The Author(s).)

Community psychology is expressly concerned with social justice. Such concern necessitates attention to race. Yet, nearly absent from the field's literature is explicit and critical attention to whiteness. Thus, community psychology's contribution to promoting social justice remains incomplete. In this article, we examine how a critical construction of whiteness can be useful for community research and action. After a brief history of the construction of whiteness in the United States, and a summary of key insights from critical whiteness studies, we present a scoping review of the nascent body of community psychology literature that addresses whiteness. That work implicates whiteness in the emergence of the field itself, frames whiteness as social location, problematizes whiteness, addresses White supremacy and institutional racism, interrogates White privilege, and employs whiteness as a theoretical standpoint. We conclude with three propositions for scholars to broker the relationship between community psychology and critical whiteness studies: (a) community psychology should become more critically conscious of whiteness, (b) community psychologists should promote critical awareness of the ways that whiteness operates as a complex system, and (c) greater critical awareness of whiteness should be applied to the development of multilevel interventions aimed at dismantling whiteness as a system of domination., (© 2020 Society for Community Research and Action.)

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent P falciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Objective: We aimed to compare general surgery emergency (GSE) volume, demographics and disease severity before and during COVID-19., Background: Presentations to the emergency department (ED) for GSEs fell during the early COVID-19 pandemic. Barriers to accessing care may be heightened, especially for vulnerable populations, and patients delaying care raises public health concerns., Methods: We included adult patients with ED presentations for potential GSEs at a single quaternary-care hospital from January 2018 to August 2020. To compare GSE volumes in total and by subgroup, an interrupted time-series analysis was performed using the March shelter-in-place order as the start of the COVID-19 period. Bivariate analysis was used to compare demographics and disease severity., Results: 3255 patients (28/week) presented with potential GSEs before COVID-19, while 546 (23/week) presented during COVID-19. When shelter-in-place started, presentations fell by 8.7/week (31%) from the previous week (p<0.001), driven by decreases in peritonitis (β=-2.76, p=0.017) and gallbladder disease (β=-2.91, p=0.016). During COVID-19, patients were younger (54 vs 57, p=0.001), more often privately insured (44% vs 38%, p=0.044), and fewer required interpreters (12% vs 15%, p<0.001). Fewer patients presented with sepsis during the pandemic (15% vs 20%, p=0.009) and the average severity of illness decreased (p<0.001). Length of stay was shorter during the COVID-19 period (3.91 vs 5.50 days, p<0.001)., Conclusions: GSE volumes and severity fell during the pandemic. Patients presenting during the pandemic were less likely to be elderly, publicly insured and have limited English proficiency, potentially exacerbating underlying health disparities and highlighting the need to improve care access for these patients., Level of Evidence: III., Competing Interests: Competing interests: TB is funded by the Agency for Healthcare Research and Quality (AHRQ) (K12HS026383). JPR receives funding from Merck & Co. for unrelated research., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null merozoites with RBCs leads to either abortive invasion with rapid RBC lysis, or successful entry but developmental arrest. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for host RBC membrane sealing following parasite internalisation and for correct functioning of merozoite surface proteins., Competing Interests: CC, FH, SH, AS, MR, LC, MB No competing interests declared, (© 2020, Collins et al.)

As part of a larger research project, 274 skeletons from three medieval Icelandic sites were evaluated for signs of infectious disease and 32 were found to have lesions at least consistent with a diagnosis of tuberculosis (TB): eight non-adults ranging in age from infancy to up to 17 years of age, and 24 adults. A higher proportion of individuals from Skeljastaðir and Keldudalur were affected than at Hofstaðir, an observation which may be compatible with Hofstaðir's higher status. A higher number of male skeletons overall (n. 17) than female skeletons (n. 8) exhibited pathological change. The sample is unique for its high numbers of well-preserved infants, and the appearance of TB in children is indicative of continual transmission in a community. The changes recorded in infant remains are marked by destruction and minimal periosteal new bone formation, while one adult skeleton exhibits the classic sign of Pott's disease. Other signs on the skeletons include evidence for past lymphadenitis and iliopsoas (cold) abscess. These cases indicate that TB was likely introduced to Iceland soon after the settlement period and became endemic in different regions from at least the late 10 th - mid 13 th centuries.

In recent years, there has been a robust racial justice movement in the United States, which has pursued power with the goal of promoting wellness and liberating people from racially and historically oppressed communities. Organizations such as Black Lives Matter and Showing Up for Racial Justice continue building power and promoting psychological and political liberation. The purpose of our study is to investigate the developmental processes by which anti-racist activists resist psychological and political oppression to approach wellness and liberation. We conducted 24 interviews from self-identified anti-racist activists in the United States and utilized thematic analysis to construct qualitative codes to identify the psychological and political strategies activists implemented in their racial justice work. We found that activists adopted four psychological strategies, two political strategies, and two bridging strategies to resist oppression. Psychologically, activists tended to examine political and historical contexts to understand the root causes of oppression and how their own oppressed and privileged identities fit within those larger systems. Politically, activists sought opportunities to enhance their capacity as activists and engage in critical actions to build power and seek liberation. Bridging these psychological and political domains, activists also formulated a theory of liberation and engaged in critical self-reflection, which guided their political actions., (© 2019 Wiley Periodicals, Inc.)

Background: Within the United States, surgical site infections (SSIs) have now become the most common hospital-acquired infection and impact 2%-5% of all surgical patients. It is estimated that approximately 60% of SSIs could be prevented through improved adherence to pre-existing practice guidelines. Methods: The myriad of contributing factors leading to SSIs highlights the need for a multi-faceted approach. Although collaboration and coordination among providers and patients represents a requirement of any sustainable solution, it also creates a space and possible role for innovative technologies and mobile applications utilizing patient-generated health data (PGHD). Results: Upon analysis of hospital practice, we have identified substantial variability in documentation, peri-operative care, and post-discharge instruction with regard to SSI prevention and incision care techniques. This variability is further exacerbated by a loss of information within each transition of patient care. As a result, a patient's risk of SSI often becomes dictated by their provider's preferred (and sometimes arbitrary) peri-operative practices and their own initiative in following poorly explained pre-operative instructions. The quality and efficiency of any subsequent SSI treatment similarly rests on a seemingly inconsistent approach with poor patient instruction for the post-discharge setting. Conclusions: Surgical site infection risk can be mitigated successfully through reliable performance of several evidence-based process measures within the operating room, which are now at the guideline level. However, optimal performance only happens when teams and patients are aligned and truly believe both that the evidence is correct, and that SSIs are preventable. The journey toward this goal will be an iterative process that may take months to years. Although technology can be complementary, it cannot replace human passion for harm prevention.

Objective: To test whether symptom generation in infants is related to the severity of gastroesophageal reflux disease (GERD) symptoms as determined by the Acid Reflux Index (ARI), stimulus media, and stimulus volume during provocative esophageal manometry., Study Design: Symptomatic neonates (n = 74) born at a median of 28.9 weeks gestation (range, 23.4-39.4 weeks) were studied at a median of 41.0 weeks (range, 34.6-48.0 weeks) postmenstrual age using 24-hour pH-impedance methods to determine ARI severity, followed by provocative esophageal manometry with graded mid-esophageal infusions (0.1-5.0 mL) of air, water, and apple juice. Peristaltic reflexes and symptom characteristics were compared among ARI severity categories using linear mixed models and generalized estimating equations., Results: The effects of 2635 separate esophageal stimuli on reflexes and symptoms were analyzed. Peristaltic reflexes occurred in 1880 infusions (71%), and physical, cardiorespiratory, sensory symptoms were seen in 439 infusions (17%). Symptom prevalence did not differ across the ARI severity categories (ARI <3, 18%; ARI 3-7, 17%; ARI >7, 16%; P = 1.0). Symptom and peristaltic responses increased with incremental stimulus volumes (all media, P < .001)., Conclusions: Symptoms and peristaltic reflexes are manifestations of the recruitment of several neurosensory and neuromotor pathways evoked by mid-esophageal infusions. ARI severity grade plays no role in symptom generation, indicating that GERD should not be diagnosed and severity should not be assigned based on symptoms alone. An increase in symptom occurrence was noted with increasing stimulus volumes, which provided increased activation of receptors, afferents, and efferents in evoking peristaltic clearance reflexes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway., Competing Interests: The authors have declared that no competing interests exist.

Apicomplexa are obligate intracellular parasites that actively invade, replicate within, and egress from host cells. The parasite actinomyosin-based molecular motor complex (often referred to as the glideosome) is considered an important mediator of parasite motility and virulence. Mature intracellular parasites often become motile just prior to egress from their host cells, and in some genera, this motility is important for successful egress as well as for subsequent invasion of new host cells. To determine whether actinomyosin-based motility is important in the red blood cell egress and invasion activities of the malaria parasite, we have used a conditional genetic approach to delete GAP45 , a primary component of the glideosome, in asexual blood stages of Plasmodium falciparum Our results confirm the essential nature of GAP45 for invasion but show that P. falciparum does not require a functional motor complex to undergo egress from the red blood cell. Malarial egress therefore differs fundamentally from induced egress in the related apicomplexan Toxoplasma gondii IMPORTANCE Clinical malaria results from cycles of replication of single-celled parasites of the genus Plasmodium in red blood cells. Intracellular parasite replication is followed by a highly regulated, protease-dependent process called egress, in which rupture of the bounding membranes allows explosive release of daughter merozoites which rapidly invade fresh red cells. A parasite actinomyosin-based molecular motor (the glideosome) has been proposed to provide the mechanical force to drive invasion. Studies of the related parasite Toxoplasma gondii have shown that induced egress requires parasite motility, mediated by a functional glideosome. However, whether the glideosome has a similar essential role in egress of malaria merozoites from red blood cells is unknown. Here, we show that although a functional glideosome is required for red blood cell invasion by Plasmodium falciparum merozoites, it is not required for egress. These findings place further emphasis on the key role of the protease cascade in malarial egress., (Copyright © 2018 Perrin et al.)

Two different classes of molecular representations for use in machine learning of thermodynamic and electronic properties are studied. The representations are evaluated by monitoring the performance of linear and kernel ridge regression models on well-studied data sets of small organic molecules. One class of representations studied here counts the occurrence of bonding patterns in the molecule. These require only the connectivity of atoms in the molecule as may be obtained from a line diagram or a SMILES string. The second class utilizes the three-dimensional structure of the molecule. These include the Coulomb matrix and Bag of Bonds, which list the inter-atomic distances present in the molecule, and Encoded Bonds, which encode such lists into a feature vector whose length is independent of molecular size. Encoded Bonds' features introduced here have the advantage of leading to models that may be trained on smaller molecules and then used successfully on larger molecules. A wide range of feature sets are constructed by selecting, at each rank, either a graph or geometry-based feature. Here, rank refers to the number of atoms involved in the feature, e.g., atom counts are rank 1, while Encoded Bonds are rank 2. For atomization energies in the QM7 data set, the best graph-based feature set gives a mean absolute error of 3.4 kcal/mol. Inclusion of 3D geometry substantially enhances the performance, with Encoded Bonds giving 2.4 kcal/mol, when used alone, and 1.19 kcal/mol, when combined with graph features.

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Egress of the malaria parasite Plasmodium falciparum from its host red blood cell is a rapid, highly regulated event that is essential for maintenance and completion of the parasite life cycle. Egress is protease-dependent and is temporally associated with extensive proteolytic modification of parasite proteins, including a family of papain-like proteins called SERA that are expressed in the parasite parasitophorous vacuole. Previous work has shown that the most abundant SERA, SERA5, plays an important but non-enzymatic role in asexual blood stages. SERA5 is extensively proteolytically processed by a parasite serine protease called SUB1 as well as an unidentified cysteine protease just prior to egress. However, neither the function of SERA5 nor the role of its processing is known. Here we show that conditional disruption of the SERA5 gene, or of both the SERA5 and related SERA4 genes simultaneously, results in a dramatic egress and replication defect characterised by premature host cell rupture and the failure of daughter merozoites to efficiently disseminate, instead being transiently retained within residual bounding membranes. SERA5 is not required for poration (permeabilization) or vesiculation of the host cell membrane at egress, but the premature rupture phenotype requires the activity of a parasite or host cell cysteine protease. Complementation of SERA5 null parasites by ectopic expression of wild-type SERA5 reversed the egress defect, whereas expression of a SERA5 mutant refractory to processing failed to rescue the phenotype. Our findings implicate SERA5 as an important regulator of the kinetics and efficiency of egress and suggest that proteolytic modification is required for SERA5 function. In addition, our study reveals that efficient egress requires tight control of the timing of membrane rupture.

Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro.

Conditional genome engineering in the human malaria pathogen Plasmodium falciparum remains highly challenging. Here we describe a strategy for facile and rapid functional analysis of genes using an approach based on the Cre/lox system and tailored for organisms with short and few introns. Our method allows the conditional, site-specific removal of genomic sequences of essential and non-essential genes by placing loxP sites into a short synthetic intron to produce a module (loxPint) can be placed anywhere in open reading frames without compromising protein expression. When duplicated, the loxPint module serves as an intragenic recombineering point that can be used for the fusion of gene elements to reporters or the conditional introduction of point mutations. We demonstrate the robustness and versatility of the system by targeting the P. falciparum merozoite surface protein 1 gene (msp1), which has previously proven refractory to genetic interrogation, and the parasite exported kinase FIKK10.1.

The malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Collective efficacy is defined as residents' perceived collective capacity to take coordinated and interdependent action on issues that affect their lives. This study explored factors associated with neighborhood collective efficacy among residents. Utilizing a national sample of 4,120 urban households provided by Annie E. Casey Foundation's Making Connection Initiative, we investigated the mediating role of residents' perceptions of bonding social capital (i.e. reciprocity, trust, and shared norms) in the association between civic engagement and collective efficacy. Multiple regression analyses revealed that civic engagement and bonding social capital were both directly related to collective efficacy. Additionally, bonding social capital partially mediated the relationship between civic engagement and collective efficacy. Specifically, residents who reported greater levels of civic engagement also reported higher levels of bonding social capital. In turn, residents who reported higher levels of bonding social capital also reported higher levels of neighborhood collective efficacy. We discuss implications of these findings for researchers and practitioners interested in associations of neighborhood collective efficacy.

1,4-Dimercapto-2,5-diphosphinobenzene and 3,6-bis(hexyloxy)-1,4-dimercapto-2,5-diphosphinobenzene were synthesized and combined with various acid chlorides to obtain a series of benzobisthiaphospholes. Electrochemical and photophysical properties of the substituted benzobisthiaphospholes have been evaluated, and the observed reductions are more facile than the related benzothiaphospholes and 2,6-diphenylbenzobisthiazole. A benzobisthiaphosphole with C6H4-p-CN substituents was reduced at E(1/2)=-1.08 V (vs. saturated calomel electrode (SCE)). X-ray diffraction data for several of these phosphorus heterocycles has been obtained, and DFT calculations at the B3LYP level have been performed., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Upon rupture of Plasmodium falciparum ( P. falciparum ) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. This shedding, which takes place even in the absence of invasion, is therefore a surrogate marker for the degree of egress taking place in a culture, and can be measured using a specific capture ELISA to quantify AMA1 levels in culture supernatants (Collins et al. , 2013). The assay uses a monoclonal antibody specific for AMA1 (called 4G2dc1) (Kocken et al ., 1998; Collins et al ., 2009) to capture and immobilize the protein from culture supernatants, then uses a specific rabbit polyclonal antiserum to detect the immobilized antigen. A phosphatase-conjugated goat anti-rabbit antibody is finally used to quantify the binding of the second antibody. Egress is absolutely dependent upon the activity of a parasite cGMP-dependent protein kinase, PKG, and so is influenced by levels of intracellular cGMP (Collins et al ., 2013). This is regulated by the interplay between guanylate cyclases and phosphodiesterases. The latter enzymes may also degrade cAMP, so it may also be informative to measure intracellular cAMP levels.

The malaria merozoite invades erythrocytes in the vertebrate host. Iterative rounds of asexual intraerythrocytic replication result in disease. Proteases play pivotal roles in erythrocyte invasion, but little is understood about their mode of action. The Plasmodium falciparum malaria merozoite surface sheddase, PfSUB2, is one such poorly characterized example. We have examined the molecular determinants that underlie the mechanisms by which PfSUB2 is trafficked initially to invasion-associated apical organelles (micronemes) and then across the surface of the free merozoite. We show that authentic promoter activity is important for correct localization of PfSUB2, likely requiring canonical features within the intergenic region 5' of the pfsub2 locus. We further demonstrate that trafficking of PfSUB2 beyond an early compartment in the secretory pathway requires autocatalytic protease activity. Finally, we show that the PfSUB2 transmembrane domain is required for microneme targeting, while the cytoplasmic domain is essential for surface translocation of the protease to the parasite posterior following discharge from micronemes. The interplay of pre- and post-translational regulatory elements that coordinate subcellular trafficking of PfSUB2 provides the parasite with exquisite control over enzyme-substrate interactions., (© 2013 The Authors. Traffic published by John Wiley & Sons Ltd.)

The overall goal of this review was to observe how the material properties of the unfused midpalatal suture have been considered by researchers in finite element analyses of maxillary expansion. Literature from Scopus, PubMed, and Biosis were all considered. Upon completion of the review, it was found that researchers assumed the suture to be vacant, have the same elastic properties as bone, or have elastic properties indicative of soft tissues. By performing a simplified analysis of the maxilla complex during expansion, it is shown that the suture may have a significant influence on treatment outcome. As a result, despite valuable contributions from previous studies, it would be ideal to incorporate a more representative model of the midpalatal suture into finite element simulations., (Copyright © 2013 CEO. Published by Elsevier Masson SAS. All rights reserved.)

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes., (© 2013 John Wiley & Sons Ltd.)

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Host cell invasion by the malaria parasite is a crucial step in its life cycle. In this issue of Cell Host & Microbe, Giovannini et al. (2011) raise questions about the validity of a widely accepted model of the tight junction, a ring-like structure through which the invading parasite passes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1.

Antibodies that inhibit red blood cell invasion by the Plasmodium merozoite block the erythrocytic cycle responsible for clinical malaria. The invasion-inhibitory monoclonal antibody (mAb) 4G2 recognizes a conserved epitope in the ectodomain of the essential Plasmodium falciparum microneme protein and vaccine candidate, apical membrane antigen 1 (PfAMA1). Here we demonstrate that purified Fab fragments of 4G2 inhibit invasion markedly more efficiently than the intact mAb, suggesting that the invasion-inhibitory activity of this mAb is not due solely to steric effects and that the epitope lies within a functionally critical region of the molecule. We have taken advantage of a synthetic gene encoding a modified form of PfAMA1, and existing x-ray crystal structure data, to fully characterize this epitope. We first validate the gene by demonstrating that it fully complements the function of the authentic gene in P. falciparum. We then use it to identify a group of residues within the previously described domain II loop of PfAMA1 that are critical for recognition by mAb 4G2 and demonstrate that the epitope lies exclusively within this loop with no contributions from residues in other domains of the molecule. This is the first complete characterization of a conserved invasion-inhibitory epitope on PfAMA1. Our results will aid in the design of subunit vaccines designed to generate a broadly effective, focused anti-PfAMA1 protective immune response and may help elucidate the function of PfAMA1.

Apical membrane antigen 1 (AMA1) is a membrane protein present in Plasmodium species and is probably common to all apicomplexan parasites. The recent crystal structure of the complete ectoplasmic region of AMA1 from Plasmodium vivax has shown that it comprises three structural domains and that the first two domains are based on the PAN folding motif. Here, we discuss the consequences of this analysis for the three-dimensional structure of AMA1 from other Plasmodium species and other apicomplexan parasites, and for the Plasmodium paralogue MAEBL. Many polar and apolar interactions observed in the PvAMA1 crystal structure are made by residues that are invariant or highly conserved throughout all Plasmodium orthologues; a subgroup of these residues is also present in other apicomplexan orthologues and in MAEBL. These interactions presumably play a key role in defining the protein fold. Previous studies have shown that the ectoplasmic region of AMA1 must be cleaved from the parasite surface for host-cell invasion to proceed. The cleavage site in the crystal structure is not readily accessible to proteases and we discuss possible consequences of this observation. The three-dimensional distribution of polymorphic sites in PfAMA1 shows that these are all on the surface and that their positions are significantly biased to one side of the ectoplasmic region. Of particular note, a flexible segment in domain II, comprising about 40 residues and devoid of polymorphism, carries an epitope recognized by an invasion-inhibitory monoclonal antibody and a T-cell epitope implicated in the human immune response to AMA1.

Apical membrane antigen 1 from Plasmodium is a leading malaria vaccine candidate. The protein is essential for host-cell invasion, but its molecular function is unknown. The crystal structure of the three domains comprising the ectoplasmic region of the antigen from P. vivax, solved at 1.8 angstrom resolution, shows that domains I and II belong to the PAN motif, which defines a superfamily of protein folds implicated in receptor binding. We also mapped the epitope of an invasion-inhibitory monoclonal antibody specific for the P. falciparum ortholog and modeled this to the structure. The location of the epitope and current knowledge on structure-function correlations for PAN domains together suggest a receptor-binding role during invasion in which domain II plays a critical part. These results are likely to aid vaccine and drug design.

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actin-independent process.

EXAFS spectra of Hg(2+) sorbed on goethite at pH 4.60 has revealed the existence of an inner-sphere complex with an immediate coordination shell of 2 O atoms at 2.04 Å and a further shell of 1 Fe atom at 3.28 Å. Using density functional calculations, we determined the optimized geometries of small clusters representing surface complexes. These results shows that adsorption of Hg(2+) occurs via two oxygen atoms bound to edge-sharing Fe sites on the (110) surface. This is the same surface site we find for Cd (1, 2) and Sr (3). The short Hg-O bond length shows that the oxygens bridging Hg and Fe atoms are deprotonated. Copyright 1999 Academic Press.

> Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p93.html

Purpose: A prospective study was designed to evaluate the use of botulinum toxin as a temporary treatment in patients awaiting surgical repair for involutional entropion and to compare its use with lid taping., Methods: Botulinum toxin was administered to 30 patients with involutional entropion (35 eyelids). These patients had all previously been using lid taping and lubricant ointment as a temporary measure whilst awaiting lid surgery. Patients' symptoms and signs were assessed before and after toxin injection. The date of entropion recurrence was recorded. Eyelid tissue from 8 patients treated with toxin and 3 control patients who had not been given toxin was obtained after surgical entropion repair and examined histologically to ensure the botulinum toxin had no potential detrimental effects on the results of surgery., Results: The toxin was simple and quick to administer. Anatomical success was achieved in 33 of the 35 eyelids with significant improvements in symptoms and signs. The mean duration of action of the toxin was 12.5 weeks. Lower lid laxity was inversely correlated with duration of toxin action. There were no consistent changes in orbicularis oculi morphology after toxin injection., Conclusion: Botulinum toxin is a highly effective temporary treatment for involutional entropion with few complications and no adverse effects on the results of surgical entropion repair.

Objective: This study sought to examine variations in the frequency of procedures performed and patterns of care of sick infants and older children by general pediatricians in different sized communities. The results of the study will be considered in developing relevant educational experiences for postgraduate trainees., Methodology: Questionnaires were sent to 1412 Texas pediatricians requesting frequency information for 29 procedures and whether they provided various levels of care to sick infants and older children. Responses were tabulated by the size of the community in which each pediatrician practiced., Results: Fifty-four percent of the questionnaires were returned. The proportion of pediatricians performing each procedure was significantly different for all but 8 of the 29 procedures between communities of less than 100,000 and more than 100,000 population. For all procedures with significant differences, the proportion of physicians performing the procedures was significantly greater for pediatricians practicing in communities of less than 100,000 population. No significant difference was found between the proportion of pediatricians providing newborn level II and III care; however, more than 65% of both groups provided level II care. Physicians in communities of less than 100,000 population were more likely to provide intermediate and intensive care beyond the newborn period., Conclusion: The general practice rotation in the community setting will not provide adequate training experiences for many of the procedures currently being performed by general pediatricians.

The purpose of this study was to determine (a) if nonstutterers prefer to interact with mild and severe stutterers who acknowledge their stuttering and (b) if nonstutterers' perceptions of mild and severe stutterers' intelligence, personality, and appearance are altered as a function of stutterers' acknowledgment of their stuttering. Acknowledgment and nonacknowledgment videotapes of two mild and two severe male stutterers were made during interview situations. Eighty-four female subjects examined the interview tapes and were then instructed to answer questions and complete adjective scales. Findings indicate that significant numbers of female nonstutterers prefer to interact with severe stutterers who acknowledged their stuttering. Mild and severe stutterers who acknowledged their stuttering receive more favorable ratings on intelligence, personality, and appearance.

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoptmetric (Cyt CIII) and chemiluminescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemiluminescence clearly demonstrated that the six-membered piperidine-1-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 x 10(4) to 1.2 x 10(5) M-1 s-1. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides, they may ultimately be more useful as superoxide oxide dismutase mimetic agents.