Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii.

The phylum Apicomplexa contains several parasitic species of medical and agricultural importance. The ubiquitination machinery remains, for the most part, uncharacterised in apicomplexan parasites, despite the important roles that it plays in eukaryotic biology. Bioinformatic analysis of the ubiquitination machinery in apicomplexan parasites revealed an expanded ovarian tumour domain-containing (OTU) deubiquitinase (DUB) family in Toxoplasma, potentially reflecting functional importance in apicomplexan parasites. This study presents comprehensive characterisation of Toxoplasma OTU DUBs. AlphaFold-guided structural analysis not only confirmed functional orthologues found across eukaryotes, but also identified apicomplexan-specific enzymes, subsequently enabling discovery of a cryptic OTU DUB in Plasmodium species. Comprehensive biochemical characterisation of 11 Toxoplasma OTU DUBs revealed activity against ubiquitin- and NEDD8-based substrates and revealed ubiquitin linkage preferences for Lys6-, Lys11-, Lys48-, and Lys63-linked chain types. We show that accessory domains in Toxoplasma OTU DUBs impose linkage preferences, and in case of apicomplexan-specific TgOTU9, we discover a cryptic ubiquitin-binding domain that is essential for TgOTU9 activity. Using the auxin-inducible degron (AID) to generate knockdown parasite lines, TgOTUD6B was found to be important for Toxoplasma growth.

Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the endoplasmic reticulum.

The voltage-dependent anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC protein, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes in the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii. This article has an associated First Person interview with the first author of the paper.

Environmental sensing and regulation of motility in Toxoplasma.

Toxoplasma and other apicomplexan parasites undergo a unique form of cellular locomotion referred to as "gliding motility." Gliding motility is crucial for parasite survival as it powers tissue dissemination, host cell invasion and egress. Distinct environmental cues lead to activation of gliding motility and have become a prominent focus of recent investigation. Progress has been made toward understanding what environmental cues are sensed and how these signals are transduced in order to regulate the machinery and cellular events powering gliding motility. In this review, we will discuss new findings and integrate these into our current understanding to propose a model of how environmental sensing is achieved to regulate gliding motility in Toxoplasma. Collectively, these findings also have implications for the understanding of gliding motility across Apicomplexa more broadly.

Protein Kinase A Is Essential for Invasion of Plasmodium falciparum into Human Erythrocytes.

Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610.IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca2+] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K+] to activate cytosolic [Ca2+] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca2+ signaling and motility in Toxoplasma.

Protein kinase A negatively regulates Ca2+ signalling in Toxoplasma gondii.

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

Somali Older Adults' and Their Families' Perceptions of Adult Home Health Services.

Many studies have identified the vulnerability of ethnic elders, and there is promising evidence indicating home health care (HHC) services can improve the health outcomes of Somali older adults. This study used a community-engaged qualitative descriptive approach with the participation of non-profit organization Refugees Helping Refugees. The purpose of this study was to explore and describe Somali older adults' and their families' perceptions of and experiences with HHC services in order to improve its use and access. Data collection included home visits (n = 15), semi-structured interviews (n = 17) and debriefing sessions (n = 16) with 19 individuals from 14 Somali families. Somali families recognized HHC services were needed and believed having services in the home facilitated learning but HHC agencies should work more with the Somali community. HHC agencies need to work with community organizations to facilitate cultural and health understanding, and better health care for Somali older adults.

Using a Clinical Outreach Project to Foster a Community-Engaged Research Partnership With Somali Families.

BACKGROUND: Community-engaged research partnerships build the capacity of community and educational organizations to work together toward addressing important health issues and disparities for vulnerable populations, such as refugees or immigrants. A critical step for building a community-engaged research partnership is the Thrst contact or entrée into the community. PURPOSE: The purpose of this paper is to describe how a successful home health community-engaged partnership became the entrée and foundation for a community-engaged research partnership to explore the home health needs of Somali older adults and their families. METHODS: A number of strategies were used to engage the Somali community, initially in a clinical home health project and subsequently in an academic research study. LESSONS LEARNED: Valuable lessons were learned on delivering home health care (HHC) services to Somali older adults and their families as well as conducting research with this population. The most important lesson was that none of the work could be done without the involvement of the Somali community. The partnership described is one of the Thrst to address the home health needs and experiences of Somali older adults and their families. The project illustrates a mutually beneThcial relationship that can occur when a community-engaged clinical project expanded to address an issue of importance to the community through research. CONCLUSIONS: This foundation served to create an opportunity for more comprehensive community-academic partnerships with the potential to improve the delivery of HHC to Somali older adults, as well as open avenues for research in other areas that are relevant to the Somali, medical, and academic communities.

Measurement Reactivity in a Randomized Clinical Trial Using Self-Reported Data.

PURPOSE: The objective of this article is to describe how self-monitoring contributed to measurement reactivity in a randomized clinical trial (RCT). The implications of measurement reactivity on self-monitoring and suggestions for minimizing its effect will be discussed. DESIGN: The study involved a secondary data analysis of responses from 145 participants who completed a 12-month long RCT. The original sample consisted of 202 community-living adults with long-term indwelling urinary catheters. METHODS: The data analyzed were from the participants' brief responses to the final study question concerning their perceived study participation value. The data were discussed and coded iteratively until three researchers reached consensus. At the end, each item was recoded into eight categories and minor codes. FINDINGS: Our belief that the calendar was the probable cause of reactivity was not fully supported as the control group also learned about paying attention to urinary catheter problems by the questions asked through the seven bimonthly interviews. CONCLUSIONS: The calendar and interview questions were reactive by increasing participants' self-monitoring of catheter problems. Specifically, the frequent interviews exposed participants to catheter-related concerns and provided them with ideas as to how to better manage them. CLINICAL RELEVANCE: This article provides insight related to the internal validity threat of measurement reactivity in RCTs and gives suggestions to reduce its effects.

Exploring relationships of catheter-associated urinary tract infection and blockage in people with long-term indwelling urinary catheters.

AIMS AND OBJECTIVES: To describe and explore relationships among catheter problems in long-term indwelling urinary catheter users, including excess healthcare use for treating catheter problems. BACKGROUND: Long-term urinary catheter users experience repeated problems with catheter-related urinary tract infection and blockage of the device, yet little has been reported of the patterns and relationships among relevant catheter variables. DESIGN: Secondary data analysis was conducted from a sample in a randomised clinical trial, using data from the entire sample of 202 persons over 12 months' participation. METHODS: Descriptive statistics were used to characterise the sample over time. Zero-inflated negative binomial models were employed for logistic regressions to evaluate predictor variables of the presence/absence and frequencies of catheter-related urinary tract infection and blockage. RESULTS: Catheter-related urinary tract infection was marginally associated with catheter blockage. Problems reported at least once per person in the 12 months were as follows: catheter-related urinary tract infection 57%, blockage 34%, accidental dislodgment 28%, sediment 87%, leakage (bypassing) 67%, bladder spasms 59%, kinks/twists 42% and catheter pain 49%. Regression analysis demonstrated that bladder spasms were significantly related to catheter-related urinary tract infection and sediment amount, and catheter leakages were marginally significantly and positively related to catheter-related urinary tract infection. Frequencies of higher levels of sediment and catheter leakage were significantly associated with higher levels of blockage, and being female was associated with fewer blockages. Persons who need help with eating (more disabled) were also more likely to have blockages. CONCLUSIONS: Catheter-related urinary tract infection and blockage appear to be related and both are associated with additional healthcare expenditures. More research is needed to better understand how to prevent adverse catheter outcomes and patterns of problems in subgroups. RELEVANCE TO CLINICAL PRACTICE: Nurses can develop care management strategies to identify catheter blockage prior to its occurrence by tracking the amount of sediment and frequency of leakage. Bladder spasms could be an early warning of catheter-related urinary tract infection.

Meeting the Needs of Older Adult Refugee Populations With Home Health Services.

The United States resettles close to 70,000 refugees each year more than any other country in the world. Adult refugees are at risk for negative health outcomes and inefficient health resource use, and meeting the multiple health needs of this vulnerable population is a challenge. The purpose of this study was to assess the impact of a home health care (HHC) pilot project on meeting the needs of older adult refugee patients. A retrospective chart review of 40 refugee adult patients who participated in an HHC pilot was done to analyze their health outcomes using OASIS-C data. Participants' pain level, anxiety level, medication management, and activities of daily living management all significantly improved over the course of their HHC episode. Results of this study indicate that HHC has great potential to improve the health of vulnerable refugee populations and assist the families involved in their care.

Feasibility of a Web-Based Self-management Intervention for Intermittent Urinary Catheter Users With Spinal Cord Injury.

PURPOSE: The purpose of this study was to evaluate the feasibility of a new Web-based intermittent catheter self-management intervention. DESIGN: We tested the acceptability and usability of intervention components, which included multiple Web-based materials (an online urinary diary adapted for mobile phone use and an educational booklet), 3 phone calls with a nurse, and a peer-led discussion forum. SUBJECTS AND SETTING: Thirty adults with spinal cord injury using intermittent catheterization for bladder drainage were enrolled; 26 participants received the nurses' phone-based consultations. METHODS: Preliminary effectiveness of new self-efficacy and self-management scales were evaluated using baseline and 3-month online surveys. Participants' perceived value of the intervention components, self-management changes, and suggestions were assessed with data from the 3-month surveys, followed by brief tape-recorded interviews. RESULTS: Several catheter practices improved somewhat over 3 months. The frequency of catheterizations every 4 to 6 hours increased from 71% to 77%. Self-management of neurogenic bladder dysfunction increased significantly (P = .032); participant comments indicated that fluid intake was the biggest change. Catheter-related self-efficacy and quality-of-life scores increased but not significantly. The frequency of urinary tract infection and pain did not change significantly. For feasibility, intervention components, with the exception of the forum, were rated highly by the majority of participants for usefulness, satisfaction (desired information), and Web-based usability. CONCLUSION: Further testing of this intervention is recommended in a multisite randomized clinical trial.

Testing a Model of Self-Management of Fluid Intake in Community-Residing Long-term Indwelling Urinary Catheter Users.

BACKGROUND: Urinary tract infection and blockage are serious and recurrent challenges for people with long-term indwelling catheters, and these catheter problems cause worry and anxiety when they disrupt normal daily activities. OBJECTIVE: The goal was to determine whether urinary catheter-related self-management behaviors focusing on fluid intake would mediate fluid intake-related self-efficacy toward decreasing catheter-associated urinary tract infection (CAUTI) and/or catheter blockage. METHODS: The sample involved data collected from 180 adult community-living, long-term indwelling urinary catheter users. The authors tested a model of fluid intake self-management related to fluid intake self-efficacy for key outcomes of CAUTI and blockage. To account for the large number of zeros in both outcomes, a zero-inflated negative binomial (ZINB) structural equation model was tested. RESULTS: Structurally, fluid intake self-efficacy was positively associated with fluid intake self-management, suggesting that higher fluid intake self-efficacy predicts more (higher) fluid intake self-management; however, fluid intake self-management was not associated with either the frequency of CAUTIs or the presence or absence of CAUTI. Fluid intake self-efficacy was positively related to fluid intake self-management, and fluid intake self-management predicted less frequency of catheter blockage, but neither fluid intake self-efficacy nor fluid intake self-management predicted the presence or absence of blockage. DISCUSSION: Further research is needed to better understand determinants of CAUTI in long-term catheter users and factors which might influence or prevent its occurrence. Increased confidence (self-efficacy) and self-management behaviors to promote fluid intake could be of value to long-term urinary catheter users to decrease catheter blockage.

Development and psychometric evaluation of the ICIQ-LTCqol: A self-report quality of life questionnaire for long-term indwelling catheter users.

AIMS: Long-term indwelling catheterisation may affect health related quality of life, but clinical assessment and monitoring of people with indwelling catheters is poorly recorded because there are no validated measures to capture these criteria. In this paper, we describe the development of the ICIQ-Long Term Catheter quality of life (ICIQ-LTCqol), one of the modules of the ICIQ series, an international project to standardise assessment of lower pelvic dysfunction: www.iciq.net. METHODS: In-depth interviews were conducted with 27 catheter-users and 4 informal carers and cognitive debriefing with a further 31 catheter-users and clinical experts to evaluate clarity and comprehensiveness. The draft 44 item questionnaire was then sent by post to 893 long-term catheter-users; the 370 completed questionnaires were used to test content validity, test re-test reliability and internal consistency (Cronbach α coefficient). Factor analysis alongside expert opinion was used to formulate the final questionnaire of 16 items. This was then sent by post to another 438 long-term catheter-users to evaluate domain scores. RESULTS: The final questionnaire consists of two scored domains: catheter function and concern (9 items) and lifestyle impact (3 items) and four standalone items, relating to pads, pain, sexual activity and bladder spasm. Levels of missing data are good (mean 3.6%) with moderate to good agreement and acceptable internal consistency (Cronbach's alpha 0.76 and 0.74 for each domain respectively), suggesting acceptability and stability of the questionnaire. CONCLUSION: The ICIQ-LTCqol is a psychometrically robust self-report questionnaire for the clinical assessment and evaluation of health related quality of life for long-term catheter users.

Self-care management questionnaire for long-term indwelling urinary catheter users.

AIMS: Self-management is believed to be an important behavioral capacity in people with chronic illness, and self-efficacy is an important component contributing to self-management. Two new and related measures for urinary catheter self-efficacy (C-SE) and self-management (C-SMG) were developed based on existing tools and tested for psychometrics for use in a randomized clinical trial (RCT) teaching urinary catheter self-management. METHODS: The instruments were evaluated at baseline (intake face to face interview) with 202 persons and with 158 of the same individuals 6 months later by telephone interviews. Exploratory factor analysis was conducted in an iterative process related to items' theoretical and statistical appropriateness. To assess construct validity and goodness of fit for model testing, confirmatory factor analysis was conducted with the samples at intake and 6 months. Also, bivariate analyses were conducted of the measures in relation to each other. RESULTS: Both scales were modified reducing the items to 13 in each, with four factors in C-SE and three factors in C-SMG. Reliability testing (Cronbach's alpha) was viewed as satisfactory for both scales, though some of the subscale (factors) reliabilities were lower in the self-management measure. Confirmatory factor analysis was adequate. Correlation of the two instruments (r = 0.25, P < 001) suggests that they are related scales. CONCLUSION: With a change in the C-SMG scale from 3- to 5-level responses and subsequent testing, both new scales are appropriate for use separately or together as a questionnaire related to catheter self-care management in intervention research with long-term indwelling urinary catheter users. Neurourol. Urodynam. 35:492-496, 2016. © 2015 Wiley Periodicals, Inc.

Development of a Web-Based Self-management Intervention for Intermittent Urinary Catheter Users With Spinal Cord Injury.

While Web-based interventions have proliferated recently, information in the literature is often lacking about how the intervention was developed. In response to that gap, this is a report of the development of a Web-based self-management intervention for intermittent urinary catheter users and pretesting with four adults with spinal cord injury living in the community. Two Web sites were created, one for recruitment and the other for the intervention itself. The intervention involved developing new Web-based technology, including an interactive urinary diary (with fluid intake/urine output and a journal), extensive catheter products information, three intervention nurse phone call consultations, and user-community discussion forums. Study participants completed an online survey and were interviewed twice about the enrollment process and their perceptions of their involvement in the intervention. Suggestions from the pretesting participants were used to revise the Web site applications prior to the next stage of research (a feasibility study). Numerous recommendations and comments were received related to content, interactivity of components, and usability. This article provides a description of how the Web sites were developed (including the technology and software programs used), issues encountered and what was done to address them, and how the Web-based intervention was modified for improvements.

A Web-Based Self-Management Intervention for Intermittent Catheter Users.

A new Web-based self-management intervention was developed for persons with spinal cord injury who use intermittent urinary catheters. Included are a description of the components, examples from the educational book, and multiple screen shots of the online urinary diary.

A hybrid process fidelity assessment in a home-based randomized clinical trial.

A process fidelity assessment was conducted as a nested study within a home-based randomized clinical trial teaching self-management to 101 long-term indwelling urinary catheter users in the treatment group. Our hybrid model combined external assessments (outside observations and tape recordings) with internal evaluation methods (through study nurse forms and notes) for a comprehensive process fidelity assessment. Barriers, patient-related issues, and nurse perspectives were identified demonstrating the complexity in home care intervention research. The complementary and synergistic approaches provided in-depth information about the context of the delivery and the impact of the intervention on study outcomes.

Self-management intervention for long-term indwelling urinary catheter users: randomized clinical trial.

BACKGROUND: People using long-term indwelling urinary catheters experience multiple recurrent catheter problems. Self-management approaches are needed to avoid catheter-related problems. OBJECTIVES: The aim was to determine effectiveness of a self-management intervention in prevention of adverse outcomes (catheter-related urinary tract infection, blockage, and accidental dislodgement). Healthcare treatment associated with the adverse outcomes and catheter-related quality of life was also studied. METHODS: A randomized clinical trial was conducted. The intervention involved learning catheter-related self-monitoring and self-management skills during home visits by a study nurse (twice during the first month and at 4 months-with a phone call at 2 months). The control group received usual care. Data were collected during an initial face-to-face home interview followed by bimonthly phone interviews. A total of 202 adult long-term urinary catheter users participated. Participants were randomized to treatment or control groups following collection of baseline data. Generalized estimating equations were used for the analysis of treatment effect. RESULTS: In the intervention group, there was a significant decrease in reported blockage in the first 6 months (p = .02), but the effect did not persist. There were no significant effects for catheter-related urinary tract infection or dislodgment. Comparison of baseline rates of adverse outcomes with subsequent periods suggested that both groups improved over 12 months. DISCUSSION: A simple-to-use catheter problems calendar and the bimonthly interviews might have functioned as a modest self-monitoring intervention for persons in both groups. A simplified intervention using a self-monitoring calendar is suggested-with optimal and consistent fluid intake likely to add value.

Defining and advancing education and conservative therapies of underactive bladder.

In contrast to other forms of voiding dysfunction, underactive bladder (UAB) has traditionally received little research or educational attention. This is changing as our understanding of the underlying mechanisms of detrusor dysfunction and other forms of underactive bladder improves. In addition, the impact of UAB on patient symptoms, general and health-related quality of life, and caregiver burden are becoming more recognized. However, there remains a paucity of data on the subject, and an extensive need for additional research and education on the topic. This paper explores the current state of knowledge about UAB with an emphasis on education regarding the condition and conservative methods of assessment and treatment. Recommendations for future work in this area are considered.