Purification of Intracellular Bacterial Communities during Experimental Urinary Tract Infection Reveals an Abundant and Viable Bacterial Reservoir.

Urinary tract infections (UTIs) are a major infection of humans, particularly affecting women. Recurrent UTIs can cause significant discomfort and expose patients to high levels of antibiotic use, which in turn contributes to the development of higher antibiotic resistance rates. Most UTIs are caused by uropathogenic Escherichia coli, which is able to form intracellular collections (termed intracellular bacterial communities [IBCs]) within the epithelial cells lining the bladder lumen. IBCs are seen in both infected mice and humans and are a potential cause of recurrent UTI. Genetic and molecular studies of IBCs have been hampered both by the low number of bacteria in IBCs relative to the number extracellular bacteria and by population bottlenecks that occur during IBC formation. We now report the development of a simple and rapid technique for isolating pure IBCs from experimentally infected mice. We verified the specificity and purity of the isolated IBCs via microscopy, gene expression, and culture-based methods. Our results further demonstrated that our isolation technique practically enables specific molecular studies of IBCs. In the first such direct measurement, we determined that a single epithelial cell containing an early IBC typically contains 103 viable bacteria. Our isolation technique complements recent progress in low-input, single-cell genomics to enable future genomic studies of the formation of IBCs and their activation pathways during recurrent UTI, which may lead to novel strategies to eliminate them from the bladder.

Enhancing Early Childhood Mental Health Primary Care Services: Evaluation of MA Project LAUNCH.

Objectives The purpose of this study was to evaluate the efficacy of an innovative early childhood mental health intervention, Massachusetts Project LAUNCH. Early childhood mental health clinicians and family partners (paraprofessionals with lived experience) were embedded within community pediatric medical homes. Methods A longitudinal study design was used to test the hypotheses that (1) children who received services would experience decreased social, emotional and behavioral problems over time and (2) caregivers' stress and depressive symptoms would decrease over time. Families who were enrolled in services and who consented to participate in the evaluation study were included in analyses (N = 225). Individual growth models were used to test longitudinal effects among MA LAUNCH participants (children and caregivers) over three time points using screening tools. Results Analyses showed that LAUNCH children who scored in age-specific clinically significant ranges of social, emotional and behavioral problems at Time 1 scored in the normal range on average by Time 3. Caregivers' stress and depressive symptoms also declined across the three time points. Results support hypotheses that the LAUNCH intervention improved social and emotional health for children and caregivers. Conclusions for Practice This study led to sustainability efforts, an expansion of the model to three additional communities across the state and development of an online toolkit for other communities interested in implementation.

NUCare: Advancing research on technological integration for self-management in the aging population.

BACKGROUND: The Center for Technology in Support of Self-Management and Health (NUCare) is an exploratory research center funded by the National Institute of Nursing Research's P20 mechanism positioned to conduct rigorous research on the integration of technology in the self-management of the older adult population. PURPOSE: The purpose of this paper is to describe the development and application of an evaluation plan and preliminary evaluation results from the first year of implementation. METHODS: This evaluation plan is derived from and is consistent with Dorsey et al.'s (2014) logic model. Dorsey's model provided guidelines for evaluating sustainability, leveraging of resources, and interdisciplinary collaboration within the center. DISCUSSION: Preliminary results and strategies for addressing findings from the first year of evaluation are discussed. A secondary aim of this paper is to showcase the relevance of this center to the advancement and maintenance of health in the aging population.

The therapist as advocate: anti-oppression advocacy in psychological practice.

In this article, we present an approach entitled Anti-Oppression Advocacy (AOA) as a means of addressing poverty in a psychotherapeutic context. AOA is an integrative approach designed to meet two goals that we argue to be closely inter-related: therapeutic change, and economic justice. In AOA, the practitioner is expected to act as an advocate and to encourage clients themselves to engage in some form of social justice action. We provide a case example of using AOA in a shelter for homeless survivors of domestic violence. We also discuss some of the challenges involved in implementing the AOA approach.

Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes.

RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 µM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

Review of Programs to Combat Elder Mistreatment: Focus on Hospitals and Level of Resources Needed.

BACKGROUND: Elder mistreatment is common and has serious social and medical consequences for victims. Though programs to combat this mistreatment have been developed and implemented for more than three decades, previous systematic literature reviews have found few successful ones. OBJECTIVE: To conduct a more comprehensive examination of programs to improve elder mistreatment identification, intervention, or prevention, including those that had not undergone evaluation. DESIGN: Systematic review. SETTING: Ovid MEDLINE, Ovid EMBASE, Cochrane Library, PsycINFO Elton B. Stephens Co. (EBSCO), AgeLine, CINAHL. MEASUREMENTS: We abstracted key information about each program and categorized programs into 14 types and 9 subtypes. For programs that reported an impact evaluation, we systematically assessed the study quality. We also systematically examined the potential for programs to be successfully implemented in environments with limited resources available. RESULTS: We found 116 articles describing 115 elder mistreatment programs. Of these articles, 43% focused on improving prevention, 50% focused on identification, and 95% focused on intervention, with 66% having multiple foci. The most common types of program were: educational (53%), multidisciplinary team (MDT) (21%), psychoeducation/therapy/counseling (15%), and legal services/support (8%). Of the programs, 13% integrated an acute-care hospital, 43% had high potential to work in low-resource environments, and 57% reported an attempt to evaluate program impact, but only 2% used a high-quality study design. CONCLUSION: Many programs to combat elder mistreatment have been developed and implemented, with the majority focusing on education and MDT development. Though more than half reported evaluation of program impact, few used high-quality study design. Many have the potential to work in low-resource environments. Acute-care hospitals were infrequently integrated into programs.

The fungal alkaloid Okaramine-B activates an L-glutamate-gated chloride channel from Ixodes scapularis, a tick vector of Lyme disease.

A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400 nA currents in response to 100 µM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ±â€¯0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 µM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 µM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ±â€¯0.04) and the phenylpyrazole fipronil (pIC50 6.90 ±â€¯0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ±â€¯0.43) and may serve as a candidate lead compound for the development of new acaricides.

Tick neurobiology: recent advances and the post-genomic era.

Increasing worldwide resistance to acaricides necessitates greater research on the identification of potential acaricide targets in ticks to aid in the control of these serious pests of medical and veterinary importance. Historically, and most likely in the future, acaricide targets are largely of neural origin, but our knowledge of tick neurobiology is surprisingly limited. The tick central nervous system is a fused nerve mass, termed the synganglion. Tick synganglion material is relatively easily accessible to most researchers and employing modern amplification methods of complementary-DNA construction is readily amenable for gene cloning investigations. The various tick neurotransmitter systems are described with emphasis on our current knowledge of both existing and potential acaricide targets at the molecular level. We describe the impact of mass gene sequencing (expressed sequence tag and genome projects), advances in bioinformatics and RNA-interference on target identification and validation.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Actions of agonists, fipronil and ivermectin on the predominant in vivo splice and edit variant (RDLbd, I/V) of the Drosophila GABA receptor expressed in Xenopus laevis oocytes.

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> ß-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and ß-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides.

Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus.

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken ß2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 µM) and choline (100 µM). Rsanα1/ß2 was insensitive to both imidacloprid (100 µM) and spinosad (100 µM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.