Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species also are among the most important and problematic, opportunistic nosocomial pathogens. Surprisingly, these two species antagonize each other's virulence in both nematode infection and in vitro biofilm models. We report here the identification of the E. faecalis bacteriocin, EntV, produced from the entV (ef1097) locus, as both necessary and sufficient for the reduction of C. albicans virulence and biofilm formation through the inhibition of hyphal formation, a critical virulence trait. A synthetic version of the mature 68-aa peptide potently blocks biofilm development on solid substrates in multiple media conditions and disrupts preformed biofilms, which are resistant to current antifungal agents. EntV68 is protective in three fungal infection models at nanomolar or lower concentrations. First, nematodes treated with the peptide at 0.1 nM are completely resistant to killing by C. albicans The peptide also protects macrophages and augments their antifungal activity. Finally, EntV68 reduces epithelial invasion, inflammation, and fungal burden in a murine model of oropharyngeal candidiasis. In all three models, the peptide greatly reduces the number of fungal cells present in the hyphal form. Despite these profound effects, EntV68 has no effect on C. albicans viability, even in the presence of significant host-mimicking stresses. These findings demonstrate that EntV has potential as an antifungal agent that targets virulence rather than viability.

Assessing the Quality of Nursing Homes in Managed Care Organizations: Integrating LTSS for Dually Eligible Beneficiaries.

Little is known about the quality of nursing homes in managed care organizations (MCOs) networks. This study (1) described decision-making criteria for selecting nursing home networks and (2) compared selected quality indicators of network and nonnetwork nursing homes. The sample was 17 MCOs participating in a California demonstration that provided integrated long-term services and supports to dually eligible enrollees in 2017. The findings showed that the MCOs established a broad network of nursing homes, with only limited attention to using quality criteria. Network nursing homes (602) scored significantly lower on 6 selected quality measures than nonnetwork (117) nursing homes. Low registered nurse and total nurse staffing were strong predictors of network nursing homes controlling for facility characteristics. Managed care organizations should consider greater transparency about the quality of their nursing homes and use specific quality criteria to improve the quality of their networks.

Impact of the Village Model: Results of a National Survey.

Villages are a relatively new, consumer-directed model that brings together older adults in a community who have a mutual interest in aging in place. These membership organizations provide social and civic engagement opportunities, volunteer provided support services, and referral to vetted community providers to achieve their primary goals of promoting independence and preventing undesired relocations. This cross sectional survey of 1,753 active Village members from 28 Villages across the US measured members' perceived impacts in the areas of social connection, civic engagement, service access, health and well-being, and ability to age in place. Results showed that involvement in the Village was a key factor associated with greater perceived impacts. Over half of members perceive that the Village has improved their sense of connection to others and their feeling that they have someone to count on. Though younger members in better health were more likely to perceive impacts in social connections, results suggest older women, living alone with some disability may be the most likely to experience improved health, quality of life, and mobility. The implications for social work practice are discussed.

Does the Village model help to foster age-friendly communities?

This article explores the potential role of the Village model, a social initiative that emphasizes member involvement and service access, in helping communities to become more age-friendly. A survey of 86.3% of operational Villages examined activities designed to help members access a variety of supports and services consistent with the World Health Organization's (WHO) Global Network of Age-Friendly Cities and Communities program model, as well as other potential contributions to community age friendliness. Analysis revealed that 85.5% of Villages provided assistance with at least six of the eight WHO domains, but only 10.1% implemented features of all eight; more than one-third were engaged in direct or indirect efforts to improve community physical or social infrastructures or improve community attitudes toward older persons. These findings suggest that Villages and other social organizations may have untapped potential for enhancing their members' ability to age in place consistent with the goals of age-friendly initiatives while also promoting constructive changes in the overall community.

Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans.

The Gram-positive bacterium Enterococcus faecalis and the fungus Candida albicans are both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using a Caenorhabditis elegans model of polymicrobial infection, we discovered that E. faecalis and C. albicans negatively impact each other's virulence. Much of the negative effect of E. faecalis on C. albicans was due to the inhibition of C. albicans hyphal morphogenesis, a developmental program crucial to C. albicans pathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence in E. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibit C. albicans filamentation in the context of an in vitro biofilm. Finally, a screen of an E. faecalis transposon mutant library identified other genes required for suppression of C. albicans hyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.

Rapid assays for Gaucher and Hurler diseases in dried blood spots using digital microfluidics.

OBJECTIVE: Easy tool for newborn screening of Gaucher and Hurler diseases. METHODS: Method comparison between fluorometric enzymatic activity assay on a digital microfluidic platform and micro-titer plate bench assay was performed on normal (n = 100), Gaucher (n = 10) and Hurler (n = 7) dried blood spot samples. RESULTS: Enzymatic activity analysis of glucocerebrosidase (Gaucher) and α-l-iduronidase (Hurler) revealed similar discrimination between normal and affected samples on both platforms. CONCLUSIONS: Digital microfluidics is suitable for Gaucher and Hurler newborn screening.

A novel fluorometric enzyme analysis method for Hunter syndrome using dried blood spots.

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate enzyme assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay compared well with a recently reported digital microfluidic method, from which it was adapted. Results show that this DBS assay is robust and reproducible using both technologies.

The Living Alone with Cognitive Impairment Project's Policy Advisory Group on Long-Term Services and Supports: Setting a Research Equity Agenda.

(1) Background: A United States national policy advisory group (PAG) was convened to identify barriers and facilitators to expand formal long-term services and support (LTSS) for people living alone with cognitive impairment (PLACI), with a focus on equitable access among diverse older adults. The PAG's insights will inform the research activities of the Living Alone with Cognitive Impairment Project, which is aimed at ensuring the equitable treatment of PLACI. (2) Methods: The PAG identified barriers and facilitators of providing effective and culturally relevant LTSS to PLACI via one-on-one meetings with researchers, followed by professionally facilitated discussions among themselves. (3) Results: The PAG identified three factors that were relevant to providing effective and culturally relevant LTSS to PLACI: (i) better characterization of PLACI, (ii) leveraging the diagnosis of cognitive impairment, and (iii) expanding and enhancing services. For each factor, the PAG identified barriers and facilitators, as well as directions for future research. (4) Conclusions: The barriers and facilitators the PAG identified inform an equity research agenda that will help inform policy change.

Digital microfluidic platform for multiplexing enzyme assays: implications for lysosomal storage disease screening in newborns.

BACKGROUND: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. METHODS: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. RESULTS: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. CONCLUSIONS: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Identifying and Understanding Transfusion Reactions in the Oncology Population.

OBJECTIVES: To provide an overview of transfusion reactions, their underlying pathophysiology, clinical presentation, and recommendations for nursing management. DATA SOURCES: We researched peer-reviewed journal articles, book chapters, Internet, and lecture proceedings. CONCLUSION: Transfusion reactions are adverse reactions to blood products frequently seen in the oncology population and can significantly vary in severity and etiology. Oncology nurses are in a critical position to assist with prevention, early detection, and time-sensitive treatment of transfusion reactions. IMPLICATIONS FOR NURSING PRACTICE: The oncology nurse's comprehensive understanding of possible transfusion reactions and management recommendations is key for optimal care of the oncology patient.

Emerging Approaches for Fluorescence-Based Newborn Screening of Mucopolysaccharidoses.

Interest in newborn screening for mucopolysaccharidoses (MPS) is growing, due in part to ongoing efforts to develop new therapies for these disorders and new screening assays to identify increased risk for the individual MPSs on the basis of deficiency in the cognate enzyme. Existing tests for MPSs utilize either fluorescence or mass spectrometry detection methods to measure biomarkers of disease (e.g., enzyme function or glycosaminoglycans) using either urine or dried blood spot (DBS) samples. There are currently two approaches to fluorescence-based enzyme function assays from DBS: (1) manual reaction mixing, incubation, and termination followed by detection on a microtiter plate reader; and (2) miniaturized automation of these same assay steps using digital microfluidics technology. This article describes the origins of laboratory assays for enzyme activity measurement, the maturation and clinical application of fluorescent enzyme assays for MPS newborn screening, and considerations for future expansion of the technology.

Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.

BACKGROUND: Decreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. METHODS: GALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. RESULTS: We present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 µmol/l/h with a limit of detection of 0.066 µmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. CONCLUSIONS: A fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from ß-galactosidase.

Participatory design of mass health communication in three languages for seniors and people with disabilities on Medicaid.

OBJECTIVES: We used participatory design methods to develop and test guidebooks about health care choices intended for 600 000 English-, Spanish-, and Chinese-speaking seniors and people with disabilities receiving Medicaid in California. METHODS: Design and testing processes were conducted with consumers and professionals; they included 24 advisory group interviews, 36 usability tests, 18 focus groups (105 participants), 51 key informant interviews, guidebook readability and suitability testing, linguistic adaptation, and iterative revisions of 4 prototypes. RESULTS: Participatory design processes identified preferences of intended audiences for guidebook content, linguistic adaptation, and format; guidebook readability was scored at the sixth- to eighth-grade level and suitability at 95%. These findings informed the design of a separate efficacy study that showed high guidebook usage and satisfaction, and better gains in knowledge, confidence, and intended behaviors among intervention participants than among control participants. CONCLUSIONS: Participatory design can be used effectively in mass communication to inform vulnerable audiences of health care choices. The techniques described can be adapted for a broad range of health communication interventions.

Phase II Study of Systemic High-dose Methotrexate and Intrathecal Liposomal Cytarabine for Treatment of Leptomeningeal Carcinomatosis From Breast Cancer.

Metastatic breast cancer frequently leads to brain metastases and, less commonly, leptomeningeal carcinomatosis (LC). Once cerebrospinal fluid involvement occurs, the prognosis is poor. There are limited treatment options available, but none offer significant survival benefit. Methotrexate, given systemically at high doses (3.5-8 gm/m2), achieves cytotoxic concentrations in the CSF and has been shown to prolong survival in patients with LC. Intrathecal liposomal cytarabine has been shown to increase time to neurologic progression in patients with breast cancer and LC. The combination of these 2 agents in LC has not been studied extensively. Here, we present the results of the phase II study with this combination showing promising efficacy and very good tolerability.

Organizational Characteristics Associated With the Predicted Sustainability of Villages.

Guided by resource dependence theory, this mixed-methods study examined organizational characteristics contributing to the perceived sustainability of Villages, a rapidly proliferating grassroots approach for promoting social participation and service access for community-dwelling older adults. Surveys conducted with leaders of 86% of Villages in the United States in 2012 found that higher predicted confidence in their Village's 10-year survival was associated with greater financial reserves, human resources, number of Village members, formal policies and procedures, and formal collaboration agreements. Respondents' explanations of their confidence ratings revealed additional themes of organizational leadership and perceived community need. Member resource inputs were not found to be as salient for Village leaders' perceptions of sustainability as was anticipated given the Village model's emphasis on consumer involvement. Despite the lack of longitudinal prospective data, study findings suggest potential limitations of consumer-driven organizational models such as Villages, including the need for a more stable resource base.

Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other's virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicansIMPORTANCEEnterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.

Phase II pilot study of bevacizumab in combination with temozolomide and regional radiation therapy for up-front treatment of patients with newly diagnosed glioblastoma multiforme: interim analysis of safety and tolerability.

PURPOSE: To assess interim safety and tolerability of a 10-patient, Phase II pilot study using bevacizumab (BV) in combination with temozolomide (TMZ) and regional radiation therapy (RT) in the up-front treatment of patients with newly diagnosed glioblastoma. METHODS AND MATERIALS: All patients received standard external beam regional RT of 60.0 Gy in 30 fractions started within 3 to 5 weeks after surgery. Concurrently TMZ was given daily at 75 mg/m(2) for 42 days during RT, and BV was given every 2 weeks at 10 mg/kg starting with the first day of RT/TMZ. After a 2-week interval upon completion of RT, the post-RT phase commenced with resumption of TMZ at 150 to 200 mg/m(2) for 5 days every 4 weeks and continuation of BV every 2 weeks. RESULTS: For these 10 patients, toxicities were compiled until study discontinuation or up to approximately 40 weeks from initial study treatment for those remaining on-study. In terms of serious immediate or delayed neurotoxicity, 1 patient developed presumed radiation-induced optic neuropathy. Among the toxicities that could be potentially treatment related, relatively high incidences of fatigue, myelotoxicity, wound breakdown, and deep venous thrombosis/pulmonary embolism were observed. CONCLUSION: The observed toxicities were acceptable to continue enrollment toward the overall target group of 70 patients. Preliminary efficacy analysis shows encouraging mean progression-free survival. At this time data are not sufficient to encourage routine off-label use of BV combined with TMZ/RT in the setting of newly diagnosed glioblastoma without longer follow-up, enrollment of additional patients, and thorough efficacy assessment.

Do Villages Promote Aging in Place? Results of a Longitudinal Study.

Villages are a new, grassroots, consumer-directed model that aims to promote aging in place and prevent unwanted relocations for older adults. In exchange for a yearly membership fee, Villages provide seniors with opportunities for social engagement (social events and classes), civic engagement (member-to-member volunteer opportunities), and an array of support services. In total, 222 Village members were surveyed at intake and 12-month follow-up to examine changes in their confidence aging in place, social connectedness, and health. The strongest positive results were in the domain of confidence, including significantly greater confidence aging in place, perceived social support, and less intention to relocate after 1 year in the Village. As most seniors were in good health and well connected at the time they joined the Village, there were not improvements in health or social connectedness. Authors discuss the importance of longer term, longitudinal studies to examine the effectiveness of Villages in preventing institutionalization over time.

Integration of Medicare and Medicaid for dually eligible beneficiaries: A focus group study examining beneficiaries' early experiences in California's dual financial alignment demonstration.

BACKGROUND: In 2014 California implemented a federal dual alignment demonstration used a capitated managed healthcare model called Cal MediConnect (CMC) to integrate medical care and long term services and supports (LTSS) for beneficiaries with both Medicare and Medicaid. These beneficiaries often have complex care needs, including multiple chronic conditions and disabilities. By 2016, 120,000 eligible beneficiaries were enrolled in the program. OBJECTIVES: Focus groups with enrolled beneficiaries were conducted to gather rich data about their early experiences with quality of care, access to care, and coordination of care in CMC plans and to identify recommendations for program improvement. METHODS: Evaluators conducted 14 focus groups with 104 beneficiaries enrolled in CMC plans in 6 demonstration counties. RESULTS: The passive enrollment process did not provide adequate information about certain aspects of CMC, leaving many beneficiaries unaware of new benefits such as care coordination, transportation, and managed LTSS. Most beneficiaries who were using the CMC care coordination benefit reported increased access to specialty care, medical equipment, and other LTSS. Changing providers and having trouble with authorization for specialty services, prescriptions, or medical equipment were common reasons for dissatisfaction. Many beneficiaries reported that early disruptions in care due to the transition of delivery system improved with time. CONCLUSION: Similar to other studies that examine beneficiaries' experiences with delivery system change, participants were confused about the passive enrollment process and demonstrated a lack of understanding of many aspects of Cal MediConnect. Analysis identified areas where beneficiaries noted improvement in their quality of care, access, and coordination of care, but also areas for improvement. Streamlining the authorization processes and extending continuity of care provisions would improve access to providers. Increasing beneficiaries' awareness of CMC plans' role in LTSS is key to improving their access to home- and community-based services.

Beneficiaries Respond To California's Program To Integrate Medicare, Medicaid, And Long-Term Services.

In 2014 California implemented a demonstration project called Cal MediConnect, which used managed care organizations to integrate Medicare and Medicaid, including long-term services and supports for beneficiaries dually eligible for Medicare and Medicaid. Postenrollment telephone surveys assessed how enrollees adjusted to Cal MediConnect over time. Results showed increased satisfaction with benefits, improved ratings of quality of care, fewer acute care visits, and increased personal care assistance hours over time. Enrollees also had somewhat better prescription medication access and lower unmet needs for personal care, compared to the comparison group. The lack of improvement in care coordination raises concerns about the implementation of the care coordination benefit, a key feature of the program. The Bipartisan Budget Act of 2018 contains provisions that permanently certify the use of managed care (such as Dual Eligible Special Needs Plans) to integrate Medicare and Medicaid, which makes the lessons learned from California's duals demonstration especially relevant for informing other integrated programs for seniors and people with disabilities.