SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle.

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.

Consumer-centric approach to enhance access to pediatric specialty care.

OBJECTIVES: Health systems must adapt to an increased consumer-centric environment to remain relevant in an ever-growing competitive health care landscape in which convenience is a key driver of patient satisfaction and loyalty. To adapt to this new environment, health systems must redesign processes to transform the delivery of ambulatory care and provide near real-time access to specialty care. STUDY DESIGN: A pediatric academic medical center in western Pennsylvania used a process-improvement approach to enhance timely access to specialty care and deliver a consumer-centric patient experience. METHODS: Critical factors in this process included engagement of key stakeholders, implementation of scheduling best practices, development of a set of scheduling guidelines, increased use of advanced practice providers, and use of data analytics to measure and benchmark performance. RESULTS: The time to schedule a new patient appointment decreased from 42 to 4 days and the patient satisfaction access domain increased by 57 percentile points. CONCLUSIONS: These factors should scale to other institutions, thereby enabling generalizable results.

Functionally uncoupled transcription-translation in Bacillus subtilis.

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

Examining the impact of modality and learning style preferences on recall of psychiatric nursing and pharmacology terms.

The purpose of this experimental research study was to explore how modality and learning style preferences impact non-prescribing, first-year Licensed Vocational Nurse (LVN) students' recall of vocabulary. Independent t-test results indicated a statistically significant mean difference in short-term recall of pharmacological and psychiatric terms, with learners receiving visual text instruction recalling significantly more vocabulary than learners receiving audio text instruction. A correlation was not found between learning preferences and vocabulary recall.

User-Centered Design of a Mobile App for Weight and Health Management in Adolescents With Complex Health Needs: Qualitative Study.

BACKGROUND: Growing research has been conducted into the deployment and evaluation of mobile technology interventions for weight management in adolescents. However, no work has yet been conducted toward the development of these technologies for adolescents with complex health needs receiving specialized tertiary-level health care. OBJECTIVE: The aim of this study was to conduct a user-centered needs assessment of adolescents interested in weight management with complex health needs requiring specialized health care services, their parents, and health care providers (HCPs) to inform the design and development of a mobile app for weight and health management. METHODS: A qualitative study design was employed. Participants were recruited from two tertiary health care centers. Separate audiotaped focus group interviews were conducted with adolescents aged 12 to 18 years, parents, and HCPs. Interviews were transcribed, and field notes were collected by research staff. Iterative simple content analysis was performed independently by 4 research team members using computer software NVivo (QSR International) 10.0. RESULTS: A total of 19 adolescents, 16 parents, and 21 HCPs were interviewed. Qualitative analysis revealed seven major themes related to app functionality: healthy eating, social support, self-monitoring, communicating with HCPs, supporting mental health, gamification and incentives, and user interface (UI) design. Adolescents provided several ideas related to each feature, whereas parents' views focused on assistance with meal planning and greater access to HCPs. HCPs viewed the app as a novel and more acceptable platform to connect remotely with adolescents than conventional methods. They also strongly endorsed the value of social support capabilities and the ability to connect with an HCP. CONCLUSIONS: This is the first study to conduct a qualitative needs assessment in adolescents receiving specialized health care services toward the design of a mobile app for weight and health management. Our results indicate that core components of the app should include tailored meal recommendations and assistance with meal planning, social networking for peer support, customized and convenient tracking, remote access to HCPs, features to support mental health, and an attractive and engaging UI. These findings will be used to develop and evaluate a mobile app targeting adolescents with complex health needs.

Early introduction of peanuts.

Peanut allergy is the most common food allergy and the leading cause of anaphylaxis and death due to food allergy. Despite previous guidelines created by the American Academy of Pediatrics in 2003, peanut allergy has continued to significantly increase over the past decade. Therefore, further research has been done to help clinicians provide more evidence-based recommendations about the timing of introduction of peanuts. The LEAP study, published in February 2015, demonstrated the value of much earlier introduction of peanuts to nonallergic patients than previously suggested. These findings have altered current practice, and recommendations supported by the American Academy of Pediatrics now allow the introduction of peanuts as early as age 4 months to reduce the likelihood of developing peanut allergy.

Communicating about obesity and weight-related topics with children with a physical disability and their families: spina bifida as an example.

PURPOSE: The purpose of this study was to explore the experiences of children with spina bifida (SB), their families and healthcare professionals (HCPs) when discussing weight-related topics. METHOD: In-depth qualitative interviews were conducted with HCPs from Canadian outpatient SB clinics (n = 13), children aged 6-18 years with SB (n = 17) and their parents (n = 20). Data were analyzed using a phenomenological approach within an interpretative paradigm. RESULTS: Many HCPs were not confident talking about weight, concerned that they would damage relationships with children and families. Parents wanted routine weight surveillance, but were worried about their children's self-esteem if their weight was discussed. They wanted HCPs to acknowledge the challenges of weight management in children with a physical disability and provide specialized solutions. Children wanted a positively framed and tailored approach to weight discussions, although this had generally not been their experience. CONCLUSIONS: Stakeholders describe therapeutic relationships that are currently disconnected around the issue of weight and obesity. However, children, parents and HCPs all believed that discussing this topic was critical. Positively framed, strengths-based and tailored approaches to weight-related discussions are warranted. Implications for Rehabilitation Rates of overweight and obesity in children and youth with physical disabilities are substantially higher than their typically developing peers. Healthcare professionals, children with physical disabilities and families often find weight-related discussions challenging and disconnected. Weight-related discussions should be tailored to the child and family's circumstances and priorities. Positively framed and strengths-based approaches to weight-related discussions are warranted.

A Cautionary Note: Ceriodaphnia dubia Inter-Laboratory Test Variability.

An inter-laboratory comparison of the three-brood survival and reproduction Ceriodaphnia dubia toxicity test was conducted involving three toxicity testing laboratories. This comparison was initiated due to sporadic toxicity with this test related to discharge of a mine effluent, which some regulators believed indicated adverse effects in the receiving environment. One mine effluent and two receiving water samples were evaluated. There were no adverse effects on C. dubia survival in any tests. However, sublethal effects on reproduction (i.e., the IC25; the concentration that causes a 25 % inhibitory effect in the measured sublethal endpoint) ranged from 35 % to >100 % among all test treatments. All laboratories were certified for this test and followed established procedures. Dilute mineral water appears to be the best medium for culturing, dilution, and control water. Variability in the C. dubia laboratory toxicity test should not be mistaken for adverse effects in receiving environments.

Double-Blind, Placebo-Controlled Pilot Study on the Use of Platelet-Rich Plasma in Women With Female Androgenetic Alopecia.

BACKGROUND: Platelet-rich plasma (PRP) has been suggested as a therapeutic intervention for female androgenetic alopecia. OBJECTIVE: To perform a pilot study on the effect of PRP scalp injections in women with female androgenetic alopecia. MATERIALS AND METHODS: This double-blind, multicenter, placebo-controlled study compared the effect of PRP with that of saline placebo as scalp injection. The endpoints were hair count and hair mass index (HMI), along with patient-opinion survey responses. Platelet-rich plasma was manufactured from patients' blood using the Angel PRP system. RESULTS: Hair mass index or hair count did not statistically significantly differ between the study and placebo groups. However, 13.3% of the treatment subjects (vs 0% of the placebo subjects) experienced substantial improvement in hair loss, rate of hair loss, hair thickness, and ease of managing/styling hair, and 26.7% (vs 18.2% of the placebo group) reported that their hair felt coarser or heavier after the treatment. CONCLUSION: Platelet-rich plasma failed to demonstrate any statistically significant improvement in HMI or hair count in women with congenital female pattern hair loss. The patient survey results suggest a therapeutic advantage of PRP as perceived by patients but not according to hair count or HMI.

Outcomes before and after implementation of a pediatric rapid-response extracorporeal membrane oxygenation program.

BACKGROUND: Rapid-response extracorporeal membrane oxygenation (RR-ECMO) has been implemented at select centers to expedite cannulation for patients placed on ECMO during extracorporeal cardiopulmonary resuscitation (ECPR). In 2008, we established such a program and used it for all pediatric venoarterial ECMO initiations. This study was designed to compare outcomes before and after program implementation. METHODS: Between 2003 and 2011, 144 pediatric patients were placed on venoarterial ECMO. Records of patients placed on ECMO before (17 ECPR and 62 non-ECPR) or after (14 ECPR and 51 non-ECPR) RR-ECMO program implementation were retrospectively compared. RESULTS: The peak performance of the ECMO team was assessed by measuring ECMO initiation times for the ECPR patient subgroup (n = 31). There was a shift toward more ECPR initiations achieved in less than 40 minutes (24% pre-RR-ECMO versus 43% RR-ECMO; p = 0.25) and fewer requiring more than 60 minutes (47% pre-RR-ECMO versus 21% RR-ECMO; p = 0.14) after program implementation, although these changes did not reach statistical significance. After multivariable risk adjustment, RR-ECMO was associated with a 52% reduction in neurologic complications for all patients (adjusted odds ratio, 0.48; 95% confidence interval, 0.23 to 0.98; p = 0.04), but the risk of in-hospital death remained unchanged (adjusted odds ratio, 0.99; 95% confidence interval, 0.50 to 1.99; p = 0.99). CONCLUSIONS: Implementation of a pediatric RR-ECMO program for venoarterial ECMO initiation was associated with reduced neurologic complications but not improved survival during the first 3 years of program implementation. These data suggest that development of a coordinated system for rapid ECMO deployment may benefit both ECPR and non-ECPR patients, but further efforts are required to improve survival.

Extracorporeal membrane oxygenation for severe refractory respiratory failure secondary to 2009 H1N1 influenza A.

BACKGROUND: Respiratory failure and acute respiratory distress syndrome secondary to H1N1 influenza infection is a source of substantial morbidity and mortality, having caused over 265,000 hospitalizations in the United States in 2009. During the H1N1 pandemic, up to 31% of the H1N1 patients required intensive care unit admission, and many were refractory to maximal conventional therapies. These most critically ill patients may require extracorporeal membrane oxygenation (ECMO) for survival. METHODS: We retrospectively reviewed the medical records of the 7 patients with refractory hypoxemia due to H1N1 influenza who were treated with ECMO in our pediatric intensive care unit. RESULTS: Five of the 7 patients survived to hospital discharge. The cohort's mean age was 21 years, and 4 were female. At admission to the pediatric intensive care unit, 6 had at least one comorbid condition, 6 were mechanically ventilated, and one was in shock. All 7 patients were treated with oral oseltamivir, high-frequency oscillatory ventilation, and inhaled nitric oxide prior to ECMO. Five received intravenous steroids, and 2 were treated with compassionate-use intravenous zanamivir. The mean duration of pre-ECMO ventilation was 8.7 days (range 14 h to 25 d). Mean oxygenation index was 50 (range 26-73) at ECMO cannulation. Six received venovenous ECMO, and one received venoarterial ECMO. The mean duration of ECMO was 432 hours (range 192-890 h). CONCLUSIONS: This series suggests that ECMO is a viable treatment for refractory hypoxemia secondary to H1N1 influenza infection in both pediatric and adult patients.

Development of a collaborative program to provide extracorporeal membrane oxygenation for adults with refractory hypoxemia within the framework of a pandemic.

OBJECTIVE: We report the process used to rapidly develop a collaborative adult respiratory extracorporeal membrane oxygenation program as a response to caring for young adult patients with refractory hypoxemia in the setting of the pH1N1 pandemic. DESIGN: Interdisciplinary response of a complex medical system to a public health crisis. PATIENTS, INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: After the successful use of extracorporeal membrane oxygenation in young adults with pH1N1-induced acute respiratory distress syndrome refractory to conventional therapies, an adult venovenous extracorporeal membrane oxygenation program was implemented over an 8-wk period. Implementation of this program involved a number of key steps that were crucial in the development process, including administrative and institutional support, multidisciplinary leadership and collaboration, extensive interdisciplinary educational initiatives, and substantial technical modifications. CONCLUSIONS: In the setting of the pH1N1 influenza pandemic, an adult respiratory extracorporeal membrane oxygenation program was successfully developed to complement an established neonatal-pediatric program. This program expansion integrated all of the necessary components involved in the development process from start to finish and confirms that a healthcare system can respond very quickly and successfully to an urgent healthcare need.

Successful use of extracorporeal membrane oxygenation for pH1N1-induced refractory hypoxemia in a child with hypoplastic left heart syndrome.

OBJECTIVE: To report the first successful use of extracorporeal membrane oxygenation for acute respiratory distress syndrome secondary to 2009 pH1N1 influenza A infection in a child status post the Fontan operation for hypoplastic left heart syndrome. DESIGN: Individual case report. SETTING: Pediatric intensive care unit. PATIENT: We describe a 7-yr-old boy with a history of the Fontan operation for hypoplastic left heart syndrome admitted with acute respiratory distress syndrome secondary to 2009 pH1N1 influenza A infection. INTERVENTION: Cannulation for venoarterial extracorporeal membrane oxygenation. MEASUREMENTS AND MAIN RESULTS: In this patient with a history of complex congenital heart disease and repair, extracorporeal membrane oxygenation was a successful rescue therapy for refractory pH1N1-induced respiratory failure. CONCLUSION: Extracorporeal membrane oxygenation can be successfully applied for refractory respiratory failure, even in the setting of significant underlying comorbidity. With emerging data to support the role of extracorporeal membrane oxygenation in improving mortality for refractory hypoxemia secondary to pH1N1, it is prudent to strongly consider the use of extracorporeal support in patients with underlying diseases or comorbidities that may have previously precluded them from being candidates for this therapy.

Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor.

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.

Changes in morphine analgesia and side effects during daily subcutaneous administration in healthy volunteers.

Tolerance to the anti-nociceptive effects of opioids develops rapidly in animals. In contrast, humans with chronic pain show little or no loss of pain relief in prospective opioid trials of 4-8 weeks duration. Employing the Brief Thermal Sensitization model to induce transient cutaneous secondary hyperalgesia, we tested the hypothesis that opioid analgesic tolerance would develop rapidly. In this outpatient randomized placebo-controlled study, subjects in the MMMMP group received two injections of subcutaneous morphine 6 mg (150 min apart) on Monday-Thursday (total 48 mg over 4 days) and matching saline placebo on Friday. Subjects in the PPPPM group received placebo on Monday-Thursday and morphine (total 12 mg) on Friday. Sixty-one healthy volunteers were enrolled; morphine side effects accounted for all nine non-completions. Compared to the first placebo day, the reduction in the area of secondary hyperalgesia on the first morphine day was significant and robust in both groups. Morphine suppression of the painfulness of skin heating and elevation of the heat pain detection threshold were also significant. During 4 days of twice-daily injections, the decline in anti-hyperalgesic effects of morphine did not reach statistical significance (p=0.06) compared to placebo. Morphine side effects did not correlate with anti-hyperalgesic effects and withdrawal symptoms did not emerge. As 4 days is the threshold for demonstrating analgesic tolerance to twice-daily morphine in animal models, a longer period of opioid exposure in healthy volunteers might be needed to detect analgesic tolerance.

Attenuation of chicken anemia virus by site-directed mutagenesis of VP2.

Chicken anemia virus (CAV) is a significant immunosuppressive pathogen of chickens, but relatively little is known about the effect of specific mutations on its virulence. In order to study the virulence of CAV, an infection model was developed in embryos. Significant growth depression, measured as a reduction in mean body weight, was found for wild-type CAV infection. Infection with wild-type CAV resulted in a significant reduction in thymic and splenic weights and consistently produced severe lesions in the thymus, spleen and bone marrow, as well as haemorrhages. CAVs mutated in the VP2 gene were infectious for embryos, but were highly attenuated with respect to growth depression and CAV-specific pathology. Relative to wild-type infection, viruses Mut C86R, Mut R101G, Mut H103Y, Mut R129G, Mut Q131P, Mut R/K/K150/151/152G/A/A, Mut D/E161/162G/G and Mut E186G were highly attenuated, and viruses Mut L163P and Mut D169G were moderately attenuated. Attenuation of the ability to produce lesions was found consistently for the thymus, spleen and bone marrow, thymic and splenic weights, and for CAV-induced haemorrhage. There was no growth depression associated with infection by the group of highly attenuated mutant viruses and a moderate reduction in mean body weight was only found for virus Mut L163P. These findings show that mutations in the VP2 gene can reduce the virulence of CAV and these mutant viruses may have value as vaccine candidates.

Depressive symptoms in early marriage: predictions from relationship confidence and negative marital interaction.

The authors proposed a model of depressive symptoms in early marriage in which relationship confidence, defined as perceived couple-level efficacy to manage conflicts and maintain a healthy relationship, mediates the effect of negative marital interactions on depressive symptoms. The model was tested in a sample of 139 couples assessed prior to marriage and 1 year later. As predicted, relationship confidence demonstrated simple negative associations with negative marital interaction and depressive symptoms for all participants. Longitudinal path analyses supported the mediational model for women only. In women but not men, negative marital interaction indirectly had an impact on depressive symptoms through the mediator of relationship confidence. Findings suggest that relationship confidence may be important to understanding links between marital distress and depressive symptoms, especially in women.