Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr286 and Thr305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients

Policy Feedback and the Politics of the Affordable Care Act

There is a large body of literature devoted to how “policies create politics” and how feedback effects from existing policy legacies shape potential reforms in a particular area. Although much of this literature focuses on self‐reinforcing feedback effects that increase support for existing policies over time, Kent Weaver and his colleagues have recently drawn our attention to self‐undermining effects that can gradually weaken support for such policies. The following contribution explores both self‐reinforcing and self‐undermining policy feedback in relationship to the Affordable Care Act, the most important health‐care reform enacted in the United States since the mid‐1960s. More specifically, the paper draws on the concept of policy feedback to reflect on the political fate of the ACA since its adoption in 2010. We argue that, due in part to its sheer complexity and fragmentation, the ACA generates both self‐reinforcing and self‐undermining feedback effects that, depending of the aspect of the legislation at hand, can either facilitate or impede conservative retrenchment and restructuring. Simultaneously, through a discussion of partisan effects that shape Republican behavior in Congress, we acknowledge the limits of policy feedback in the explanation of policy stability and change

Factors associated with health-seeking behavior among migrant workers in Beijing, China

<p>Abstract</p> <p>Background</p> <p>Migrant workers are a unique phenomenon in the process of China's economic transformation. The household registration system classifies them as temporary residents in cities, putting them in a vulnerable state with an unfair share of urban infrastructure and social public welfare. The amount of pressure inflicted by migrant workers in Beijing, as one of the major migration destinations, is currently at a threshold. This study was designed to assess the factors associated with health-seeking behavior and to explore feasible solutions to the obstacles migrant workers in China faced with when accessing health-care.</p> <p>Methods</p> <p>A sample of 2,478 migrant workers in Beijing was chosen by the multi-stage stratified cluster sampling method. A structured questionnaire survey was conducted via face-to-face interviews between investigators and subjects. The multilevel methodology (MLM) was used to demonstrate the independent effects of the explanatory variables on health seeking behavior in migrant workers.</p> <p>Results</p> <p>The medical visitation rate of migrant workers within the past two weeks was 4.8%, which only accounted for 36.4% of those who were ill. Nearly one-third of the migrant workers chose self-medication (33.3%) or no measures (30.3%) while ill within the past two weeks. 19.7% of the sick migrants who should have been hospitalized failed to receive medical treatment within the past year. According to self-reported reasons, the high cost of health service was a significant obstacle to health-care access for 40.5% of the migrant workers who became sick. However, 94.0% of the migrant workers didn't have any insurance coverage in Beijing. The multilevel model analysis indicates that health-seeking behavior among migrants is significantly associated with their insurance coverage. Meanwhile, such factors as household monthly income per capita and working hours per day also affect the medical visitation rate of the migrant workers in Beijing.</p> <p>Conclusion</p> <p>This study assesses the influence of socio-demographic characteristics on the migrant workers' decision to seek health care services when they fall ill, and it also indicates that the current health service system discourages migrant workers from seeking appropriate care of good quality. Relevant policies of public medical insurance and assistance program should be vigorously implemented for providing affordable health care services to the migrants. Feasible measures need to be taken to reduce the health risks associated with current hygiene practices and equity should be assured in access to health care services among migrant workers.</p

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs

Preconception Care Between Pregnancies: The Content of Internatal Care

For more than two decades, prenatal care has been a cornerstone of our nation’s strategy for improving pregnancy outcomes. In recent years, however, a growing recognition of the limits of prenatal care and the importance of maternal health before pregnancy has drawn increasing attention to preconception and internatal care. Internatal care refers to a package of healthcare and ancillary services provided to a woman and her family from the birth of one child to the birth of her next child. For healthy mothers, internatal care offers an opportunity for wellness promotion between pregnancies. For high-risk mothers, internatal care provides strategies for risk reduction before their next pregnancy. In this paper we begin to define the contents of internatal care. The core components of internatal care consist of risk assessment, health promotion, clinical and psychosocial interventions. We identified several priority areas, such as FINDS (family violence, infections, nutrition, depression, and stress) for risk assessment or BBEEFF (breastfeeding, back-to-sleep, exercise, exposures, family planning and folate) for health promotion. Women with chronic health conditions such as hypertension, diabetes, or weight problems should receive on-going care per clinical guidelines for their evaluation, treatment, and follow-up during the internatal period. For women with prior adverse outcomes such as preterm delivery, we propose an internatal care model based on known etiologic pathways, with the goal of preventing recurrence by addressing these biobehavioral pathways prior to the next pregnancy. We suggest enhancing service integration for women and families, including possibly care coordination and home visitation for selected high-risk women. The primary aim of this paper is to start a dialogue on the content of internatal care

Efficacy of Increased Ube3a Protein Levels in the Brain in Rescuing the Phenotype of an Angelman Syndrome Mouse

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes an E6-AP ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. Although it was previously believed that UBE3A was imprinted in a brain region-specific manner, primarily in the hippocampus and cortex, recent evidence indicates that there is a widespread knockdown of Ube3a protein throughout the AS mouse brain. As a result, it became necessary to evaluate AS human brain samples to verify the relevance and accuracy of the AS mouse model. It was determined that Ube3a is deficient throughout all major brain regions in humans with AS. The remainder of this dissertation work was focused on determining if increased UBE3A expression in the AS mouse brain would be sufficient to rescue the AS phenotype. The results show that adeno-associated virus-mediated UBE3A delivery is not effective in the AS neonatal brain. In the adult AS mouse brain, however, it increased Ube3a in the hippocampus to near wild-type levels. This was sufficient to rescue the associative fear conditioning learning deficit in the AS mouse and improve learning and memory in the Morris water maze. These studies are the first to demonstrate that increased protein production in the adult AS mouse is sufficient to improve the AS phenotype, indicating that the symptoms of AS are not necessarily embryonic developmental

AS TR2-UBE3A mice had significant improvements in the Morris water maze.

<p>(A) Escape latency to reach the platform during 5 days of training in Morris water maze. The only significant differences seen were an increase in latency for the AS TR2-GFP mice on day 3 and a decrease in latency for wildtype mice on day 4 compared to the other two groups (2-way ANOVA Bonferroni: Interaction [F(8,100) = 1.01, <i>P>0.05</i>]; Treatment [F(2,100) = 5.30, <i>P<0.05</i>]; Time [F(4,100) = 53.49, <i>P<0.0001</i>]; Matching [F(25,100) = 5.37, <i>P<0.0001</i>]). The target platform is indicated by the black circles. (B) Quantification of the number of platform crossings in the target (T), opposite (O), right (R), and left (L) quadrants during the probe trial of the Morris water maze 24 hours after training indicate no significant differences among any of the groups in comparing target platform crossings to opposite platform crossings (ANOVA Tukey WT: [F(3,35) = 9.546, <i>P<0.0005</i>]; AS TR2-GFP: [F(3,35) = 3.186, <i>P<0.01</i>]; AS TR2-UBE3A: [F(3,39) = 2.814, <i>P<0.05</i>]). All three groups learned the platform location based on a spatial bias as indicated by the time spent in the target quadrants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027221#pone-0027221-g005" target="_blank">Fig. 5D</a> 24 h ANOVA Tukey [F(2,26) = 1.027, <i>P>0.05</i>]) (C) A probe test 72 hours after training indicate that the WT and AS TR2-UBE3A groups had significantly more target platform crossings compared to the number of opposite platform crossings (ANOVA Tukey WT: [F(3,35) = 6.086, <i>P<0.005</i>]; AS TR2-GFP: [F(3,35) = 0.5650, <i>P>0.05</i>]; AS TR2-UBE3A: [F(3,39) = 2.679, <i>P<0.05</i>]), but this improvement was not spatially biased as seen by the time spent in the target quadrant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027221#pone-0027221-g005" target="_blank">Fig. 5D</a> 72 h ANOVA Tukey [F(2,25) = 5.067, <i>P<0.02</i>]). Results shown represent the mean with standard error.</p

There were no changes in motor coordination, activity levels, or anxiety.

<p>(A) There was no change in latency to fall of the rotorod in either AS group. (B) Total distance travelled during the open field test revealed no significant difference between any of the treatment groups. (C) Time spent in the open arms of the elevated plus maze was used to determine general anxiety. (D) Time spent immobile in the elevated plus maze was not significantly different in any of the groups. Results shown represent the mean with standard error.</p

E6-AP protein levels were restored to wildtype levels in the TR2-UBE3A treated AS mice.

<p>(A) Representative coronal slices through the hippocampus from each group stained for E6-AP. (B) Quantitative analysis of the IHC revealed a significant increase in E6-AP expression in the WT and AS-TR2-UBE3A mice compared to the AS TR2-GFP group, while there was no significant change between the WT and AS TR2-UBE3A mice. Results shown represent the mean with standard error.</p

Increasing E6-AP in the AS mouse results in improvements in early phase LTP.

<p>(A) AS TR2-GFP mice have significant deficits in hippocampal synaptic plasticity. LTP was induced following 20 min of baseline recordings. (B) Immediately following TBS, acute hippocampal slices taken from AS TR2-GFP mice had significant deficits in the average PTP (average of first 5 min recordings of fEPSPs slopes). To compare late phase LTP, the last 5 min recordings of fEPSPs slopes were averaged, and there was no significant difference between any of the groups. (C) There were no significant differences between any of the groups in either PPF or (D) PTP, indicating that short term synaptic plasticity mechanisms are unaffected. Results shown represent the mean with standard error.</p