The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2).

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (Aß) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aß aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aß is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aß. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aß effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aß1-42, the most cytotoxic and aggregate-prone amongst the Aß family. Furthermore, exposure to physiological amounts of Aß1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aß persisted as discrete puncta for as long as 72 h. To assess whether Aß could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aß1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aß deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aß aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aß to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD.

Lot quality assurance sampling to monitor supplemental immunization activity quality: an essential tool for improving performance in polio endemic countries.

Monitoring the quality of supplementary immunization activities (SIAs) is a key tool for polio eradication. Regular monitoring data, however, are often unreliable, showing high coverage levels in virtually all areas, including those with ongoing virus circulation. To address this challenge, lot quality assurance sampling (LQAS) was introduced in 2009 as an additional tool to monitor SIA quality. Now used in 8 countries, LQAS provides a number of programmatic benefits: identifying areas of weak coverage quality with statistical reliability, differentiating areas of varying coverage with greater precision, and allowing for trend analysis of campaign quality. LQAS also accommodates changes to survey format, interpretation thresholds, evaluations of sample size, and data collection through mobile phones to improve timeliness of reporting and allow for visualization of campaign quality. LQAS becomes increasingly important to address remaining gaps in SIA quality and help focus resources on high-risk areas to prevent the continued transmission of wild poliovirus.

Single-entity hydrocodone extended-release capsules in opioid-tolerant subjects with moderate-to-severe chronic low back pain: a randomized double-blind, placebo-controlled study.

OBJECTIVE: A single-agent, extended-release formulation of hydrocodone (HC) has been developed for treatment of chronic moderate-to-severe pain. This study was designed to examine the safety and efficacy of HC extended release in opioid-experienced adults with moderate-to-severe chronic low back pain (CLBP). METHODS: This multicenter, enriched enrollment, randomized withdrawal study comprised an open-label conversion/titration phase (≤6 weeks) followed by placebo-controlled, double-blind treatment (12 weeks). During the conversion/titration phase, subjects (N = 510) converted from their current opioid and were titrated to a stabilized dose of HC extended release (20-100 mg every 12 hours). During treatment, subjects (N = 151 per group) received HC extended release or placebo; rescue medication was permitted. The primary efficacy end point was mean change in average pain intensity from baseline to day 85. Response rates (30% pain improvement) and satisfaction (Subject Global Assessment of Medication) were assessed. RESULTS: Demographic and baseline characteristics were similar between groups. Mean ± SD change in average pain intensity score from baseline to day 85 was significantly lower in the HC extended-release treatment group vs placebo (0.48 ± 1.56 vs 0.96 ± 1.55; P = 0.008). Significantly more responders were in the treatment group (68% vs 31%; P < 0.001). Mean Subject Global Assessment of Medication scores increased significantly (0.8 ± 1.3 vs 0.0 ± 1.4; P < 0.0001), indicating greater satisfaction with HC extended release. The adverse event profile was consistent with other opioids. CONCLUSIONS: Extended-release HC is well tolerated and effective, without acetaminophen-associated risks of liver toxicity, for treatment of CLBP.

Mutations on the N-terminal edge of the DELSEED loop in either the α or ß subunit of the mitochondrial F1-ATPase enhance ATP hydrolysis in the absence of the central γ rotor.

F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)ß(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)ß(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)ß(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and ß subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the ß subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the ß subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or ß subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)ß(3) subcomplex.

The impact of geographic inequality in federal research funding: A comparative longitudinal study of research and scholarly outputs in EPSCoR versus non-EPSCoR states.

Some states in the U.S. have traditionally received less federal research funding than other states. The National Science Foundation (NSF) created a program in 1979, called the Experimental Program to Stimulate Competitive Research (EPSCoR) to enhance the research competitiveness in such states. While the geographic disparity in federal research funding is well known, the overall impact of federal funding on the research performance of EPSCoR and non-EPSCoR has not been previously studied. In the current study, we compared the combined research productivity of Ph.D. granting institutions in EPSCoR versus the non-EPSCoR states to better understand the scientific impact of federal investments in sponsored research across all states. The research outputs we measured included journal articles, books, conference papers, patents, and citation count in academic literature. Unsurprisingly, results indicated that the non-EPSCoR states received significantly more federal research funding than their EPSCoR counterparts, which correlated with a higher number of faculty members in the non-EPSCoR versus EPSCoR states. Also, in the overall research productivity expressed on a per capita, the non-EPSCoR states fared better than EPSCoR states. However, when the research output was measured based on per $1M investment of federal research funding, EPSCoR states performed significantly better than the non-EPSCoR states in many research productivity indicators, with the notable exception of patents. Together, this study found preliminary evidence that EPSCoR states achieved a high degree of research productivity despite receiving significantly fewer federal research dollars. The limitations and next steps of this study are also discussed.

Binary Classification of the Endocrine Disrupting Chemicals by Artificial Neural Networks.

We develop a machine learning framework that integrates high content/high throughput image analysis and artificial neural networks (ANNs) to model the separation between chemical compounds based on their estrogenic receptor activity. Natural and man-made chemicals have the potential to disrupt the endocrine system by interfering with hormone actions in people and wildlife. Although numerous studies have revealed new knowledge on the mechanism through which these compounds interfere with various hormone receptors, it is still a very challenging task to comprehensively evaluate the endocrine disrupting potential of all existing chemicals and their mixtures by pure in vitro or in vivo approaches. Machine learning offers a unique advantage in the rapid evaluation of chemical toxicity through learning the underlying patterns in the experimental biological activity data. Motivated by this, we train and test ANN classifiers for modeling the activity of estrogen receptor-α agonists and antagonists at the single-cell level by using high throughput/high content microscopy descriptors. Our framework preprocesses the experimental data by cleaning, scaling, and feature engineering where only the middle 50% of the values from each sample with detectable receptor-DNA binding is considered in the dataset. Principal component analysis is also used to minimize the effects of experimental noise in modeling where these projected features are used in classification model building. The results show that our ANN-based nonlinear data-driven framework classifies the benchmark agonist and antagonist chemicals with 98.41% accuracy.

Unveiling the mystery of mitochondrial DNA replication in yeasts.

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing.

Mental health law in Ghana.

In 2012 Ghana passed a new Mental Health Act, which aimed to create a new system of mental healthcare in Ghana. The Act includes provisions for the creation of a modern, community-based mental health system and for the protection of the rights of persons with mental disorders. This article discusses the implications of the Act and the progress which has been made towards its implementation.

Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds.

Unintended inhibition of the cardiac potassium channel human ether-a-go-go-related gene (hERG) is considered the main culprit in drug-induced arrhythmias known as torsades de pointes. Electrophysiology is the most reliable in vitro screening method for identifying potential cardiac hERG liabilities, but only the recent advent of planar electrode-based voltage clamp electrophysiology promises sufficient throughput to support the drug testing needs of most drug discovery programs. We have assessed the reliability of this new format of the voltage clamp technology in measuring the activity of small molecules on the hERG channel. Based on the results herein of a screening against a panel of well-characterized hERG-active and -inactive molecules, we demonstrate that planar electrode electrophysiology, utilizing the Sealchip and PatchXpress technology platform (AVIVA Biosciences Corp., San Diego, CA), is comparable to traditional electrophysiology based on glass micropipettes in its reliability and data content. The new technology will allow significantly higher throughput and more thorough testing of pharmaceutical compounds.

The N-terminal intrinsically disordered domain of Mgm101p is localized to the mitochondrial nucleoid.

The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted.

Bacterial degradation of risperidone and paliperidone in decomposing blood.

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high-performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole-cleaved degradation products for both drugs were identified as 2-hydroxybenzoyl-risperidone and 2-hydroxybenzoyl-paliperidone utilizing liquid chromatography quadrupole-time-of-flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.

The F1-ATPase inhibitor Inh1 (IF1) affects suppression of mtDNA loss-lethality in Kluyveromyces lactis.

Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of Inh1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of Inh1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing Inh1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of Inh1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of Inh1p.

A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.

Analysis of Mgm101p isolated from mitochondria shows that the mature protein of 27.6 kDa lacks 22 amino acids from the N-terminus. This mitochondrial targeting sequence has been incorporated in the design of oligonucleotides used to determine a functional core of Mgm101p. Progressive deletions, although retaining the targeting sequence, reveal that 76 N-terminal and six C-terminal amino acids of Mgm101p can be removed without altering the ability to complement an mgm101-1(ts) temperature-sensitive mutant. However, this active core is unable to complement mgm101 null mutants, suggesting that the Mgm101p might need to form a dimer or multimer to be functional in vivo. The active core, enriched in basic residues, contains 165 amino acids with a pI of 9.2. Alignment with 22 Mgm101p sequences from other lower eukaryotes shows that a number of amino acids are highly conserved in this region. Random mutagenesis confirms that certain critical amino acids required for function are invariant across the 23 proteins. Searches in the PFAM database revealed a low level of structural similarity between the active core and the Rad52 protein family.

Bacteriology, Water Activity and Moisture/Salt Ratio of Six Brands of Precooked Canned Bacon.

Six commercial brands of precooked canned bacon, comprising 101 cans, were examined to determine if they complied with military specifications for a moisture-to-salt (M/S) ratio (percent moisture divided by percent salt) of ⩽ 9.0. Three brands were found in compliance with expected lot average values (ELAV) for M/S ratio of 4.70, 5.58 and 6.10. Water activity ELAVs of samples from these three brands were 0.82, 0.89 and 0.91; aerobic plates counts (APCs) ranged from <100 (64%) to 1500/g. Brands not in compliance had M/S ratio ELAVs of 11.24, 12.00 and 12.83; water activity ELAVs of 0.93, 0.97 and 0.99; and APCs as high as 1.7 × 107/g.

Does aromatherapy massage benefit patients with cancer attending a specialist palliative care day centre?

A randomised controlled pilot study was carried out to examine the effects of adjunctive aromatherapy massage on mood, quality of life and physical symptoms in patients with cancer attending a specialist unit. Participants were randomised to conventional day care alone or day care plus weekly aromatherapy massage using a standardised blend of oils for four weeks. At baseline and at weekly intervals, patients rated their mood, quality of life and the intensity and bother of two symptoms most important to them. Forty-six patients were recruited to the study. Due to a large number of withdrawals, only 11 of 23 (48%) patients in the aromatherapy group and 18 of 23 (78%) in the control group completed all four weeks. Mood, physical symptoms and quality of life improved in both groups. There was no statistically significant difference between groups in any of the outcome measures. Despite a lack of measurable benefit, all patients were satisfied with the aromatherapy and wished to continue. Whilst this pilot study has shown that a randomised controlled trial of complementary therapy is feasible, it has also identified several areas that would require further consideration when designing future studies, e.g., the recruitment and retention of appropriate numbers of patients and the outcome measures used.