Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel.

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.

Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

Einstein@Home discovers a radio-quiet gamma-ray millisecond pulsar.

Millisecond pulsars (MSPs) are old neutron stars that spin hundreds of times per second and appear to pulsate as their emission beams cross our line of sight. To date, radio pulsations have been detected from all rotation-powered MSPs. In an attempt to discover radio-quiet gamma-ray MSPs, we used the aggregated power from the computers of tens of thousands of volunteers participating in the Einstein@Home distributed computing project to search for pulsations from unidentified gamma-ray sources in Fermi Large Area Telescope data. This survey discovered two isolated MSPs, one of which is the only known rotation-powered MSP to remain undetected in radio observations. These gamma-ray MSPs were discovered in completely blind searches without prior constraints from other observations, raising hopes for detecting MSPs from a predicted Galactic bulge population.

Open DeveloperSpace: An Enabling Infrastructure for Stakeholders to Generate New Access Solutions.

The DeveloperSpace, one of the core components of GPII, is a self-sustainable infrastructure and collaborative environment, where developers, implementers, consumers, prosumers and other directly and indirectly involved actors (e.g. teachers, caregivers, clinicians) may interact with and play a role in its viability and the development of new access solutions.

Use Model for a User Centred Design in Multidisciplinary Teams.

The Use Model identifies user groups who will be using services and products the Prosperity4All infrastructure offers. The Model provides developers a tool to keep in mind the full diversity of users while building and designing the infrastructure.

Judgments of learning as memory modifiers.

A frequent procedure used to study how individuals monitor their own learning is to collect judgments of learning (JOLs) during acquisition, considered to be important, in part, because such judgments are assumed to guide how individuals allocate their future learning resources. In such research, however, a tacit assumption is frequently made: Namely, that asking for such metacognitive judgments does not affect the learning process per se. In 3 experiments, the present research addressed the accuracy of this assumption and tested a possible account--based on aspects of Koriat's cue-utilization approach to JOLs (Koriat, 1997) and de Winstanley, Bjork, and Bjork's (1996) transfer-appropriate multifactor account of generation effects--for why the mere act of making JOLs might enhance later memory for the information so judged. Potential implications of the present findings for the future conduction of research using metacognitive measures as well as for students studying for exams is discussed.

Frontal fibrosing alopecia: a review of 60 cases.

BACKGROUND: Frontal fibrosing alopecia (FFA) is a variant of lichen planopilaris primarily affecting postmenopausal women, with a predilection for the frontotemporal hairline. OBJECTIVES: We sought to examine possible causal associations and review the clinical features, natural history, and response to treatment of patients with FFA attending a specialist hair clinic. METHODS: This was a case note review of 60 patients with FFA. RESULTS: The number of patients with FFA seen has increased over the last decade. All were Caucasian women, with significantly above-average affluence scores and were less likely to be smokers. The mean age at presentation was 64 years and average disease duration was 3.4 years (range: 6 months-30 years). Three patients were premenopausal. All patients had frontotemporal involvement, with follicular hyperkeratosis, scarring, and variable perifollicular erythema. Several patients had more unusual patterns: 8 had extensive parietal involvement, 4 had occipital involvement, 1 had asymmetric frontal involvement, and 5 had typical FFA associated with diffuse scalp lichen planopilaris. Eyebrow loss was documented in 73%, eyelash loss in 3%, and body hair loss in 25%. Almost all patients had been treated with superpotent topical steroids. Other treatments included topical calcineurin inhibitors; intralesional triamcinolone acetate; phototherapy; hydroxychloroquine; lymecycline; and prednisolone. Although some treatments may reduce inflammation, their efficacy in controlling the progress of the alopecia was uncertain. LIMITATIONS: This is a retrospective review. CONCLUSIONS: FFA is a clinically distinctive condition, the prevalence of which appears to be increasing. It has a generally poor response to treatment. The origin remains uncertain.

Using mass reconstruction along a four-site transect as a method to interpret PM10 in west-central Scotland, United Kingdom.

Concurrent 24-hr samples of particulate matter of median aerodynamic diameter less than 10 microm (PM10) were collected over a 10-day period in August 2000 at four sites along a transect in west-central Scotland, UK (passing from the coast through the city of Glasgow) in line with the prevailing southwesterly wind. Each sample was analyzed for chloride (Cl(-)), nitrate (NO3(-)), sulfate (SO4(2-)), ammonium (NH4(+)), calcium (Ca(2+)), iron (Fe), and organic hydrocarbon material (OHM). The contribution from elemental carbon (EC) was estimated. Sampling days were categorized according to local wind direction, synoptic flow, and air mass back trajectories. Chemical mass balance (CMB) reconstruction of the following PM10 components was derived for each wind direction group and at each transect location: ammonium sulfate ((NH4)2SO4), ammonium nitrate (NH4NO3), sodium chloride (NaCl), gypsum (CaSO4), OHM, EC, soil/surface dusts, and particle-bound water. The results showed that PM10 at the coastal site was dominated by the marine background (NaCl) compared with the urban sites, which were dominated by local primary (EC and soil/resuspension) and secondary sources (NH4NO3, (NH4)2SO4, and OHM). There was evidence of Cl(-) depletion as NaCl aerosol passes over urban areas. There was also evidence of long-range transport of primary PM10 (EC and OHM); for example, at the coastal site from transport from Ireland. The work demonstrates how the general approach of combining mass reconstruction along a transect with other information such as wind/air-mass direction generates insight into the sources contributing to PM10 over a more extended spatial scale than at a single receptor.

Identification of a secreted fatty acid and retinol-binding protein (Hp-FAR-1) from Heligmosomoides polygyrus.

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L4) H. polygyrus cDNA expression library using antibodies raised against L4 stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.

Homicides committed by youth assailants: a retrospective study.

The pediatric population has received considerable attention in the forensic community; the youth assailant of homicide, however, is understudied. The authors retrospectively reviewed all cases referred to the Forensic Pathology Section of the Medical University of South Carolina between January 1991 and May 2006. Cases included in the study were homicides in which 1 or more assailants were 19 years of age or younger. The cases were examined as to the cause and manner of death, victim age, gender, race, incident location, weapon used, assailant-victim relationship, assailant age, gender, race, motive, and postmortem toxicology results. Assailant information was obtained from forensic records at Medical University of South Carolina, police department records, and online search engines of South Carolina State newspaper archives confirmed by law enforcement reports. The youth assailants were predominantly black men, 15 to 19 years of age (range, 4-19 years). Most victims were black male acquaintances, and the motive was most often an argument. The most common cause of death was cerebral laceration because of a gunshot wound. The incident occurred in the home in 41% of cases, followed by the street in 31%. Victim toxicology was frequently positive for cocaine, marijuana, and alcohol.

Parental effort and reproductive skew in coalitions of brood rearing female common eiders.

Members of breeding groups face conflicts over parental effort when balancing antipredatory vigilance and feeding. Empirical evidence has shown disparate responses to manipulations of parental effort. We develop a model in which we determine the evolutionarily stable effort of partners given their body conditions, allowing the benefits of shared care to be unevenly divided, and we test this model's predictions with data on common eiders (Somateria mollissima). Eiders show uniparental female care; females may share brood rearing, or they may tend alone, and their body condition at hatching of the young shows large environmentally induced variation. The model predicts that parental effort (vigilance) in a coalition is lower than when tending alone, controlling for parental condition; this prediction is supported by the data. The parental effort in a coalition should be positively correlated with body condition, and this prediction is also supported. Finally, parental effort should increase when partner condition decreases and vice versa; this prediction is partially supported. The Nash bargaining game may provide promising avenues by which to determine the precise settlement of reproductive skew and effort between coalition partners in the future.

The characteristics of erythema induced by topical 5-aminolaevulinic acid photodynamic therapy.

BACKGROUND: Topical photodynamic therapy (PDT) is increasingly used to treat superficial non-melanoma skin cancers. Knowledge of the characteristics of 5-aminolaevulinic acid (ALA)-induced phototoxicity will increase our understanding of PDT and may facilitate optimisation of treatment regimes. METHODS: We examined the characteristics of ALA-induced erythema in 10 healthy subjects and investigated the effect of light source and body site. RESULTS AND CONCLUSIONS: Maximal erythema occurred within 1-2 h of PDT and inter-individual variation in ALA-induced phototoxicity was seen. No detectable differences were seen in the phototoxicity on back or leg sites or between coherent and non-coherent light sources. These data provide further information to allow us to optimise topical PDT regimes.

Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation.

There are unpredictable inter-individual differences in response to ultraviolet radiation, used in the treatment of psoriasis and other common skin diseases. It is therefore essential that we attempt to identify phenotypic markers that correlate with individual treatment outcomes. Exposure of human skin to ultraviolet radiation results in the generation of reactive intermediates and oxidative stress. Hepatic drug metabolizing and cytoprotective genes are induced as an adaptive response to xenobiotics and reactive intermediates; as several of these genes are present in skin, we hypothesized that their cutaneous expression and regulation may be implicated in responses to ultraviolet radiation. We used quantitative real-time reverse transcription-polymerase chain reaction to investigate interindividual differences in the cutaneous expression of a variety of drug metabolizing and cytoprotective genes, including cytochrome P450s, glutathione S-transferases and drug transporters, and investigated the regulation of gene expression by ultraviolet radiation and in lesional psoriatic skin. We confirmed significant induction of cyclooxygenase 2 (mean 3.63-fold, range 0.14-22.6, p<0.0001) by ultraviolet radiation and showed more modest (approximately 2-fold) inductions of glutathione peroxidase, and novel inductions of glutathione S-transferase P1 and the drug transporter multidrug resistance associated protein-1. Glutathione S-transferase P1 (3.74-fold, 1.3-33.1, p<0.0001) and multidrug resistance associated protein-1 (4.06-fold, 1.3-24.8, p<0.0001) were also significantly increased in psoriatic plaque, as were P450 CYP2E1 (3.64-fold, 1-28.9 p<0.0001) and heme oxygenase-1 (10.19-fold, 2.9-49.7, p<0.0001), implying a differential adaptive response to oxidant exposure in lesional psoriatic skin. We found considerable interindividual variation in constitutive gene expression and inducibility, indicating that these genes may be associated with individuality in response to ultraviolet radiation.