Calibration process for rechargeable cell and battery test systems.

Coulombic efficiency is a powerful metric for evaluating the performance of materials in rechargeable cells and batteries. The ideal Coulombic efficiency, the ratio of charge removed to charge inserted, is unity. Some specialized systems can accurately measure cell capacity and Coulombic efficiency within 0.001%, which requires precise control and measurement of current, voltage, time, and temperature. Most battery electrode and electrolyte research is not performed with such precise but complex systems. The purpose of this paper is to demonstrate a simple, robust procedure to measure and possibly improve the accuracy of capacity and Coulombic efficiency measurements on standard systems in their as-used state. This approach is built on a commercially available thin film rechargeable cell for micro or milliampere currents and can be extended to, e.g., 18 650, cells for higher currents. An improved method to display Coulombic efficiency data is also presented. Regular, consistent calibration of testing systems and reporting of system resolution at specified test conditions is encouraged.

Large-scale pharmacogenomic study of sulfonylureas and the QT, JT and QRS intervals: CHARGE Pharmacogenomics Working Group.

Sulfonylureas, a commonly used class of medication used to treat type 2 diabetes, have been associated with an increased risk of cardiovascular disease. Their effects on QT interval duration and related electrocardiographic phenotypes are potential mechanisms for this adverse effect. In 11 ethnically diverse cohorts that included 71 857 European, African-American and Hispanic/Latino ancestry individuals with repeated measures of medication use and electrocardiogram (ECG) measurements, we conducted a pharmacogenomic genome-wide association study of sulfonylurea use and three ECG phenotypes: QT, JT and QRS intervals. In ancestry-specific meta-analyses, eight novel pharmacogenomic loci met the threshold for genome-wide significance (P<5 × 10-8), and a pharmacokinetic variant in CYP2C9 (rs1057910) that has been associated with sulfonylurea-related treatment effects and other adverse drug reactions in previous studies was replicated. Additional research is needed to replicate the novel findings and to understand their biological basis.

Preclinical Testing of a Novel Thin Film Nitinol Flow-Diversion Stent in a Rabbit Elastase Aneurysm Model.

BACKGROUND AND PURPOSE: Thin film nitinol can be processed to produce a thin microporous sheet with a low percentage of metal coverage (<20%) and high pore attenuation (∼70 pores/mm(2)) for flow diversion. We present in vivo results from the treatment of experimental rabbit aneurysms by using a thin film nitinol-based flow-diversion device. MATERIALS AND METHODS: Nineteen aneurysms in the rabbit elastase aneurysm model were treated with a single thin film nitinol flow diverter. Devices were also placed over 17 lumbar arteries to model perianeurysmal branch arteries of the intracranial circulation. Angiography was performed at 2 weeks (n = 7), 1 month (n = 8), and 3 months (n = 4) immediately before sacrifice. Aneurysm occlusion was graded on a 3-point scale (grade I, complete occlusion; grade II, near-complete occlusion; grade III, incomplete occlusion). Toluidine blue staining was used for histologic evaluation. En face CD31 immunofluorescent staining was performed to quantify neck endothelialization. RESULTS: Markedly reduced intra-aneurysmal flow was observed on angiography immediately after device placement in all aneurysms. Grade I or II occlusion was noted in 4 (57%) aneurysms at 2-week, in 6 (75%) aneurysms at 4-week, and in 3 (75%) aneurysms at 12-week follow-up. All 17 lumbar arteries were patent. CD31 staining showed that 75% ± 16% of the aneurysm neck region was endothelialized. Histopathology demonstrated incorporation of the thin film nitinol flow diverter into the vessel wall and no evidence of excessive neointimal hyperplasia. CONCLUSIONS: In this rabbit model, the thin film nitinol flow diverter achieved high rates of aneurysm occlusion and promoted tissue in-growth and aneurysm neck healing, even early after implantation.

Novel loci associated with usual sleep duration: the CHARGE Consortium Genome-Wide Association Study.

Usual sleep duration is a heritable trait correlated with psychiatric morbidity, cardiometabolic disease and mortality, although little is known about the genetic variants influencing this trait. A genome-wide association study (GWAS) of usual sleep duration was conducted using 18 population-based cohorts totaling 47 180 individuals of European ancestry. Genome-wide significant association was identified at two loci. The strongest is located on chromosome 2, in an intergenic region 35- to 80-kb upstream from the thyroid-specific transcription factor PAX8 (lowest P=1.1 × 10(-9)). This finding was replicated in an African-American sample of 4771 individuals (lowest P=9.3 × 10(-4)). The strongest combined association was at rs1823125 (P=1.5 × 10(-10), minor allele frequency 0.26 in the discovery sample, 0.12 in the replication sample), with each copy of the minor allele associated with a sleep duration 3.1 min longer per night. The alleles associated with longer sleep duration were associated in previous GWAS with a more favorable metabolic profile and a lower risk of attention deficit hyperactivity disorder. Understanding the mechanisms underlying these associations may help elucidate biological mechanisms influencing sleep duration and its association with psychiatric, metabolic and cardiovascular disease.

The genetics of common variation affecting platelet development, function and pharmaceutical targeting.

Common variant effects on human platelet function and response to anti-platelet treatment have traditionally been studied using candidate gene approaches involving a limited number of variants and genes. These studies have often been undertaken in clinically defined cohorts. More recently, studies have applied genome-wide scans in larger population samples than prior candidate studies, in some cases scanning relatively healthy individuals. These studies demonstrate synergy with some prior candidate gene findings (e.g., GP6, ADRA2A) but also uncover novel loci involved in platelet function. Here, I summarise findings on common genetic variation influencing platelet development, function and therapeutics. Taken together, candidate gene and genome-wide studies begin to account for common variation in platelet function and provide information that may ultimately be useful in pharmacogenetic applications in the clinic. More than 50 loci have been identified with consistent associations with platelet phenotypes in ≥ 2 populations. Several variants are under further study in clinical trials relating to anti-platelet therapies. In order to have useful clinical applications, variants must have large effects on a modifiable outcome. Regardless of clinical applications, studies of common genetic influences, even of small effect, offer additional insights into platelet biology including the importance of intracellular signalling and novel receptors. Understanding of common platelet-related genetics remains behind parallel fields (e.g., lipids, blood pressure) due to challenges in phenotype ascertainment. Further work is necessary to discover and characterise loci for platelet function, and to assess whether these loci contribute to disease aetiologies or response to therapeutics.

Pharmacogenomics of the RNA world: structural RNA polymorphisms in drug therapy.

The use of pharmacogenomic biomarkers can enhance treatment outcomes. Regulatory polymorphisms are promising biomarkers that have proven difficult to uncover. They come in two flavors: those that affect transcription (regulatory single-nucleotide polymorphisms (rSNPs)) and those that affect RNA functions such as splicing, turnover, and translation (termed structural RNA SNPs (srSNPs)). This review focuses on the role of srSNPs in drug metabolism, transport, and response. An understanding of the nature and diversity of srSNPs and rSNPs enables clinical scientists to evaluate genetic biomarkers.

Promoter polymorphisms in ACE (angiotensin I-converting enzyme) associated with clinical outcomes in hypertension.

Genetic variants of ACE are suspected risk factors in cardiovascular disease, but the alleles responsible for the variations remain unidentified. To search for regulatory polymorphisms, allelic angiotensin I-converting enzyme (ACE) mRNA expression was measured in 65 heart tissues, followed by genotype scanning of the ACE locus. Marked allelic expression imbalance (AEI) detected in five African-American subjects was associated with single-nucleotide polymorphisms (SNPs) (rs7213516, rs7214530, and rs4290) residing in conserved regions 2-3 kb upstream of ACE. Moreover, each of the SNPs affected transcription in reporter gene assays. SNPs rs4290 and rs7213516 were tested for associations with adverse cardiovascular outcomes in hypertensive patients with coronary disease (International Verapamil SR Trandolapril Study Genetic Substudy (INVEST-GENES), n = 1,032). Both SNPs were associated with adverse cardiovascular outcomes, largely attributable to nonfatal myocardial infarction in African Americans, showing an odds ratio of 6.16 (2.43-15.60) (P < 0.0001) for rs7213516. The high allele frequency in African Americans (16%) compared to Hispanics (4%) and Caucasians (<1%) suggests that these alleles contribute to variation between populations in cardiovascular risk and treatment outcomes.

Dynorphin-containing axons directly innervate noradrenergic neurons in the rat nucleus locus coeruleus.

Stress causes increased dynorphin (DYN) expression in limbic brain regions and antagonism of kappa-opioid receptors may offer therapeutic potential for the treatment of depression. A potential site of DYN action relevant to stress and related neuropsychiatric disorders is the locus coeruleus (LC), the primary source of forebrain norepinephrine. Therefore, using immunofluorescence and immunoelectron microscopic analyses, we characterized the cellular substrates for interactions between DYN and tyrosine hydroxylase (TH), a catecholamine synthesizing enzyme in single sections through the rat LC. Light microscopic analysis of DYN immunoreactivity indicated that DYN fibers are distributed within the core and pericoerulear subregions of the LC. Using electron microscopy, immunoperoxidase labeling for DYN was primarily found in axon terminals, although in some cases was diffusely localized to somatodendritic processes. When DYN-containing axons formed synaptic contacts, they typically (89%) exhibited an asymmetric morphology. Almost a third (28%) of the postsynaptic targets of DYN-containing axons contained immunogold labeling for TH. These findings reveal some diversity as to the localization of DYN in the LC within axons that contact both TH and non-TH containing dendrites. However, the present data provide the first ultrastructural evidence that DYN-containing axon terminals directly innervate catecholaminergic LC dendrites. Moreover, DYN axon terminals targeting catecholaminergic LC dendrites via asymmetric synapses are consistent with localization within excitatory type afferents to the LC. Therefore, direct modulation of catacholaminergic LC neurons maybe an important site of action for DYN relevant to stress and stress-related disorders.

Mating in Candida albicans and the search for a sexual cycle.

Candida albicans is a normal part of the human microflora, but it is also an opportunistic fungal pathogen that causes both mucosal infections and life-threatening systemic infections. Until recently, C. albicans was thought to be asexual, existing only as an obligate diploid. However, a mating locus was identified that was homologous to those in sexually reproducing fungi, and mating of C. albicans strains was subsequently demonstrated in the laboratory. In this review, we compare and contrast the mating process in C. albicans with that of other fungi, particularly Saccharomyces cerevisiae, whose mating has been most intensively studied. Several features of the mating pathway appear unique to C. albicans, including aspects of gene regulation and cell biology, as well as the involvement of "white-opaque" switching, an alteration between two quasi-stable inheritable states. These specializations of the mating process may have evolved to promote the survival of C. albicans in the hostile environment of a mammalian host.

The attachment, proliferation, and wound healing of endothelial and smooth muscle cells on nitinol thin film in vitro.

Nitinol (TiNi) thin film is a potentially useful material for various clinical applications, e.g., vascular stents. In this study we examined the biological functions of bovine endothelial cell (BAEC) and bovine smooth muscle cells (BSMC) on 1-mum thick amorphous TiNi films. BAEC and BSMC both attached on TiNi film, and exhibited increased cell spreading in the presence of serum containing media. Both cell types had decreased cell proliferation on TiNi as compared to glass control; however, BAEC had significantly greater proliferation rate than BSMC on TiNi. In an in vitro wound model, BSMC migrated faster than BAEC, resulting in a quicker closure of the monolayer. These results provide important insights into the interactions of vascular cells and TiNi thin film, and will help us to optimize the surface properties of TiNi film for applications in the vascular system.

Opioid circuits originating from the nucleus paragigantocellularis and their potential role in opiate withdrawal.

Neurons in the rat nucleus paragigantocellularis (PGi), located in the ventrolateral medulla, send collateral projections to the locus coeruleus (LC) and to the nucleus of the solitary tract (NTS). The present study examined whether neurons in the PGi that project to both the LC and NTS contain leucine(5)-enkephalin (ENK), and also whether opioid-containing neurons in the PGi are activated following withdrawal from opiates. Retrograde transport of Fluoro-Gold (FG) from the LC and transport of a protein-gold tracer from the NTS was combined with detection of an antibody directed against ENK in the PGi. Using fluorescence and brightfield microscopy, it was established that more than half of the neurons containing both FG and the protein-gold tracer, also exhibited immunolabeling for ENK. The most frequent location of triply labeled neurons was the retrofacial portion of the PGi. In a separate series, rats were chronically implanted with morphine or placebo pellets and, on the fifth day, were subjected to an intraperitoneal injection of naltrexone. Two hours following initiation of withdrawal, rat brains were obtained and processed for detection of c-fos and in situ hybridization labeling of preproenkephalin (PPE) mRNA. Naltrexone injections into morphine-dependent rats caused a dramatic increase in c-fos as compared to control rats. Approximately 66% of the c-fos-labeled neurons exhibited labeling for PPE mRNA. These were also enriched in the retrofacial portion of the PGi. Taken together, the present data indicate that withdrawal from opiates engages opioid neurons in the PGi, some of which may coordinate activity of neurons in both the NTS and the LC.

Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus.

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.

Axbrn-1: a maternal transcript encodes a POU transcription factor that is later expressed in the developing central nervous system of axolotl embryos.

Axbrn-1 encodes a class III POU protein with a POU-specific domain and a POU homeodomain that is most similar to RHS2 from rat. In embryos Axbrn-1 transcripts are maternally inherited and persist through cleavage. New transcripts accumulate beginning at the mid-blastula transition. In gastrulating embryos Axbrn-1 RNA is in the dorsal marginal zone and by neurula it is in the neural folds and neural plate. Later, it is specific to the developing brain and anterior spinal cord. Transcriptional activation at the mid-blastula transition indicates that Axbrn-1 is among the earliest genes to be expressed in axolotl embryos.

EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development.

The Pax gene egl-38 plays an important role in the development of several organs in C. elegans. To understand how a Pax transcription factor influences distinct developmental choices in different cells and tissue types, we have characterized a second gene, lin-48. lin-48 functions with egl-38 in the development of one structure, the hindgut, but not in other tissues such as the egg-laying system. We show that lin-48 encodes a C2H2 zinc-finger protein that is similar to the product of the Drosophila gene ovo and is expressed in the hindgut cells that develop abnormally in lin-48 mutants. We present evidence that lin-48 is a target for EGL-38 in hindgut cells. We show that lin-48 requires egl-38 for its expression in the hindgut. Using deletion analysis, we have identified two elements in the lin-48 promoter that are necessary for lin-48 expression. We demonstrate that EGL-38 binds with high affinity to one of these elements. In addition, we have observed genetic interactions between mutations in the lin-48 promoter and specific alleles of egl-38. These experiments demonstrate a functional link between Pax and Ovo transcription factors, and provide a model for how Pax transcription factors can regulate different target genes in different cells.

NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction.

In response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and filaments composed of elongated cells attached end-to-end. Here we identify a DNA-binding protein, Nrg1, that represses filamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly filamentous under non-filament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two filament-specific genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under non-inducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37 degrees C, a potent inducer of filamentous growth, causes a reduction of NRG1 mRNA, suggesting that filamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of filamentous growth.

Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad.

How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, we cloned a DAZ-like sequence from axolotls, Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells.

Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans.

Candida albicans, the major fungal pathogen in humans, can undergo a reversible transition from ellipsoidal single cells (blastospores) to filaments composed of elongated cells attached end to end. This transition is thought to allow for rapid colonization of host tissues, facilitating the spread of infection. Here, we report the identification of Rfg1, a transcriptional regulator that controls filamentous growth of C. albicans in an environment-dependent manner. Rfg1 is important for virulence of C. albicans in a mouse model and is shown to control a number of genes that have been implicated in this process. The closest relative to Rfg1 in Saccharomyces cerevisiae is Rox1, a key repressor of hypoxic genes. However, Rfg1 does not appear to play a role in the regulation of hypoxic genes in C. albicans. These results demonstrate that a regulatory protein that controls the hypoxic response in S. cerevisiae controls filamentous growth and virulence in C. albicans. The observations described in this paper raise new and intriguing questions about the evolutionary relationship between these processes.

Expression of the cardiac actin gene in axolotl embryos.

Axolotis are an important model system for studying heart development. Patterning of the somitic mesoderm occurs in axolotis in a manner that is much more similar to the pattern observed in higher vertebrates than in Xenopus. For these reasons we cloned the axolotl cardiac actin gene, since this gene is expressed during the development of both somitic and cardiac muscle in other vertebrates. In this paper we characterize its expression. Expression of cardiac actin RNA is switched on during gastrula stages and appears in the somitic mesoderm when it is formed; expression is later activated in the embryonic heart. In adults the gene is expressed only in the heart. The results demonstrate that the clone encoding cardiac actin provides a useful marker for studying development of both skeletal and cardiac muscle development in axolotls.

Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster.

Myogenic satellite cells were isolated from control and dystrophic hamster diaphragms to examine cellular mechanisms involved in the physiology of muscular dystrophy. The Bio 14.6 dystrophic hamster, which possesses a defect in the delta-sarcoglycan gene, develops biochemical and physical symptoms of Duchenne-like and limb girdle muscular dystrophies. Because primary cultures of the control and dystrophic satellite cells became extensively contaminated with non-myogenic cells during proliferation, cell clones were developed to provide pure cultures for study. Cell culture conditions were optimized with the use of Ham's F-12K medium containing 10% fetal bovine serum +5% horse serum + 10 ng/mL basic fibroblast growth factor + 50 microg/mL porcine gelatin. Proliferation rates of the two clonal cultures were similar between the two lines. Satellite cell-derived myotubes from both primary cultures and clones differed between control and dystrophic animals. Dystrophic myotubes tended to be long and narrow, while the control-derived myotubes were broader. Measurement of muscle-specific creatine kinase during differentiation revealed that the dystrophic myotubes possessed higher creatine kinase levels than control myotubes (up to 146-fold at 168 h). The results demonstrate that satellite cells can be isolated from the hamster and may provide a useful tool to study muscular dystrophies associated with defects in the sarcoglycan complex and the involvement of sarcoglycans in normal skeletal muscle growth and development.

Identification and characterization of TUP1-regulated genes in Candida albicans.

TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans. Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1. One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5. These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins. The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2. The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions. We produced knockout strains in C. albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media. However, two animal models for C. albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Delta and rbt4Delta strains had significantly reduced virulence. TUP1 appears, therefore, to regulate many genes in C. albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.