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Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

Our senses define our view of the world. They allow us to adapt to environmental stimuli and are essential for communication and social behaviour. For most humans, seeing and hearing are central senses for their daily life. Our eyes and ears respond to an extraordinary broad range of stimuli covering about 12 log units of light intensity or acoustic power, respectively. The cellular basis is represented by sensory cells (photoreceptors in the retina and inner hair cells in the cochlea) that convert sensory inputs into electrical signals. Photoreceptors and inner hair cells have developed a specific pre-synaptic structure, termed synaptic ribbon, that is decorated with numerous vesicles filled with the excitatory neurotransmitter glutamate. At these ribbon synapses, glutamatergic signal transduction is guided by distinct sets of metabotropic glutamate receptors (mGluRs). MGluRs belong to group II and III of the receptor classification can inhibit neuronal activity, thus protecting neurons from overstimulation and subsequent degeneration. Consequently, dysfunction of mGluRs is associated with vision and hearing disorders. In this review, we introduce the principle characteristics of ribbon synapses and describe group II and III mGluRs in these fascinating structures in the retina and cochlea.

The ionoSolv pretreatment is a new technique employing protic low-cost ionic liquids and has previously been applied to successfully fractionate switchgrass and the grass Miscanthus giganteus. This study investigates the effect of using the protic ionic liquid solution [N 2220 ][HSO 4 ] 80% with two different acid/base ratios (1.02 and 0.98) at 120, 150 and 170 °C on the pretreatment outcome of the hardwood willow. The ionic liquid solution was able to fractionate willow, and a pulp and lignin fraction were recovered after treatment. The pretreatment success was determined via enzymatic hydrolysis of the pulp, which showed that the ionoSolv pretreatment was able to increase enzymatic glucose yields compared to untreated willow biomass. The pretreatment produced a cellulose-rich pulp with high hemicellulose and lignin removal. The pulp composition and glucose yield after saccharification were greatly influenced by the acidity of the ionic liquid solution, temperature and pretreatment time. The extracted lignin was analysed via 2-D HSQC NMR spectroscopy and GPC to investigate the changes in the lignin structure induced by the pretreatment severity. The lignin structure (in terms of inter-unit linkages and S/G ratio) and molecular weight varied significantly depending on the pretreatment conditions used.

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

Transport is associated with environmental problems, economic losses, health and social inequalities. A number of European and US cities have implemented initiatives to promote multimodal modes of transport. In Latin America changes are occurring in public transport systems and a number of projects aimed at stimulating non-motorized modes of transport (walking and cycling) have already been implemented. Based on articles from peer-reviewed academic journals, this paper examines experiences in Bogotá (Colombia), Curitiba (Brazil), and Santiago (Chile), and identifies how changes to the transport system contribute to encourage active transportation. Bus rapid transit, ciclovias, bike paths/lanes, and car use restriction are initiatives that contribute to promoting active transportation in these cities. Few studies have been carried out on the relationship between transport and physical activity. Car ownership continues to increase. The public health sector needs to be a stronger activist in the transport policy decision-making process to incorporate health issues into the transport agenda in Latin America.

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.

We have previously reported that vaccination with CpG oligodeoxynucleotides delivered concomitantly with live Leishmania major (Lm/CpG) eliminates lesions associated with live vaccination in C57BL/6 mice. The absence of lesions is at least in part a result of the CpG DNA-mediated activation of dermal dendritic cells to produce cytokines such as interleukin (IL)-6. Wild-type C57BL/6 mice and IL-6-/- mice were immunized with the Lm/CpG vaccine and monitored for the development of lesions. IL-6-/- mice developed extensive, nonhealing lesions following live vaccination. The analysis of the inoculation site and draining lymph nodes of the IL-6-/- mice revealed a constitutive reduction in lymphocyte numbers, particularly CD4+ T cells. Live vaccination resulted in the specific expansion of CD4+Foxp3+ regulatory T cells in the knockout mice, and in a decrease of CD4+ IFN-gamma -producing cells. These results indicate that IL-6-/- mice may have collateral immune defects that could influence the development of the natural immune response to pathogens, vaccines, or other inflammatory stimuli.

This case study is about the politics of incorporating active-living elements into a concept plan for a new community of about 68,000 people on the edge of the Portland, Oregon, metropolitan area. Development on the rural-urban fringe is ongoing in metropolitan areas around the United States. In this article, we evaluate the product of the concept-planning process from the standpoint of the extent to which environmental elements conducive to active living were included. We also analyze four issues in which challenges to the incorporation of active-living features surfaced: choices related to transportation facilities, the design and location of retail stores, the location of schools and parks, and the location of a new town center. Overall, the Damascus/Boring Concept Plan positions the area well to promote active living. Analyses of the challenges that emerged yielded lessons for advocates regarding ways to deal with conflicts between facilitating active living and local economic development and related tax-base concerns and between active-living elements and school-district planning autonomy as well as the need for advocates to have the capacity to present alternatives to the usual financial and design approaches taken by private- and public-sector investors.

Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca(2+)](i). To determine whether MLCK and ROK antagonists alter Ca(2+) signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca(2+)](i), Ca(2+) entry, and Ca(2+) release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca(2+)](i) in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca(2+) release from the sarcoplasmic reticulum and Ca(2+) entry through store- and voltage-operated Ca(2+) channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca(2+) signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca(2+) signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.

The inoculation of live Leishmania major to produce a lesion that heals (leishmanization) is to date the only vaccine against cutaneous leishmaniasis that has proven effective in humans, but it still has an unacceptable frequency of large ulcerating lesions that are slow to heal or, in rare cases, non-healing. We have previously shown that C57BL/6 mice vaccinated intradermally with 10(4) L. major/50 microg CpG oligodeoxynucleotides develop little or no dermal lesions and show early containment of parasite growth in the vaccination site, eliminating safety concerns related to the inoculation of live organisms. The addition of CpG to the live vaccine resulted in early activation of dermal dendritic cells and increased IL-6 production, as well as in a reduction in the accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T (T(reg)) cells that naturally occurs in the skin following Leishmania infection. Neutralization of IL-6 caused the development of larger lesions and increased local T(reg) cell numbers. Transfer of vaccine-primed dendritic cells into IL-6-deficient mice mitigated lesion development, indicating that IL-6 reconstitution limited pathology in the vaccination site.

Chronic hypoxia (CH) causes pulmonary vasoconstriction because of increased pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation. We previously demonstrated that intracellular Ca(2+) concentration ([Ca(2+)](i)) was elevated in PASMCs from chronically hypoxic rats because of Ca(2+) influx through pathways other than L-type Ca(2+) channels and that development of hypoxic pulmonary hypertension required full expression of the transcription factor hypoxia inducible factor 1 (HIF-1). In this study, we examined the effect of CH on the activity and expression of store-operated Ca(2+) channels (SOCCs) and the regulation of these channels by HIF-1. Capacitative Ca(2+) entry (CCE) was enhanced in PASMCs from intrapulmonary arteries of rats exposed to CH (10% O(2); 21 days), and exposure to Ca(2+)-free extracellular solution or SOCC antagonists (SKF96365 or NiCl(2)) decreased resting [Ca(2+)](i) in these cells. Expression of TRPC1 and TRPC6, but not TRPC4, mRNA and protein was increased in PASMCs from rats and wild-type mice exposed to CH, in PASMCs from normoxic animals cultured under hypoxic conditions (4% O(2); 60 hours), and in PASMCs in which HIF-1 was overexpressed under nonhypoxic conditions. Hypoxia-induced increases in basal [Ca(2+)](i) and TRPC expression were absent in mice partially deficient for HIF-1. These results suggest that increased TRPC expression, leading to enhanced CCE through SOCCs, may contribute to hypoxic pulmonary hypertension by facilitating Ca(2+) influx and increasing basal [Ca(2+)](i) in PASMCs and that this response is mediated by HIF-1.

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.

Previous studies indicated that acute hypoxia increased intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) influx, and capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca(2+)-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl(2), and LaCl(3)) on pulmonary arterial pressor responses to 2% O(2) and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca(2+)](i) responses to hypoxia in PASMC, SKF-96365 and NiCl(2) prevented and reversed HPV but did not alter pressor responses to KCl. At 10 microM, LaCl(3) had similar effects, but higher concentrations (30 and 100 microM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca(2+)-free perfusate and the voltage-operated Ca(2+) channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca(2+) through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca(2+) on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.

In pulmonary arterial smooth muscle cells (PASMCs), voltage-gated K+ (Kv) channels play an important role in regulating membrane potential, cytoplasmic free Ca2+ concentration, and pulmonary vasomotor tone. Previous studies demonstrated that exposure of rats to chronic hypoxia decreased Kv channel function in PASMCs from distal pulmonary arteries (dPA). To determine whether this decrease in function was due to decreased expression of Kv channel proteins and which Kv proteins might be involved, we analyzed Kv channel gene expression in intact, endothelium-denuded dPAs obtained from rats exposed to 10% O2 for 3 wk. Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.3, and Kv9.3 channel alpha-subunits and Kv1, Kv2, and Kv3 beta-subunits were expressed in rat dPAs. Exposure to chronic hypoxia decreased mRNA and protein levels of Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv4.3 alpha-subunits in dPAs but did not alter gene or protein expression of these channels in aorta. Furthermore, chronic hypoxia did not alter the mRNA levels of beta-subunits in dPAs. These results suggest that diminished transcription of Kv alpha-subunits may reduce the number of functional Kv channels in dPAs during prolonged hypoxia, causing the decreased Kv current previously observed in PASMCs and leading to pulmonary artery vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.