In vivo somatic delivery of plasmid DNA and retrograde transport to obtain cell-specific gene expression in the central nervous system

We utilised the retrograde transport machinery of neurones to deliver naked plasmid DNA into the central nervous system. A 5.4-kb fragment of the glycine receptor (GlyR) alpha1 subunit gene was cloned and used to drive the expression of a construct encoding for the enhanced green fluorescent protein (EGFP). Injections of the plasmid DNA in the tongue of mice resulted in the expression of the marker protein in hypoglossal motor neurones, showing that the GlyRalpha1 promoter sequence is sufficient to drive expression of the transgene. In order to determine the specificity of expression of the 5.4-kb fragment of the GlyR alpha1 subunit gene promoter, we subsequently injected the plasmid DNA into the mouse central nucleus of the amygdala. This nucleus receives projections from the parabrachial nucleus, a brainstem area that has a high density of GlyRs, and from the insular cortex, a forebrain structure devoid of GlyRs. We observed EGFP-labelled neurones in the parabrachial nucleus, but not in the insular cortex, indicating that the 5.4-kb GlyR alpha1 subunit gene promoter confers specificity of expression. This approach provides a simple and rapid way to identify, in vivo, promoter elements that mediate neurone-specific gene expression.     

Structural and functional characteristics of the Val44Met insulin-like growth factor I missense mutation: correlation with effects on growth and development

We have previously described the phenotype resulting from a missense mutation in the IGF-I gene, which leads to expression of IGF-I with a methionine instead of a valine at position 44 (Val44Met IGF-I). This mutation caused severe growth and mental retardation as well as deafness evident at birth and growth retardation in childhood, but is relatively well tolerated in adulthood. We have conducted a biochemical and structural analysis of Val44Met IGF-I to provide a molecular basis for the phenotype observed. Val44Met IGF-I exhibits a 90-fold decrease in type 1 IGF receptor (IGF-1R) binding compared with wild-type human IGF-I and only poorly stimulates autophosphorylation of the IGF-1R. The ability of Val44Met IGF-I to signal via the extracellular signal-regulated kinase 1/2 and Akt/protein kinase B pathways and to stimulate DNA synthesis is correspondingly poorer. Binding or activation of both insulin receptor isoforms is not detectable even at micromolar concentrations. However, Val44Met IGF-I binds IGF-binding protein-2 (IGFBP-2), IGFBP-3, and IGFBP-6 with equal affinity to IGF-I, suggesting the maintenance of overall structure, particularly in the IGFBP binding domain. Structural analysis by nuclear magnetic resonance confirms retention of near-native structure with only local side-chain disruptions despite the significant loss of function. To our knowledge, our results provide the first structural study of a naturally occurring mutant human IGF-I associated with growth and developmental abnormalities and identifies Val44 as an essential residue involved in the IGF-IGF-1R interaction.     

Differential regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCalpha, -epsilon, and -zeta isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCdelta significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCdelta inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCdelta (deltaTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.     

The Chlamydomonas Genome Project,version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating-type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism.

A major update to the Chlamydomonas reference genome and structural annotations is presented, revealing large genomic changes that have occurred in laboratory culture.

IN A NUTSHELL Background: Chlamydomonas reinhardtii (Chlamydomonas) is an important reference organism. The first draft genome for the species was sequenced 20 years ago. The current assembly, version 5, contains many gaps and some misassemblies. Although the structural annotations are generally of high quality, some gene models are missing, and many genes have misleading names. Finally, the reference genome has always been based on CC-503, a mating-type plus strain that was mutagenized to achieve a cell wall-less phenotype. Question: We aimed to update the Chlamydomonas reference genome and annotations using long-read sequencing. We also sequenced a second strain, the mating-type minus CC-4532. Via comparison, we tested whether the mutagenesis of CC-503 resulted in any gene-disrupting structural mutations. Findings: We produced highly contiguous genome assemblies for CC-503 and CC-4532 that were validated by linkage analyses. Most of the filled gaps were in genic regions, leading to substantial improvements in the gene models. Gene symbols were manually overhauled, providing a reliable nomenclature that can serve as a template for green algal genome projects. We discovered that the CC-503 genome harbors many large mutations and is unstable, making it an unsuitable reference. Genomic instability may stem from the deletion of the helicase RECQ3. We also fnd that the CC-4532 genome is experiencing an ongoing proliferation of transposable elements. We expect that all strains carry some large laboratory mutations. Next steps: The CC-4532 genome is presented as the new Chlamydomonas reference. The assembly and annotation bring great advancements and will serve as a foundation for future updates. However, no single strain can provide a perfect reference, and we anticipate a truly representative Chlamydomonas pan-genome.     

Conservation planning in a changing world

Conservation planning is the process of locating, configuring, implementing and maintaining areas that are managed to promote the persistence of biodiversity and other natural values. Conservation planning is inherently spatial. The science behind it has solved important spatial problems and increasingly influenced practice. To be effective, however, conservation planning must deal better with two types of change. First, biodiversity is not static in time or space but generated and maintained by natural processes. Second, humans are altering the planet in diverse ways at ever faster rates.     

Tetrahydrobiopterin improves endothelial function and decreases arterial stiffness in estrogen-deficient postmenopausal women

The mechanisms mediating arterial stiffening with aging and menopause are not completely understood. We determined whether administration of tetrahydrobiopterin (BH(4)), a critical cofactor for endothelial nitric oxide synthase to produce nitric oxide, would increase vascular endothelial-dependent vasodilatory tone and decrease arterial stiffness in estrogen-deficient postmenopausal women. Additionally, we examined whether the beneficial effects of estrogen on vascular function were possibly related to BH(4). Arterial stiffness (carotid artery compliance) and endothelial-dependent vasodilation [brachial artery flow-mediated dilation (FMD)] were measured in postmenopausal (n = 24; 57 ± 1 yr, mean ± SE) and eumenorrheic premenopausal (n = 9; 33 ± 2 yr) women before and 3 h after the oral administration of BH(4). Subsequently, in postmenopausal women, vascular testing (before and after BH(4)) was repeated following randomization to either 2 days of transdermal estradiol or placebo. Baseline carotid artery compliance and brachial artery FMD were lower in postmenopausal than in premenopausal women (P < 0.0001). BH(4) administration increased carotid artery compliance (0.61 ± 0.05 to 0.73 ± 0.04 mm(2)·mmHg(-1)·10(-1) vs. baseline, P < 0.0001) and brachial artery FMD (P < 0.001) in postmenopausal women but had no effect in premenopausal women (P = 0.62). Carotid artery compliance (0.59 ± 0.05 to 0.78 ± 0.06 mm(2)·mmHg(-1)·10(-1), P < 0.001) and FMD increased in postmenopausal women in response to estradiol (P = 0.02) but were not further improved with the coadministration of BH(4), possibly because estrogen increased BH(4) bioavailability. Carotid artery compliance and FMD increased with BH(4) in the placebo group (P = 0.02). Although speculative, these results suggest that reduced vascular BH(4) may be an important contributor to arterial stiffening in estrogen-deficient postmenopausal women, related in part to reduced endothelial-dependent vasodilatory tone.     

Bayesian Estimation of Small Effects in Exercise and Sports Science

The aim of this paper is to provide a Bayesian formulation of the so-called magnitude-based inference approach to quantifying and interpreting effects, and in a case study example provide accurate probabilistic statements that correspond to the intended magnitude-based inferences. The model is described in the context of a published small-scale athlete study which employed a magnitude-based inference approach to compare the effect of two altitude training regimens (live high-train low (LHTL), and intermittent hypoxic exposure (IHE)) on running performance and blood measurements of elite triathletes. The posterior distributions, and corresponding point and interval estimates, for the parameters and associated effects and comparisons of interest, were estimated using Markov chain Monte Carlo simulations. The Bayesian analysis was shown to provide more direct probabilistic comparisons of treatments and able to identify small effects of interest. The approach avoided asymptotic assumptions and overcame issues such as multiple testing. Bayesian analysis of unscaled effects showed a probability of 0.96 that LHTL yields a substantially greater increase in hemoglobin mass than IHE, a 0.93 probability of a substantially greater improvement in running economy and a greater than 0.96 probability that both IHE and LHTL yield a substantially greater improvement in maximum blood lactate concentration compared to a Placebo. The conclusions are consistent with those obtained using a ‘magnitude-based inference’ approach that has been promoted in the field. The paper demonstrates that a fully Bayesian analysis is a simple and effective way of analysing small effects, providing a rich set of results that are straightforward to interpret in terms of probabilistic statements.     

Classification of the Universe of Immune Epitope Literature: Representation and Knowledge Gaps

Background

A significant fraction of the more than 18 million scientific articles currently indexed in the PubMed database are related to immune responses to various agents, including infectious microbes, autoantigens, allergens, transplants, cancer antigens and others. The Immune Epitope Database (IEDB) is an online repository that catalogs immune epitope reactivity data derived from articles listed in the National Library of Medicine PubMed database. The IEDB is maintained and continually updated by monitoring PubMed for new, potentially relevant references.

Methodology

Herein we detail the classification of all epitope-specific literature in over 100 different immunological domains representing Infectious Diseases and Microbes, Autoimmunity, Allergy, Transplantation and Cancer. The relative number of references in each category reflects past and present areas of research on immune reactivities. In addition to describing the overall landscape of data distribution, this particular characterization of the epitope reference data also allows for the exploration of possible correlations with global disease morbidity and mortality data.

Conclusions/Significance

While in most cases diseases associated with high morbidity and mortality rates were amongst the most studied, a number of high impact diseases such as dengue, Schistosoma, HSV-2, B. pertussis and Chlamydia trachoma, were found to have very little coverage. The data analyzed in this fashion represents the first estimate of how reported immunological data corresponds to disease-related morbidity and mortality, and confirms significant discrepancies in the overall research foci versus disease burden, thus identifying important gaps to be pursued by future research. These findings may also provide a justification for redirecting a portion of research funds into some of the underfunded, critical disease areas.     

A Genome-Wide Scan for Evidence of Selection in a Maize Population Under Long-Term Artificial Selection for Ear Number

A genome-wide scan to detect evidence of selection was conducted in the Golden Glow maize long-term selection population. The population had been subjected to selection for increased number of ears per plant for 30 generations, with an empirically estimated effective population size ranging from 384 to 667 individuals and an increase of more than threefold in the number of ears per plant. Allele frequencies at >1.2 million single-nucleotide polymorphism loci were estimated from pooled whole-genome resequencing data, and F_ST values across sliding windows were employed to assess divergence between the population preselection and the population postselection. Twenty-eight highly divergent regions were identified, with half of these regions providing gene-level resolution on potentially selected variants. Approximately 93% of the divergent regions do not demonstrate a significant decrease in heterozygosity, which suggests that they are not approaching fixation. Also, most regions display a pattern consistent with a soft-sweep model as opposed to a hard-sweep model, suggesting that selection mostly operated on standing genetic variation. For at least 25% of the regions, results suggest that selection operated on variants located outside of currently annotated coding regions. These results provide insights into the underlying genetic effects of long-term artificial selection and identification of putative genetic elements underlying number of ears per plant in maize.