Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane
Roberta Lane de Oliveira Silva
Manassés Daniel Silva
José Ribamar Costa Ferreira Neto
Claudia Huerta de Nardi
Sabrina Moutinho Chabregas
William Lee Burnquist
Ana Maria Benko-Iseppon
Ederson Akio Kido
- One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.
IL-17A influences essential functions of the monocyte/macrophage lineage and is involved in advanced murine and human atherosclerosis
Kristina M. Little
Thomas J. Dengler
Hugo A. Katus
Christian A. Gleissner
- Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. IL-17A is a proinflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study, we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. The 26-wk-old apolipoprotein E–deficient mice were fed a standard chow diet and treated either with IL-17A mAb (n = 15) or irrelevant Ig (n = 10) for 16 wk. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p = 0.001) by reducing inflammatory burden and cellular infiltration (p = 0.01) and improved lesion stability (p = 0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, and sensitization of APCs toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a proinflammatory milieu and upregulation of molecules expressed by the IL-17A–induced macrophage subtype. In this study, we show that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in apolipoprotein E–deficient mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments underline the relevance for the human system.
Risk stratification for sudden cardiac death: current status and challenges for the future
Hein J.J. Wellens
Peter J. Schwartz
Fred W. Lindemans
Alfred E. Buxton
Jeffrey J. Goldberger
Stefan H. Hohnloser
Heikki V. Heikki V.Huikuri
Maria Teresa La Rovere
Robert J. Myerburg
Maarten L. Simoons
Adriaan A. Voors
Arthur A. Wilde
- Sudden cardiac death (SCD) remains a daunting problem. It is a major public health issue for several reasons: from its prevalence (20% of total mortality in the industrialized world) to the devastating psycho-social impact on society and on the families of victims often still in their prime, and it represents a challenge for medicine, and especially for cardiology. This text summarizes the discussions and opinions of a group of investigators with a long-standing interest in this field. We addressed the occurrence of SCD in individuals apparently healthy, in patients with heart disease and mild or severe cardiac dysfunction, and in those with genetically based arrhythmic diseases. Recognizing the need for more accurate registries of the global and regional distribution of SCD in these different categories, we focused on the assessment of risk for SCD in these four groups, looking at the significance of alterations in cardiac function, of signs of electrical instability identified by ECG abnormalities or by autonomic tests, and of the progressive impact of genetic screening. Special attention was given to the identification of areas of research more or less likely to provide useful information, and thereby more or less suitable for the investment of time and of research funds.
Bears in a Forest of Gene Trees: Phylogenetic Inference Is Complicated by Incomplete Lineage Sorting and Gene Flow
Verena E. Kutschera
Julia L.. Rodi
Steven R. Fain
- Ursine bears are a mammalian subfamily that comprises six morphologically and ecologically distinct extant species. Previous phylogenetic analyses of concatenated nuclear genes could not resolve all relationships among bears, and appeared to conflict with the mitochondrial phylogeny. Evolutionary processes such as incomplete lineage sorting and introgression can cause gene tree discordance and complicate phylogenetic inferences, but are not accounted for in phylogenetic analyses of concatenated data. We generated a high-resolution data set of autosomal introns from several individuals per species and of Y-chromosomal markers. Incorporating intraspecific variability in coalescence-based phylogenetic and gene flow estimation approaches, we traced the genealogical history of individual alleles. Considerable heterogeneity among nuclear loci and discordance between nuclear and mitochondrial phylogenies were found. A species tree with divergence time estimates indicated that ursine bears diversified within less than 2 My. Consistent with a complex branching order within a clade of Asian bear species, we identified unidirectional gene flow from Asian black into sloth bears. Moreover, gene flow detected from brown into American black bears can explain the conflicting placement of the American black bear in mitochondrial and nuclear phylogenies. These results highlight that both incomplete lineage sorting and introgression are prominent evolutionary forces even on time scales up to several million years. Complex evolutionary patterns are not adequately captured by strictly bifurcating models, and can only be fully understood when analyzing multiple independently inherited loci in a coalescence framework. Phylogenetic incongruence among gene trees hence needs to be recognized as a biologically meaningful signal.
Synaptic Conductance Estimates of the Connection Between Local Inhibitor Interneurons and Pyramidal Neurons in Layer 2/3 of a Cortical Column
Jochen H.O. Hoffmann
H. S. Meyer
Arno C. Schmitt
- Stimulation of a principal whisker yields sparse action potential (AP) spiking in layer 2/3 (L2/3) pyramidal neurons in a cortical column of rat barrel cortex. The low AP rates in pyramidal neurons could be explained by activation of interneurons in L2/3 providing inhibition onto L2/3 pyramidal neurons. L2/3 interneurons classified as local inhibitors based on their axonal projection in the same column were reported to receive strong excitatory input from spiny neurons in L4, which are also the main source of the excitatory input to L2/3 pyramidal neurons. Here, we investigated the remaining synaptic connection in this intracolumnar microcircuit. We found strong and reliable inhibitory synaptic transmission between intracolumnar L2/3 local-inhibitor-to-L2/3 pyramidal neuron pairs [inhibitory postsynaptic potential (IPSP) amplitude -0.88 ± 0.67 mV]. On average, 6.2 ± 2 synaptic contacts were made by L2/3 local inhibitors onto L2/3 pyramidal neurons at 107 ± 64 µm path distance from the pyramidal neuron soma, thus overlapping with the distribution of synaptic contacts from L4 spiny neurons onto L2/3 pyramidal neurons (67 ± 34 µm). Finally, using compartmental simulations, we determined the synaptic conductance per synaptic contact to be 0.77 ± 0.4 nS. We conclude that the synaptic circuit from L4 to L2/3 can provide efficient shunting inhibition that is temporally and spatially aligned with the excitatory input from L4 to L2/3.
Multimodal Imaging of Dynamic Functional Connectivity
- The study of large-scale functional interactions in the human brain with functional magnetic resonance imaging (fMRI) extends almost to the first applications of this technology. Due to historical reasons and preconceptions about the limitations of this brain imaging method, most studies have focused on assessing connectivity over extended periods of time. It is now clear that fMRI can resolve the temporal dynamics of functional connectivity, like other faster imaging techniques such as electroencephalography and magnetoencephalography (albeit on a different temporal scale). However, the indirect nature of fMRI measurements can hinder the interpretability of the results. After briefly summarizing recent advances in the field, we discuss how the simultaneous combination of fMRI with electrophysiological activity measurements can contribute to a better understanding of dynamic functional connectivity in humans both during rest and task, wakefulness, and other brain states
Parasite diversity of European Myotis species with special emphasis on Myotis myotis (Microchiroptera, Vespertilionidae) from a typical nursery roost
Bats belong to one of the most species-rich orders within the Mammalia. They show a worldwide distribution, a high degree of ecological diversification as well as a high diversity of associated parasites and pathogens. Despite their prominent and unique role, the knowledge of their parasite-host-relationships as well as the mechanisms of co-evolutionary processes are, partly due to strict conservation regulations, scarce.
Juvenile specimens of the greater mouse-eared bat (Myotis myotis) from a roosting colony in Gladenbach (Hesse, Germany) were examined for their metazoan endo-and ectoparasite infections and pathogens. Morphometric data were recorded and the individuals were checked for Lyssavirus-specific antigen using a direct immunofluorescence test. For unambiguous species identification, the bats were analysed by cyt-b sequence comparison.
Myotis myotis were parasitized by the six insect and arachnid ectoparasite species, i.e. Ixodes ricinus, Ischnopsyllus octactenus, Ichoronyssus scutatus, Steatonyssus periblepharus, Spinturnix myoti and Cimex dissimilis. Additionally, the nematode Molinostrongylus alatus and the cestode Vampirolepis balsaci were recorded. Each bat was parasitized by at least four species. The parasites showed partially extreme rates of infection, never recorded before, with more than 1,440 parasites per single host. Ichoronyssus scutatus, Steatonyssus periblepharus, Vampirolepis balsaci and Molinostrongylus alatus are recorded for the first time in Germany. A checklist for Europe is presented containing records of 98 parasite species of 14 Myotis species.
The Myotis myotis from Gladenbach (Hesse, Germany) were parasitized by a diverse parasite fauna with high infestation rates. We assume that in juvenile Myotis the number of parasites is generally higher than in adults due to only later acquired immune competence and behavioural adaptations. Our results revealed new insights into parasite fauna of M. myotis and European bats in general. The finding of endoparasitic cyclophyllidean cestodes that have a two-host lifecycle is, considering the stationary behaviour of the juvenile bats, rather unusual and suggests a non-predatory transmission mechanism (e.g. via autoinfection).
A new insight gained from the collated literature was that the European wide composition of the Myotis parasite fauna is dominated by a few specific taxonomic groups in Europe
Risk Factors for the Presence of Chikungunya and Dengue Vectors (Aedes aegypti and Aedes albopictus), Their Altitudinal Distribution and Climatic Determinants of Their Abundance in Central Nepal
Hari Datt Joshi
Robert B. O’Hara
Adventitial Vessel Growth and Progenitor Cells Activation in an Ex Vivo Culture System Mimicking Human Saphenous Vein Wall Strain after Coronary Artery Bypass Grafting
Maria Cristina Vinci
Gianfranco Beniamino Fiore
- Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation.
Contrasting Taxonomic and Phylogenetic Diversity Responses to Forest Modifications: Comparisons of Taxa and Successive Plant Life Stages in South African Scarp Forest
Eike Lena Neuschulz
- The degradation of natural forests to modified forests threatens subtropical and tropical biodiversity worldwide. Yet, species responses to forest modification vary considerably. Furthermore, effects of forest modification can differ, whether with respect to diversity components (taxonomic or phylogenetic) or to local (α-diversity) and regional (β-diversity) spatial scales. This real-world complexity has so far hampered our understanding of subtropical and tropical biodiversity patterns in human-modified forest landscapes. In a subtropical South African forest landscape, we studied the responses of three successive plant life stages (adult trees, saplings, seedlings) and of birds to five different types of forest modification distinguished by the degree of within-forest disturbance and forest loss. Responses of the two taxa differed markedly. Thus, the taxonomic α-diversity of birds was negatively correlated with the diversity of all plant life stages and, contrary to plant diversity, increased with forest disturbance. Conversely, forest disturbance reduced the phylogenetic α-diversity of all plant life stages but not that of birds. Forest loss neither affected taxonomic nor phylogenetic diversity of any taxon. On the regional scale, taxonomic but not phylogenetic β-diversity of both taxa was well predicted by variation in forest disturbance and forest loss. In contrast to adult trees, the phylogenetic diversity of saplings and seedlings showed signs of contemporary environmental filtering. In conclusion, forest modification in this subtropical landscape strongly shaped both local and regional biodiversity but with contrasting outcomes. Phylogenetic diversity of plants may be more threatened than that of mobile species such as birds. The reduced phylogenetic diversity of saplings and seedlings suggests losses in biodiversity that are not visible in adult trees, potentially indicating time-lags and contemporary shifts in forest regeneration. The different responses of taxonomic and phylogenetic diversity to forest modifications imply that biodiversity conservation in this subtropical landscape requires the preservation of natural and modified forests.