Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

BACKGROUND: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells

The relationship between the error catastrophe, survival of the flattest, and natural selection

<p>Abstract</p> <p>Background</p> <p>The quasispecies model is a general model of evolution that is generally applicable to replication up to high mutation rates. It predicts that at a sufficiently high mutation rate, quasispecies with higher mutational robustness can displace quasispecies with higher replicative capacity, a phenomenon called "survival of the flattest". In some fitness landscapes it also predicts the existence of a maximum mutation rate, called the error threshold, beyond which the quasispecies enters into error catastrophe, losing its genetic information. The aim of this paper is to study the relationship between survival of the flattest and the transition to error catastrophe, as well as the connection between these concepts and natural selection.</p> <p>Results</p> <p>By means of a very simplified model, we show that the transition to an error catastrophe corresponds to a value of zero for the selective coefficient of the mutant phenotype with respect to the master phenotype, indicating that transition to the error catastrophe is in this case similar to the selection of a more robust species. This correspondence has been confirmed by considering a single-peak landscape in which sequences are grouped with respect to their Hamming distant from the master sequence. When the robustness of a classe is changed by modification of its quality factor, the distribution of the population changes in accordance with the new value of the robustness, although an error catastrophe can be detected at the same values as in the general case. When two quasispecies of different robustness competes with one another, the entry of one of them into error catastrophe causes displacement of the other, because of the greater robustness of the former. Previous works are explicitly reinterpreted in the light of the results obtained in this paper.</p> <p>Conclusions</p> <p>The main conclusion of this paper is that the entry into error catastrophe is a specific case of survival of the flattest acting on phenotypes that differ in the trade-off between replicative ability and mutational robustness. In fact, entry into error catastrophe occurs when the mutant phenotype acquires a selective advantage over the master phenotype. As both entry into error catastrophe and survival of the flattest are caused by natural selection when mutation rate is increased, we propose differentiating between them by the level of selection at which natural selection acts. So we propose to consider the transition to error catastrophe as a phenomenon of intra-quasispecies selection, and survival of the flattest as a phenomenon of inter-quasispecies selection.</p

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

Background - Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes.Methodology/Principal Findings - We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis.Conclusions/Significance - The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this populatio

Plio-Pleistocene climatic change had a major impact on the assembly and disassembly processes of Iberian rodent communities

Comprehension of changes in community composition through multiple spatio-temporal scales is a prime challenge in ecology and palaeobiology. However, assembly, structuring and disassembly of biotic metacommunities in deep-time is insufficiently known. To address this, we used the extensively sampled Iberian Plio-Pleistocene fossil record of rodent faunas as our model system to explore how global climatic events may alter metacommunity structure. Through factor analysis, we found five sets of genera, called faunal components, which co-vary in proportional diversity over time. These faunal components had different spatio-temporal distributions throughout the Plio-Pleistocene, resulting in non-random changes in species assemblages, particularly in response to the development of the Pleistocene glaciations. Three successive metacommunities with distinctive taxonomic structures were identified as a consequence of the differential responses of their members to global climatic change: (1) Ruscinian subtropical faunas (5.3–3.4 Ma) dominated by a faunal component that can be considered as a Miocene legacy; (2) transition faunas during the Villafranchian–Biharian (3.4–0.8 Ma) with a mixture of different faunal components; and (3) final dominance of the temperate Toringian faunas (0.8–0.01 Ma) that would lead to the modern Iberian assemblage. The influence of the cooling global temperature drove the reorganisation of these rodent metacommunities. Selective extinction processes due to this large-scale environmental disturbance progressively eliminated the subtropical specialist species from the early Pliocene metacommunity. This disassembly process was accompanied by the organisation of a diversified metacommunity with an increased importance of biome generalist species, and finally followed by the assembly during the middle–late Pleistocene of a new set of species specialised in the novel environments developed as a consequence of the glaciations

Insights into pathogenic events of HIV-associated Kaposi sarcoma and immune reconstitution syndrome related Kaposi sarcoma

A decrease in the incidence of human immune deficiency virus-associated Kaposi sarcoma (HIV-KS) and regression of some established HIV-KS lesions is evident after the introduction of highly active anti-retroviral treatment (HAART), and is attributed to generalized immune restoration, to the reconstitution of human herpesvirus (HHV)-8 specific cellular immune responses, and to the decrease in HIV Tat protein and HHV-8 loads following HAART. However, a small subset of HIV-seropositive subjects with a low CD4+ T cell count at the time of introduction of HAART, may develop HIV-KS as immune reconstitution inflammatory syndrome (IRIS) within 8 weeks thereafter

Induction chemotherapy followed by chemoradiotherapy versus chemoradiotherapy alone as neoadjuvant treatment for locally recurrent rectal cancer: study protocol of a multicentre, open-label, parallel-arms, randomized controlled study (PelvEx II)

Background A resection with clear margins (R0 resection) is the most important prognostic factor in patients with locally recurrent rectal cancer (LRRC). However, this is achieved in only 60 per cent of patients. The aim of this study is to investigate whether the addition of induction chemotherapy to neoadjuvant chemo(re)irradiation improves the R0 resection rate in LRRC. Methods This multicentre, international, open-label, phase III, parallel-arms study will enrol 364 patients with resectable LRRC after previous partial or total mesorectal resection without synchronous distant metastases or recent chemo- and/or radiotherapy treatment. Patients will be randomized to receive either induction chemotherapy (three 3-week cycles of CAPOX (capecitabine, oxaliplatin), four 2-week cycles of FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) or FOLFORI (5-fluorouracil, leucovorin, irinotecan)) followed by neoadjuvant chemoradiotherapy and surgery (experimental arm) or neoadjuvant chemoradiotherapy and surgery alone (control arm). Tumours will be restaged using MRI and, in the experimental arm, a further cycle of CAPOX or two cycles of FOLFOX/FOLFIRI will be administered before chemoradiotherapy in case of stable or responsive disease. The radiotherapy dose will be 25 × 2.0 Gy or 28 × 1.8 Gy in radiotherapy-naive patients, and 15 × 2.0 Gy in previously irradiated patients. The concomitant chemotherapy agent will be capecitabine administered twice daily at a dose of 825 mg/m2 on radiotherapy days. The primary endpoint of the study is the R0 resection rate. Secondary endpoints are long-term oncological outcomes, radiological and pathological response, toxicity, postoperative complications, costs, and quality of life. Discussion This trial protocol describes the PelvEx II study. PelvEx II, designed as a multicentre, open-label, phase III, parallel-arms study, is the first randomized study to compare induction chemotherapy followed by neoadjuvant chemo(re)irradiation and surgery with neoadjuvant chemo(re)irradiation and surgery alone in patients with locally recurrent rectal cancer, with the aim of improving the number of R0 resections