High temperatures limit plant growth but hasten flowering in root chicory (Cichorium intybus) independently of vernalisation

An increase in mean and extreme summer temperatures is expected as a consequence of climate changes and this might have an impact on plant development in numerous species. Root chicory (Cichorium intybus L.) is a major crop in northern Europe, and it is cultivated as a source of inulin. This polysaccharide is stored in the tap root during the first growing season when the plant grows as a leafy rosette, whereas bolting and flowering occur in the second year after winter vernalisation. The impact of heat stress on plant phenology, water status, photosynthesis-related parameters, and inulin content was studied in the field and under controlled phytotron conditions. In the field, plants of the Crescendo cultivar were cultivated under a closed plastic-panelled greenhouse to investigate heat-stress conditions, while the control plants were shielded with a similar, but open, structure. In the phytotrons, the Crescendo and Fredonia cultivars were exposed to high temperatures (35 °C day/28 °C night) and compared to control conditions (17 °C) over 10 weeks. In the field, heat reduced the root weight, the inulin content of the root and its degree of polymerisation in non-bolting plants. Flowering was observed in 12% of the heat stressed plants during the first growing season in the field. In the phytotron, the heat stress increased the total number of leaves per plant, but reduced the mean leaf area. Photosynthesis efficiency was increased in these plants, whereas osmotic potential was decreased. High temperature was also found to induced flowering of up to 50% of these plants, especially for the Fredonia cultivar. In conclusion, high temperatures induced a reduction in the growth of root chicory, although photosynthesis is not affected. Flowering was also induced, which indicates that high temperatures can partly substitute for the vernalisation requirement for the flowering of root chicory.     

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method''s potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.A protein''s function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI)¹ maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra- and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow studying PPI modulation because they do not offer the proper context for mammalian PPI analysis, e.g. they operate in yeast cells (Y2H) or make use of cell lysates (affinity purification-based methods). Moreover, because these interactome mapping tools are biased against interactions that involve transmembrane proteins, the latter are underrepresented in current interactome network versions (4). Yet, membrane-associated proteins constitute around one third of the entire proteome and their significance is underscored by the fact that over half of currently marketed drugs target membrane proteins (5). These observations support the need for approaches that allow PPIs, including those involving transmembrane proteins, to be assayed in their native cellular environment.Apart from the high-throughput methods mentioned above, a diverse arsenal of other PPI technologies has been developed, a number of which actually operate in mammalian cells. FRET and BRET, which rely on fluorescence or bioluminescence energy transfer between interacting fusion proteins, make assays with high spatiotemporal resolution (6, 7). A variety of PCAs have been reported, including split fluorescent protein or reporter enzyme technologies, that are able to capture aspects of PPI dynamics in a mammalian background (8, 9). A recent addition is an infrared fluorescent PCA that, unlike previous fluorescent PCAs, exhibits reversible complementation, thus enabling spatiotemporal analysis of dynamic PPIs (10). Another binary interaction assay, luminescence-based mammalian interactome mapping (LUMIER), has been applied to map TGFβ induced modulation of PPIs with components of the TGFβ signaling pathway (11). MaMTH, a mammalian version of the split ubiquitin approach, was designed particularly for the analysis of PPIs involving integral membrane proteins, also allowing the detection of functional PPI modulation (12). Efforts to apply purification-based methods for detecting context-dependent PPI modulation recently resulted in the development of AP-SRM (13) and AP-SWATH (14).Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) (supplemental Fig. S1A) (15, 16), a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis (17, 18), screening for small molecule PPI disruptors (19, 20) and drug target profiling (21, 22). Although MAPPIT operates in intact human cells, thus providing the natural environment for human protein analysis, the interaction sensor is anchored to the plasma membrane, precluding the analysis of PPIs at their native subcellular localization. In addition, MAPPIT is incompatible with full size transmembrane proteins. Here we describe KInase Substrate Sensor (KISS), a novel binary PPI mapping approach that enables in situ analysis in living mammalian cells of protein interactions and their responses to physiological or pharmacological challenges.     

Accuracy of Ultrasonography and Magnetic Resonance Imaging in the Diagnosis of Placenta Accreta

Purpose

To evaluate the accuracy of ultrasonography and magnetic resonance imaging (MRI) in the diagnosis of placenta accreta and to define the most relevant specific ultrasound and MRI features that may predict placental invasion.

Material and Methods

This study was approved by the institutional review board of the French College of Obstetricians and Gynecologists. We retrospectively reviewed the medical records of all patients referred for suspected placenta accreta to two university hospitals from 01/2001 to 05/2012. Our study population included 42 pregnant women who had been investigated by both ultrasonography and MRI. Ultrasound images and MRI were blindly reassessed for each case by 2 raters in order to score features that predict abnormal placental invasion.

Results

Sensitivity in the diagnosis of placenta accreta was 100% with ultrasound and 76.9% for MRI (P = 0.03). Specificity was 37.5% with ultrasonography and 50% for MRI (P = 0.6). The features of greatest sensitivity on ultrasonography were intraplacental lacunae and loss of the normal retroplacental clear space. Increased vascularization in the uterine serosa-bladder wall interface and vascularization perpendicular to the uterine wall had the best positive predictive value (92%). At MRI, uterine bulging had the best positive predictive value (85%) and its combination with the presence of dark intraplacental bands on T2-weighted images improved the predictive value to 90%.

Conclusion

Ultrasound imaging is the mainstay of screening for placenta accreta. MRI appears to be complementary to ultrasonography, especially when there are few ultrasound signs.     

Alternatives to the Six-Minute Walk Test in Pulmonary Arterial Hypertension

Introduction

The physiological response during the endurance shuttle walk test (ESWT), the cycle endurance test (CET) and the incremental shuttle walk test (ISWT) remains unknown in PAH. We tested the hypothesis that endurance tests induce a near-maximal physiological demand comparable to incremental tests. We also hypothesized that differences in respiratory response during exercise would be related to the characteristics of the exercise tests.

Methods

Within two weeks, twenty-one PAH patients (mean age: 54(15) years; mean pulmonary arterial pressure: 42(12) mmHg) completed two cycling exercise tests (incremental cardiopulmonary cycling exercise test (CPET) and CET) and three field tests (ISWT, ESWT and six-minute walk test (6MWT)). Physiological parameters were continuously monitored using the same portable telemetric device.

Results

Peak oxygen consumption (VO_2peak) was similar amongst the five exercise tests (p = 0.90 by ANOVA). Walking distance correlated markedly with the VO_2peak reached during field tests, especially when weight was taken into account. At 100% exercise, most physiological parameters were similar between incremental and endurance tests. However, the trends overtime differed. In the incremental tests, slopes for these parameters rose steadily over the entire duration of the tests, whereas in the endurance tests, slopes rose sharply from baseline to 25% of maximum exercise at which point they appeared far less steep until test end. Moreover, cycling exercise tests induced higher respiratory exchange ratio, ventilatory demand and enhanced leg fatigue measured subjectively and objectively.

Conclusion

Endurance tests induce a maximal physiological demand in PAH. Differences in peak respiratory response during exercise are related to the modality (cycling vs. walking) rather than the progression (endurance vs. incremental) of the exercise tests.     

Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes

Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.     

Targeting head and neck tumoral stem cells: From biological aspects to therapeutic perspectives

Head and neck squamous cell cancer(HNSCC) is the sixth most common cancer in the world. Effective therapeutic modalities such as surgery, radiation, chemotherapy and combinations of each are used in the management of the disease. In most cases, treatment fails to obtain total cancer cure. In recent years, it appears that one of the key determinants of treatment failure may be the presence of cancer stem cells(CSCs) that escape currently available therapies. CSCs form a small portion of the total tumor burden but may play a disproportionately important role in determining outcomes. CSCs have stem features such as self-renewal, high migration capacity, drug resistance, high proliferation abilities. A large body of evidence points to the fact that CSCs are particularly resistant to radiotherapy and chemotherapy. In HNSCC, CSCs have been increasingly shown to have an integral role in tumor initiation, disease progression, metastasis and treatment resistance. In the light of such observations, the present review summarizes biological characteristics of CSCs in HNSCC, outlines targeted strategies for the successful eradication of CSCs in HNSCC including targeting the self-renewal controlling pathways, blocking epithelial mesenchymal transition, niche targeting, immunotherapy approaches and highlights the need to better understand CSCs biology for new treatments modalities.     

Browsing repeats in genomes: Pygram and an application to non-coding region analysis

Background  

A large number of studies on genome sequences have revealed the major role played by repeated sequences in the structure, function, dynamics and evolution of genomes. In-depth repeat analysis requires specialized methods, including visualization techniques, to achieve optimum exploratory power.     

HPV16-E7 Expression in Squamous Epithelium Creates a Local Immune Suppressive Environment via CCL2- and CCL5- Mediated Recruitment of Mast Cells

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.     

Impact of high temperature on sucrose translocation,sugar content and inulin yield in <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. var. <Emphasis Type="Italic">sativum</Emphasis>

Aims

Dianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.

Methods

ipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.

Results

Inoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high titres.

Conclusion

Klebsiella SGM 81 effectively improves root traits of D. caryophyllus in a dose dependent manner, likely through tryptophan dependent IAA production in the rhizoplane and potentially within the intercellular spaces of root tissue. Under optimal plant growth promoting conditions in non-sterile soil, the high total microbial titre in the rhizosphere supports a mutualistic relationship between Klebsiella SGM 81 and carnation that potentially extends to the wider rhizosphere microbiota.