Inhibition of heterotrimeric Gs proteins by FR900359 - more than a simple transfer of the inhibitor binding site

A plethora of vital functions of the human body, including blood pressure, cell proliferation and metabolism are regulated by G protein-mediated signaling cascades. These cascades form a complex network of cause and effect that allows our bodies to adapt to, function and thrive in a wide range of environments and circumstances. However, this network of signals does not always run flawlessly, and when off balance, pathological changes and diseases are often the consequence such as cancer or hypertension. To treat these diseases, the thorough study of G protein-mediated signaling cascades is an important component of basic research, to understand their influence or contribution to certain cellular events. Such in depth investigations could be accomplished, for example, with specific G protein inhibitors. However, despite the discovery of G proteins about 30 years ago, only specific inhibition of Gi and Gq, two of the four G protein families, could be achieved by PTX or FR900359 (FR) / YM-25489 (YM), respectively. To bridge the lack of such highly cell permeable specific inhibitors of a third G protein family, Gas (Gs) proteins with artificial FR/YM-binding sites, such as Gαs11 (Gs 11), Gαs10 (Gs 10), respectively, have been developed. To this end, we investigated the suitability of Gs 11 and Gs 10 as chemogenetic tools for further possible studies. The idea behind chemogenetics is generally described as the approach in which engineered molecules interact with previously unrecognized molecules that are pharmacologically inert in the absence of the designed protein. Using CRISPR/Cas9-generated Gαs-null or Gαs/olf/q/11/12/13/z - null cells along with several cutting-edge tools such as label-free whole-cell biosensing, HTRF based cAMP accumulation or real-time BRET-based G protein activation, we determined conditions for the use of Gs 10 as a possible chemogenetic tool. However, intriguingly despite the transfer of the inhibitor binding site, FR´s inhibitory properties could not be entirely transferred. While Gq protein- dependent signaling events could be abolished completely, comparable conditions, such as the maximum activation of the overexpressed β2 adrenergic receptor (β2AR) resulted in partial inhibition for the artificial FR-sensitive Gs proteins. Moreover, we noted biologically distinct activities for FR and YM, despite close structural similarities. Thus, the lack of full inhibition was pronounced for the studies done with YM as compared with FR. Our results suggest that the simple transfer of the inhibitor binding site is not sufficient to transfer the properties of inhibition. Thus, possible FR scaffold-based inhibitors which mimic the interaction of FR with Ga proteins accommodating engineered FR-binding sites are not sufficient to perform studies on the same scale as would be possible with Gq. As a result, our study provides an important basis for the understanding of G protein activation and inhibition thus demonstrating the uniqueness of each individual G protein family since although they share an identical inhibitor binding site, Gq but not Gs 11 and Gs 10 could be fully inhibited by FR

Providing Multilingual Access to Health-Oriented Content

Finding health-related content is not an easy task. People have to know what to search for, which medical terms to use, and where to find accurate information. This task becomes even harder when people such as immigrants wish to find information in their country of residence and do not speak the national language very well. In this paper, we present a new health information system that allows users to search for health information using natural language queries composed of multiple languages. We present the technical details of the system and outline the results of a preliminary user study to demonstrate the usability of the system

Providing multilingual access to health-related content

Finding health-related content is not an easy task. People have to know what to search for, which medical terms to use, and where to find accurate information. This task becomes even harder when people such as immigrants wish to find information in their country of residence and do not speak the national language very well. In this paper, we present a new health information system that allows users to search for health information using natural language queries composed of multiple languages. We present the technical details of the system and outline the results of a preliminary user study to demonstrate the usability of the system

The Effect of Increasing Humic Acid Applications on Some Nutrient Contents of Cress (Lepidium sativum L.) Plant

This research was done to determine the effect of increasing Humic acid application on some nutrient element contents of cress (Lepidium sativum L.) plant. For this purpose according to randomize block experimental design, an experiment was done with three replications in greenhouse conditions. Cress plant seed was sowed 1.5 g/ m2. Four humic acid doses (I. dose: 0 mL /m2, II. dose: 8 mL /m2, III. dose: 16 mL /m2 and IV. dose: 24 mL /m2) were applied to cress plant. Then cress plants were harvested 30 days after planting. Dry matter yield and some nutrient (N, P, K, Ca, Mg, S, Fe, Cu, Zn and Mn) contents of plants were determined. According to the results, important increases some nutrient element contents of plants were determined with increasing humic acid applications. These increases were found 1. dose 5.52%, and 4. dose 6.04%, for N element, respectively. Other macro elements P (0.70%, and 0. 82%); K (6.85%, and 7.67%); Ca (1.72%, and 2.01%); Mg (0.13% and 0.15%) and S (1.04%, and 1.17%), respectively. Some micro element (Fe, Cu, Zn and Mn) contents of cress plant, 1. dose: 89.86, 9.59, 59.50 and 56.20; 4. dose: 102.17, 11.03, 67.67 and 76.63 mgkg-1, respectively. These increases were found statistically significant at the level of 5% for each nutrient element, except Mg

Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid– dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other G subunits, we here set out to rationally design G16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gq/G11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold– based inhibitors, provided that these inhibitors mimic the interaction of FR with G proteins harboring engineered FR-binding sites

The Effect of Increasing Mycorrhiza Applications on Nutrition of Pak Choi (Brassica rapa L. subsp. chinensis L.) Plant

The study was done to determine the effect of increasing mycorrhiza application on some macro and micro nutrient element contents of pak choi (Brassica rapa L. subsp. chinensis L.) plant. According to the experiment results, important increases in some macro and micro nutrient element contents of pak choi plant were determined with increasing mycorrhiza applications. The contents were determined as P (0.38 %, 0.42 %, 0.45 %, 0. 49 % and 0.51 %), K (4.01 %, 4.30 %, 4.41 %, 4.56 % and 4.70 %), Ca (1.83 %, 2.01 %, 2.06 %, 2.20 % and 2.36 %), Mg (0.14 %, 0.15 %, 0.15 %, 0.16 % and 0.16 %), Fe (309 mgkg-1, 417 mgkg-1, 678 mgkg-1, 1009 mgkg-1and 1696 mgkg-1), Cu (5.49 mgkg-1, 6.10 mgkg-1, 6.53 mgkg-1, 7.05 mgkg-1and 7.63 mgkg-1), Mn (45.90 mgkg-1, 52.23 mgkg-1, 60.20 mgkg-1, 70.40 mgkg-1and 80.00 mgkg-1) and Zn (32.23 mgkg-1, 35.40 mgkg-1, 37.00 mgkg-1, 40.70 mgkg-1and 46.86 mgkg-1) at I. dose, (control): 0 ml plant-1, II. dose: 15 ml plant-1, III. dose: 20 ml plant-1, IV. dose: 30 ml plant-1  and V. dose: 40 ml plant-1, respectively. These P, K, Ca and Mg contents increases were determined significant at the level of P<0.05, statistically. The highest nutrient element contents of pak choi plant were obtained V. dose: 40 ml plant-1applications for P, K, Ca, Mg, Fe, Cu, Mn and Zn nutrient elements

The Effect of Increasing Humic Acid Applications on Some Nutrient Contents of Cress (Lepidium sativum L.) Plant

This research was done to determine the effect of increasing Humic acid application on some nutrient element contents of cress (Lepidium sativum L.) plant. For this purpose according to randomize block experimental design, an experiment was done with three replications in greenhouse conditions. Cress plant seed was sowed 1.5 g/ m2. Four humic acid doses (I. dose: 0 mL /m2, II. dose: 8 mL /m2, III. dose: 16 mL /m2 and IV. dose: 24 mL /m2) were applied to cress plant. Then cress plants were harvested 30 days after planting. Dry matter yield and some nutrient (N, P, K, Ca, Mg, S, Fe, Cu, Zn and Mn) contents of plants were determined. According to the results, important increases some nutrient element contents of plants were determined with increasing humic acid applications. These increases were found 1. dose 5.52%, and 4. dose 6.04%, for N element, respectively. Other macro elements P (0.70%, and 0. 82%); K (6.85%, and 7.67%); Ca (1.72%, and 2.01%); Mg (0.13% and 0.15%) and S (1.04%, and 1.17%), respectively. Some micro element (Fe, Cu, Zn and Mn) contents of cress plant, 1. dose: 89.86, 9.59, 59.50 and 56.20; 4. dose: 102.17, 11.03, 67.67 and 76.63 mgkg-1, respectively. These increases were found statistically significant at the level of 5% for each nutrient element, except Mg