A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5–Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response

A study of lipid profile and glycemic status in patients with chronic kidney disease of unknown etiology in Sri Lanka

Background: Dyslipidemia and impaired glucose tolerance are common complications of chronic kidney disease (CKD) and are responsible for increased cardiovascular risk. Studies on lipid profile and glycemic status in CKD of unknown origin (CKDu) are scarce. The objective of this study was to evaluate the lipid profile and glycemic status of the patients with CKDu and to aid in preventing morbidity and mortality.Methods: The descriptive, cross sectional study was conducted in a rural CKDu endemic area, Girandurukotte.  Data was collected from February 2018 to June 2019. For the diagnosis of CKDu, history and clinical features with supportive biochemical, renal biopsy and radiological evidence were taken as criteria. Blood samples were taken for serum creatinine, lipid profile and HbA1C. Already diagnosed patients with diabetes mellitus and dyslipidemia were excluded.Results: A total of 168 patients within the age range of 32-66 years (mean 50.3±7.7) were participated. There were 106 males (63%) 46.4% were farmers. Majority of the patients (65.5%) had normal body mass index (BMI) (mean 22.9 kg/m2, normal range 18.5-23.5%) followed by overweight (23.5-30 kg/m2) in 55 (32.7%) patients. The prevalence of dyslipidemia in CKDu was found to be 55.9%.  Majority of the abnormality was seen in the HDL group with 68 (40.5%) patients having low HDL cholesterol (mean 44.7 mg/dl, SD=12.3).  There was a significant rise in the serum triglyceride concentration (>150 mg/dl) in 53 (31.5%) (mean 152.4 mg/dl, SD=73.5) and total cholesterol (>200 mg/dl) in 30 (18%) patients (mean 182 mg/dl, SD=36.9). LDL cholesterol abnormality (>130 mg/dl) was seen in only 9 patients (mean 88.7 7658mg/dl, SD=25.4). From the total, 144 (85.7%) patients had abnormal HbA1C levels; 27 (16.1%) patients had HbA1C levels between 5.7% and 6.4% (pre-diabetes), and 117 (69.6%) patients had HbA1C level more than 6.5% (diabetes mellitus). There was no statistically significant association between HbA1C levels and BMI (p=0.29) or HbA1C and lipid abnormalities (p=0.32)Conclusions: The high prevalence of dyslipidemia, pre-diabetes and diabetes mellitus in patients with CKDu may accelerate the progression of chronic kidney disease and increase the risk of cardiovascular disease. Early detection, initiation of appropriate medication and early referral to the expertise will ameliorate morbidity and mortality.

Investigation of the Mechanisms Underlying the Gastroprotective Effect of Cymbopogon Citratus Essential Oil

Cymbopogon citratus is a medicinal plant popularly used in Brazil for the treatment of various diseases, and the research interest in this plant is justifiable because of its potential medicinal value in stomachache and gastric ulcer. This study was aimed to test the validity of this practice by using experimental models of gastric ulcer and to clarify the mechanisms of gastroprotection by C. citratus leaves essential oil (EOCC). EOCC was evaluated for the ability to protect the gastric mucosa against injuries caused by necrotizing agents (absolute ethanol and aspirin) in rodents. The results of this study revealed that EOCC posses a dose-independent anti-ulcer effect against the different experimental models. EOCC pretreatment depicted a higher preventive index in ethanol-(88%) and aspirin-induced (76%) acute ulceration. On pretreatment of mice with indomethacin, the cyclooxygenase inhibitor slightly suppressed the gastroprotective effect of EOCC (48.5%). Furthermore, EOCC gastroprotection was not attenuated in mice pretreated with L-NAME (85.2%), glibenclamide (100%), or yohimbine (79.7%), the respective inhibitors of nitric oxide synthase, K+ATP channel activation, and α2 receptors. These results confirmed the traditional use of C. citratus for the treatment of gastric ulcer. Thus, we provide the first evidence that EOCC reduces gastric damage induced by ethanol, at least in part, by mechanisms that involve endogenous prostaglandins

Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)-Aux/IAA co-receptor complex formation.

Structure-activity profiles for the phytohormone auxin have been collected for over 70 years, and a number of synthetic auxins are used in agriculture. Auxin classification schemes and binding models followed from understanding auxin structures. However, all of the data came from whole plant bioassays, meaning the output was the integral of many different processes. The discovery of Transport Inhibitor-Response 1 (TIR1) and the Auxin F-Box (AFB) proteins as sites of auxin perception and the role of auxin as molecular glue in the assembly of co-receptor complexes has allowed the development of a definitive quantitative structure-activity relationship for TIR1 and AFB5. Factorial analysis of binding activities offered two uncorrelated factors associated with binding efficiency and binding selectivity. The six maximum-likelihood estimators of Efficiency are changes in the overlap matrixes, inferring that Efficiency is related to the volume of the electronic system. Using the subset of compounds that bound strongly, chemometric analyses based on quantum chemical calculations and similarity and self-similarity indices yielded three classes of Specificity that relate to differential binding. Specificity may not be defined by any one specific atom or position and is influenced by coulomb matrixes, suggesting that it is driven by electrostatic forces. These analyses give the first receptor-specific classification of auxins and indicate that AFB5 is the preferred site for a number of auxinic herbicides by allowing interactions with analogues having van der Waals surfaces larger than that of indole-3-acetic acid. The quality factors are also examined in terms of long-standing models for the mechanism of auxin binding

The Role of Phe82 and Phe351 in Auxin-Induced Substrate Perception by TIR1 Ubiquitin Ligase: A Novel Insight from Molecular Dynamics Simulations

It is well known that Auxin plays a key role in controlling many aspects of plant growth and development. Crystal structures of Transport inhibitor response 1 (TIR1), a true receptor of auxin, were very recently determined for TIR1 alone and in complexes with auxin and different synthetic analogues and an Auxin/Indole-3-Acetic Acid (Aux/IAA) substrate peptide. However, the dynamic conformational changes of the key residues of TIR1 that take place during the auxin and substrate perception by TIR1 and the detailed mechanism of these changes are still unclear. In the present study, various computational techniques were integrated to uncover the detailed molecular mechanism of the auxin and Aux/IAA perception process; these simulations included molecular dynamics (MD) simulations on complexes and the free enzyme, the molecular mechanics Poisson Boltzmann surface area (MM-PBSA) calculations, normal mode analysis, and hydrogen bond energy (HBE) calculations. The computational simulation results provided a reasonable explanation for the structure-activity relationships of auxin and its synthetic analogues in view of energy. In addition, a more detailed model for auxin and Aux/IAA perception was also proposed, indicating that Phe82 and Phe351 played a pivotal role in Aux/IAA perception. Upon auxin binding, Phe82 underwent conformational changes to accommodate the subsequent binding of Aux/IAA. As a result, auxin enhances the TIR1-Aux/IAA interactions by acting as a “molecular glue”. Besides, Phe351 acts as a “fastener” to further improve the substrate binding. The structural and mechanistic insights obtained from the present study will provide valuable clues for the future design of promising auxin analogues

The small angle tile calorimeter in the DELPHI experiment

The {\bf S}mall angle {\bf TI}le {\bf C}alorimeter ({\bf STIC}) provides calorimetric coverage in the very forward region of the DELPHI experiment at the CERN LEP collider. The structure of the calorimeters, built with a so-called ``shashlik'' technique, gives a perfectly hermetic calorimeter and still allows for the insertion of tracking detectors within the sampling structure to measure the direction of the showering particle. A charged-particle veto system, composed of two scintillator layers, makes it possible to trigger on single photon events and provides e-$\gamma$ separat ion. Results are presented from the extensive studies of these detectors in the CERN testbeams prior to installation and of the detector performance at LEP

CSN-mediated deneddylation differentially modulates Ci155 proteolysis to promote Hedgehog signalling responses

The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCFSlimb-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCFSlimb-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCFSlimb is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate–E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

AbstractTroponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 ± 86 Å2 for apo-cNTnC to 3108 ± 71 Å2 for Ca2+-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca2+-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis