Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

Cell-to-cell variability in troponin I phosphorylation in a porcine model of pacing-induced heart failure

We tested the hypothesis that myocardial contractile protein phosphorylation and the Ca2+ sensitivity of force production are dysregulated in a porcine model of pacing-induced heart failure (HF). The level of protein kinase A (PKA)-dependent cardiac troponin I (TnI) phosphorylation was lower in the myocardium surrounding the pacing electrode (pacing site) of the failing left ventricle (LV) than in the controls. Immunohistochemical assays of the LV pacing site pointed to isolated clusters of cardiomyocytes exhibiting a reduced level of phosphorylated TnI. Flow cytometry on isolated and permeabilized cardiomyocytes revealed a significantly larger cell-to-cell variation in the level of TnI phosphorylation of the LV pacing site than in the opposite region in HF or in either region in the controls: the interquartile range (IQR) on the distribution histogram of relative TnI phosphorylation was wider at the pacing site (IQR = 0.53) than that at the remote site of HF (IQR = 0.42; P = 0.0047) or that of the free wall of the control animals (IQR = 0.36; P = 0.0093). Additionally, the Ca2+ sensitivities of isometric force production were higher and appeared to be more variable in single permeabilized cardiomyocytes from the HF pacing site than in the healthy myocardium. In conclusion, the level of PKA-dependent TnI phosphorylation and the Ca2+ sensitivity of force production exhibited a high cell-to-cell variability at the LV pacing site, possibly explaining the abnormalities of the regional myocardial contractile function in a porcine model of pacing-induced HF

Designed polyelectrolyte shell on magnetite nanocore for dilution-resistant biocompatible magnetic fluids.

Magnetite nanoparticles (MNPs) coated with poly(acrylic acid-co-maleic acid) polyelectrolyte (PAM) have been prepared with the aim of improving colloidal stability of core-shell nanoparticles for biomedical applications and enhancing the durability of the coating shells. FTIR-ATR measurements reveal two types of interaction of PAM with MNPs: hydrogen bonding and inner-sphere metal-carboxylate complex formation. The mechanism of the latter is ligand exchange between uncharged -OH groups of the surface and -COO(-) anionic moieties of the polyelectrolyte as revealed by adsorption and electrokinetic experiments. The aqueous dispersion of PAM@MNP particles (magnetic fluids - MFs) tolerates physiological salt concentration at composition corresponding to the plateau of the high-affinity adsorption isotherm. The plateau is reached at small amount of added PAM and at low concentration of nonadsorbed PAM, making PAM highly efficient for coating MNPs. The adsorbed PAM layer is not desorbed during dilution. The performance of the PAM shell is superior to that of poly(acrylic acid) (PAA), often used in biocompatible MFs. This is explained by the different adsorption mechanisms; metal-carboxylate cannot form in the case of PAA. Molecular-level understanding of the protective shell formation on MNPs presented here improves fundamentally the colloidal techniques used in core-shell nanoparticle production for nanotechnology applications

Evidence for diagnosis of early chronic pancreatitis after three episodes of acute pancreatitis: a cross-sectional multicentre international study with experimental animal model

Chronic pancreatitis (CP) is an end-stage disease with no specific therapy; therefore, an early diagnosis is of crucial importance. In this study, data from 1315 and 318 patients were analysed from acute pancreatitis (AP) and CP registries, respectively. The population from the AP registry was divided into AP (n = 983), recurrent AP (RAP, n = 270) and CP (n = 62) groups. The prevalence of CP in combination with AP, RAP2, RAP3, RAP4 and RAP5 + was 0%, 1%, 16%, 50% and 47%, respectively, suggesting that three or more episodes of AP is a strong risk factor for CP. Laboratory, imaging and clinical biomarkers highlighted that patients with RAP3 + do not show a significant difference between RAPs and CP. Data from CP registries showed 98% of patients had at least one AP and the average number of episodes was four. We mimicked the human RAPs in a mouse model and found that three or more episodes of AP cause early chronic-like morphological changes in the pancreas. We concluded that three or more attacks of AP with no morphological changes to the pancreas could be considered as early CP (ECP).The new diagnostic criteria for ECP allow the majority of CP patients to be diagnosed earlier. They can be used in hospitals with no additional costs in healthcare

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction. © 2015 Elsevier B.V. All rights reserved

Ins<i>P</i>₃ sensors in use for single-cell fluorescent energy transfer-based applications.

<p>K_D values represent the Ins<i>P</i>₃ concentrations required to reach 50% of the dynamic range of the appropriate sensor. ND means not determined.</p><p>*Ins<i>P</i>₃ insensitive mutant of LIBRA</p><p>**non-binding mutant for control experiments</p><p>Ins<i>P</i>₃ sensors in use for single-cell fluorescent energy transfer-based applications.</p

Comparison of the activation properties of the wild type and low-affinity Ins<i>P</i>₃ sensors.

<p>(A) Normalized BRET ratio (reciprocal of I/I_<b>0</b>) measured in HEK 293T cells transiently transfected with the AT1 angiotensin receptor and the wild type or R265K mutant Ins<i>P</i>_<b>3</b> biosensors upon Ang II stimulation (10^-12-10^-7 M) added manually. To avoid desensitization of the receptor a non-internalizing receptor was used (AT1R-Δ319). Error bars show standard error values from three independent measurements performed in triplicate. (B) Concentration-response curves for Ang II. Ins<i>P</i>_<b>3</b> responses were measured 5 minutes after stimulation. The right shift caused by the R265K mutation corresponds to the lower ligand binding affinity of the Ins<i>P</i>_<b>3</b> biosensor.</p

Characterization of the newly developed Ins<i>P</i>₃ sensor.

<p>(A) Schematic drawing of the inositol 1,4,5-trisphosphate (Ins<i>P</i>_<b>3</b>)-binding core domain based on its crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125601#pone.0125601.ref018" target="_blank">18</a>]. Residues in the alpha helical domain and the beta-trefoil domain are highlighted in green and yellow, respectively. Ins<i>P</i>_<b>3</b> is highlighted in blue. The hinge region is shown in purple. Arginine to lysine mutations were introduced in the ligand binding residues at positions 265, 269, 504 and 568 (red arrows). (B) <i>In vitro</i> characterization of the inositol phosphate binding of recombinant mRFP-tagged Ins<i>P</i>_<b>3</b> binding domains. The assays were performed using [³H]Ins<i>P</i>_<b>3</b> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125601#sec002" target="_blank">methods</a>) in the presence of the indicated concentrations of the respective unlabeled ligand. Two separate experiments in triplicates. (C) Schematic representations of domain structures of the different Ins<i>P</i>_<b>3</b> biosensors and a modified Cameleon D3 BRET sensor for measuring the changes in cytoplasmic Ca²⁺ concentration. The Ins<i>P</i>_<b>3</b> sensors contain Cerulean (FRET) or Sluc (BRET) on the N-terminus, the ligand-binding domain of the human type-I Ins<i>P</i>_<b>3</b>-receptor and either Venus or circularly permuted Venus (cp173) and Venus on the C-terminus. The blue lines show the approximate location of the designed mutations (R265K). The Ca²⁺ sensor contains the MLCK calmodulin binding peptide M13 and the D3 variant of calmodulin. (D) Representative images of Ins<i>P</i>_<b>3</b> or Ca²⁺ biosensor-containing COS-7 cells. (E) Measurements of FRET in individual HEK 293T cells expressing the FRET Ins<i>P</i>_<b>3</b> biosensor and the AT1 angiotensin receptor. Ins<i>P</i>_<b>3</b> production of the cells was triggered by 1 μM angiotensin II (Ang II). Note that binding of Ins<i>P</i>_<b>3</b> resulted in a decrease of the energy transfer. (F) Measurement of BRET in HEK 293T cells expressing two types of BRET Ins<i>P</i>_<b>3</b> biosensors containing a single or a tandem fluorescent protein (Venus or cp173-Venus and Venus). Ins<i>P</i>_<b>3</b> production was induced by ionomycin (10 μM). The curves indicate the raw BRET ratios of individual wells of a 96-well white tissue culture plate used in the BRET measurements.</p