Optogenetic reporters

The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live‐cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.     

Contrasting calcium localization and speciation in leaves of the Medicago truncatula mutant cod5 analyzed via synchrotron X–ray techniques

Oxalate‐producing plants accumulate calcium oxalate crystals (CaOx_(c)) in the range of 3–80% w/w of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca concentrations to wild‐type (WT) plants, but lower oxalate and CaOx_(c) concentrations. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X–ray fluorescence mapping (SXRF). We observed a difference in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, i.e. the opposite of what is observed in WT. X–ray microdiffraction (μXRD) of M. truncatula leaves confirmed that crystalline CaOx_(c) (whewellite; CaC₂O₄·H₂O) was present in the WT only, in cells sheathing the secondary veins. Together with μXRD, microbeam Ca K–edge X–ray absorption near‐edge structure spectroscopy (μXANES) indicated that, among the forms of CaOx, i.e. crystalline or amorphous, only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets.     

Investigation of an F-18 oxytocin receptor selective ligand via PET imaging

The compound 1-(1-(2-(2-(2-fluoroethoxy)-4-(piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one (1) was synthesized and positively evaluated in vitro for high potency and selectivity with human oxytocin receptors. The positron emitting analogue, [F-18]1, was synthesized and investigated in vivo via PET imaging using rat and cynomolgus monkey models. PET imaging studies in female Sprague–Dawley rats suggested [F-18]1 reached the brain and accumulated in various regions of the brain, but washed out too rapidly for adequate quantification and localization. In vivo PET imaging studies in a male cynomolgus monkey suggested [F-18]1 had limited brain penetration while specific uptake of radioactivity significantly accumulated within the vasculature of the cerebral ventricles in areas representative of the choroid plexus.     

8-Aminoquinoline-based ratiometric zinc probe: Unexpected binding mode and its application in living cells

PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn²⁺-induced red-shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoichiometry in PMQA–Zn²⁺, PMQA–Cu²⁺ shows 1:1 composition. Both nitrogen atoms from the aminoquinoline are missing in binding of zinc, while they are critically involved in Cu²⁺ chelation. The structure difference between PMQA–Zn²⁺ and PMQA–Cu²⁺ might shed light in designing novel zinc probes without suffering from copper interference.     

Synthesis and characterization of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, ^99mTc sestamibi and ^99mTc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. ¹²³I has a similar energy as ^99mTc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an ¹²³I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with ¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog ¹²⁷I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n = 6) and 24 h (n = 8) post injection (p.i.). Results: ¹²³I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8–111 GBq/μmol; 400–3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7′-position, with removal of the 6′,7′-double bond, and addition of a hydroxy group at 6′-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01 ± 0.48%ID/g) and high heart to liver (2.98 ± 0.93), heart to lung (4.11 ± 1.04) and heart to blood (8.37 ± 3.97) ratios. At 24 h p.i., the majority of ¹²³I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. Conclusion: ¹²³I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.     

Measurement and characteristics of neurotransmitter receptor surface trafficking (Review)

Neurotransmitter receptor trafficking in and out synapses has emerged as a key process to regulate synaptic transmission during synaptic development and plasticity both at excitatory and inhibitory synapses. Lateral diffusion of surface neurotransmitter receptors has recently emerged as a key pathway to regulate receptor trafficking to and from synapses. Receptors enter and exit synapses mainly by lateral diffusion within the plane of the membrane while their retrieval and addition from and to the plasma membrane by endo and exocytotic processes occur largely at extrasynaptic sites. As a consequence, regulation of receptor surface trafficking is likely to be a major process to regulate receptor numbers at synapses. Measurement of receptor surface diffusion has required the development of new experimental approaches to specifically label and track surface receptor with appropriate time- and space-resolutions. In this review, we first discuss the approaches that have been used to measure receptor surface diffusion, such as the ensemble approach that measure average diffusion of a defined surface receptor population and the single molecule/particle approaches that measure the surface diffusion of isolated receptors. To date, surface diffusion has been described for a variety of neurotransmitter receptors that exhibit common as well as specific features. These points are discussed in a comparative manner and emerging rules of surface trafficking as well as potential interplay between receptor classes are further commented. Because our knowledge on neurotransmitter receptor surface diffusion is fairly recent, open questions and experimental challenges facing the field are highlighted throughout the review.     

Utility of the NavX® Electroanatomic Mapping System for Permanent Pacemaker Implantation in a Pregnant Patient with Chagas Disease

Chagas disease is a highly prevalent zoonosis in Mexico, Central, and South America. Early cardiac involvement is one of the most serious complications of this disease, and conduction disturbances may occur at an early age. We describe a young pregnant woman with Chagas disease and a high degree atrioventricular block, who required implantation of a permanent dual chamber pacemaker. Using an electroanatomic navigation EnSite NavX® system the pacemaker was successfully implanted with minimal fluoroscopic exposure. This case demonstrates the safety and feasibility of using an electroanatomic navigation system to guide permanent pacemaker implantation minimizing x-ray exposure in pregnant patients.     

Visualization of Gluten and Starch Distributions in Dough by Fluorescence Fingerprint Imaging

A novel method combining imaging techniques and fluorescence fingerprint (FF) data measurement was developed to visualize the distributions of gluten and starch in dough without any preprocessing. Fluorescence images of thin sections of gluten, starch, and dough were acquired under 63 different combinations of excitation and emission wavelengths, resulting in a set of data consisting of the FF data for each pixel. Cosine similarity values between the FF of each pixel in the dough and those of gluten and starch were calculated. Each pixel was colored according to the cosine similarity value to obtain a pseudo-color image showing the distributions of gluten and starch. The dough sample was then fluorescently stained for gluten and starch. The stained image showed patterns similar to the pseudo-color FF image, validating the effectiveness of the FF imaging method. The method proved to be a powerful visualization tool, applicable in fields other than food technology.     

Ischémie myocardique du diabétique : apports de la scintigraphie myocardique de perfusion

Diabetes is an important risk factor of myocardial ischemia. Myocardial perfusion imaging (MPI) is used for diagnostic, prognostic assessment and for post-therapeutic follow-up of coronary artery disease (CAD). Its usefulness has been documented extensively in the general population. However, in diabetic population, little studies have been published. CAD is more severe and more frequent among diabetic patients. The goal of this work was to assess usefulness of MPI among diabetic patients. This work includes 44 diabetic patients (22 women, 22 men), investigated by stress MPI in nuclear medicine department of Ibn Rochd UH of Casablanca. The studied parameters concerned: age, antecedents of CAD, risk factors of CAD associated to diabetes, duration of the diabetes, diabetes complications, diabetes treatment, indication of MPI, presence or not of anomaly on the ECG performed at rest, existence or not of typical or atypical clinical signs of CAD, investigations and therapy prescribed as well as the occurrence or not of cardiac event during monitoring after MPI. Results of MPI have been compared to clinical, therapeutic and monitoring data of patients. Mean age of patients was 55 years (39 to 75 years), mean diabetes duration was 8.6 years (1 to 30 years), at least one diabetes complication has been noted in 18 patients. The most frequent complication was diabetic retinopathy. MPI has been achieved for diagnosis of ischemia in 37 patients and assessment of anti-ischemic treatment in 7 cases. Treadmill exercise has been achieved in 34 cases and a pharmacological stress in 10 others. During follow-up, which was between 1 and 36 months (mean: 14.9 months), 6 cardiac events occurred among the 44 patients. Patients with abnormal findings at stress MPI had two cardiovascular risk factors or more associated to diabetes (91.3% vs. 9.5% among patients having normal findings, P < 0.001) and had diabetic retinopathy more often (56.5% vs. 9.5% among patients with normal findings, P < 0.001). Cardiac events were more frequent among men (100% vs. 43.2% of patients who did not have a coronary event, P < 0.01). Patients with stress MPI showing ischemia in 3/17 segments or more have presented a cardiac event more often during the follow-up (4/12 Vs 2/32 among patients with normal MPI or defect in less than 3/17 segments, P < 0.01). In this series, coronary artery disease was found more frequently among patients having more than 2 cardiovascular risk factors. In addition, risk of cardiac event seems related to extent of uptake decrease.