Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

Positron Emission Tomography Techniques to Measure Active Inflammation, Fibrosis and Angiogenesis: Potential for Non-invasive Imaging of Hypertensive Heart Failure

Heart failure, which is responsible for a high number of deaths worldwide, can develop due to chronic hypertension. Heart failure can involve and progress through several different pathways, including: fibrosis, inflammation, and angiogenesis. Early and specific detection of changes in the myocardium during the transition to heart failure can be made via the use of molecular imaging techniques, including positron emission tomography (PET). Traditional cardiovascular PET techniques, such as myocardial perfusion imaging and sympathetic innervation imaging, have been established at the clinical level but are often lacking in pathway and target specificity that is important for assessment of heart failure. Therefore, there is a need to identify new PET imaging markers of inflammation, fibrosis and angiogenesis that could aid diagnosis, staging and treatment of hypertensive heart failure. This review will provide an overview of key mechanisms underlying hypertensive heart failure and will present the latest developments in PET probes for detection of cardiovascular inflammation, fibrosis and angiogenesis. Currently, selective PET probes for detection of angiogenesis remain elusive but promising PET probes for specific targeting of inflammation and fibrosis are rapidly progressing into clinical use.

CALIPER: A software for blood-free parametric Patlak mapping using PET/MRI input function.

Routine clinical use of absolute PET quantification techniques is limited by the need for serial arterial blood sampling for input function and more importantly by the lack of automated pharmacokinetic analysis tools that can be readily implemented in clinic with minimal effort. PET/MRI provides the ability for absolute quantification of PET probes without the need for serial arterial blood sampling using image-derived input functions (IDIFs). Here we introduce CALIPER, a tool for simplified pharmacokinetic modelling of PET probes with irreversible uptake or binding based on and PET/MR IDIFs and Patlak Plot analysis. CALIPER generates regional values or parametric maps of net influx rate (Ki) for tracers using reconstructed dynamic PET images and anatomical MRI for IDIF vessel delineation. We evaluated the performance of CALIPER for blood-free region-based and pixel-wise Patlak analyses of [18F]FDG. IDIFs corrected for partial volume errors including spill-out and spill-in effects were similar to AIF with a general bias of around 6-8%, even for arteries <5 mm. The Ki and cerebral metabolic rate of glucose estimated using IDIF were similar to estimates using blood sampling (<2%) and within limits of whole brain values reported in literature. Overall, CALIPER is a promising tool for irreversible PET tracer quantification and can simplify the ability to perform parametric analysis in clinical settings without the need for blood sampling.

NHS-Functionalized THP Derivative for Efficient Synthesis of Kit-Based Precursors for 68Ga Labeled PET Probes

Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe3+ at very low iron concentrations and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the radiometal gallium-68 (68Ga). THP-peptide bioconjugates rapidly and quantitatively complex 68Ga at room temperature, neutral pH, and micromolar ligand concentrations, making them amenable to kit-based radiosynthesis of 68Ga PET radiopharmaceuticals. With the aim to produce an N-hydroxysuccinimide-(NHS)-THP reagent for kit-based 68Ga-labeling and PET imaging, THP-derivatives were designed and synthesized to exploit the advantages of NHS chemistry for coupling with peptides, proteins, and antibodies. The more stable five-carbon atoms linker product was selected for a proof-of-concept conjugation and radiolabeling study with an anti-programmed death ligand 1 (PD-L1) camelid single domain antibody (sdAb) under mild conditions and further evaluated for site-specific amide bond formation with a synthesized glucagon-like peptide-1 (GLP-1) targeting peptide using solid-phase synthesis. The obtained THP-GLP-1 conjugate was tested for its 68Ga chelating ability, demonstrating to be a promising candidate for the detection and monitoring of GLP-1 aberrant malignancies. The obtained sdAb-THP conjugate was radiolabeled with 68Ga under mild conditions, providing sufficient labeling yields after 5 min, demonstrating that the novel NHS-THP bifunctional chelator can be widely used to easily conjugate the THP moiety to different targeting molecules (e.g., antibodies, anticalins, or peptides) under mild conditions, paving the way to the synthesis of different imaging probes with all the advantages of THP radiochemistry.

Detection Sensitivity Enhancement of Naphthalimide PET Fluorescent Probes by 4-Methoxy-Substitution

Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example, 4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of 4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and fluorescence emission spectra of BPNM and is further conducive to the enhancement of molar extinction coefficient ε and fluorescence quantum yield Φf of BPNM. Compared with BPN, BPNM shows lower Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays a good foundation for the synthesis of PET probe with high sensitivity.

The utility of pharmacological and radiological interventions to optimize diagnostic information from PET/CT

Background Positron Emission Tomography with Computed Tomography (PET/CT) is widely used in the assessment of many diseases, particularly including cancer. However, many factors can affect image quality and diagnostic performance of PET scans using FDG or other PET probes. Main body The aim of this pictorial essay is to review PET/CT protocols that can be useful to overcome these confounding factors in routine clinical situations, with a particular focus on pharmacological interventions and problem-oriented CT acquisition protocols. Conclusion Imaging protocols and representative cases will be discussed, in addition to potential contraindications and precautions to be taken.