Artificial Intelligence and Deep Learning for Advancing PET Image Reconstruction: State-of-the-Art and Future Directions.

Positron emission tomography (PET) is vital for diagnosing diseases and monitoring treatments. Conventional image reconstruction (IR) techniques like filtered backprojection and iterative algorithms are powerful but face limitations. PET IR can be seen as an image-to-image translation. Artificial intelligence (AI) and deep learning (DL) using multilayer neural networks enable a new approach to this computer vision task. This review aims to provide mutual understanding for nuclear medicine professionals and AI researchers. We outline fundamentals of PET imaging as well as state-of-the-art in AI-based PET IR with its typical algorithms and DL architectures. Advances improve resolution and contrast recovery, reduce noise, and remove artifacts via inferred attenuation and scatter correction, sinogram inpainting, denoising, and super-resolution refinement. Kernel-priors support list-mode reconstruction, motion correction, and parametric imaging. Hybrid approaches combine AI with conventional IR. Challenges of AI-assisted PET IR include availability of training data, cross-scanner compatibility, and the risk of hallucinated lesions. The need for rigorous evaluations, including quantitative phantom validation and visual comparison of diagnostic accuracy against conventional IR, is highlighted along with regulatory issues. First approved AI-based applications are clinically available, and its impact is foreseeable. Emerging trends, such as the integration of multimodal imaging and the use of data from previous imaging visits, highlight future potentials. Continued collaborative research promises significant improvements in image quality, quantitative accuracy, and diagnostic performance, ultimately leading to the integration of AI-based IR into routine PET imaging protocols.

LILBID-MS: using lasers to shed light on biomolecular architectures.

Structural Biology has moved beyond the aim of simply identifying the components of a cellular subsystem towards analysing the dynamics and interactions of multiple players within a cell. This focal shift comes with additional requirements for the analytical tools used to investigate these systems of increased size and complexity, such as Native Mass Spectrometry, which has always been an important tool for structural biology. Scientific advance and recent developments, such as new ways to mimic a cell membrane for a membrane protein, have caused established methods to struggle to keep up with the increased demands. In this review, we summarize the possibilities, which Laser Induced Liquid Bead Ion Desorption (LILBID) mass spectrometry offers with regard to the challenges of modern structural biology, like increasingly complex sample composition, novel membrane mimics and advanced structural analysis, including next neighbor relations and the dynamics of complex formation.

Assembly and Functional Role of PACE Transporter PA2880 from Pseudomonas aeruginosa.

The recently identified proteobacterial antimicrobial compound efflux (PACE) transporters are multidrug transporters energized by the electrochemical gradient of protons. Here, we present the results of phylogenetic and functional studies on the PACE family transporter PA2880 from Pseudomonas aeruginosa. A phylogenetic analysis of the PACE family revealed that PA2880 and AceI from Acinetobacter baumannii are classified into evolutionarily distinct clades, although they both transport chlorhexidine. We demonstrate that PA2880 mainly exists as a dimer in solution, which is independent of pH, and its dimeric state is essential for its proper function. Electrogenicity studies revealed that the chlorhexidine/H+ antiport process is electrogenic. The function of several highly conserved residues was investigated. These findings provide further insights into the functional features of PACE family transporters, facilitating studies on their transport mechanisms. IMPORTANCE Pseudomonas aeruginosa is a pathogen that causes hospital-acquired (nosocomial) infections, such as ventilator-associated pneumonia and sepsis syndromes. Chlorhexidine diacetate is a disinfectant used for bacterial control in various environments potentially harboring P. aeruginosa. Therefore, investigation of the mechanism of the efflux of chlorhexidine mediated by PA2880, a PACE family transporter from P. aeruginosa, is of significance to combat bacterial infections. This study improves our understanding of the transport mechanism of PACE family transporters and will facilitate the effective utilization of chlorhexidine for P. aeruginosa control.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6.

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

Molecular and Low-Resolution Structural Characterization of the Na+-Translocating Glutaconyl-CoA Decarboxylase From Clostridium symbiosum.

Some anaerobic bacteria use biotin-dependent Na+-translocating decarboxylases (Bdc) of ß-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na+ across the membrane with the exergonic decarboxylation of glutaconyl-CoA (ΔG 0' ≈-30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family.

Peptide Nucleic Acid Conjugates of Quinone Methide Precursors Alkylate Ribonucleic Acid after Activation with Light.

Quinone methide precursors 2 and 3 were protected with a photoreactive 2-nitrobenzyl group and conjugated to peptide nucleic acids (PNA) using a Huisgen click reaction. After brief irradiation at 365 nm, cross-linking with complementary RNA strands started and was analyzed with an ALFexpress sequencer. When this method was used, the gel temperature had a major influence on apparent rates. Quinone methides are known to form transient as well as stable bonds with nucleotides. Although both were detected at 25 °C, analysis at 57 °C only recorded the stable types of cross-links, suggesting much slower alkylation kinetics. Linker 11 allowed us to attach quinone methides to internal positions of the PNA/RNA duplex and to capture a model of miR-20a with good efficiency.

Structure of the Human TRPML2 Ion Channel Extracytosolic/Lumenal Domain.

TRPML2 is the least structurally characterized mammalian transient receptor potential mucolipin ion channel. The TRPML family hallmark is a large extracytosolic/lumenal domain (ELD) between transmembrane helices S1 and S2. We present crystal structures of the tetrameric human TRPML2 ELD at pH 6.5 (2.0 Å) and 4.5 (2.95 Å), corresponding to the pH values in recycling endosomes and lysosomes. Isothermal titration calorimetry shows Ca2+ binding to the highly acidic central pre-pore loop which is abrogated at low pH, in line with a pH-dependent channel regulation model. Small angle X-ray scattering confirms the ELD dimensions in solution. Changes in pH or Ca2+ concentration do not affect the protein's secondary structure, but can influence ELD oligomer integrity according to native mass spectrometry. Our data thus complete the set of high-resolution views of human TRPML channel ELDs and reveal some structural responses to the conditions the TRPML2 ELD encounters as the channel traffics through the endolysosomal system.

LILBID and nESI: Different Native Mass Spectrometry Techniques as Tools in Structural Biology.

Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes. Graphical Abstract ᅟ.

Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs.

Other than more widely used methods, the use of styrene maleic acid allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding. Here we present the combined use of SMALPs and LILBID-MS, allowing determination of oligomeric states of membrane proteins of different functionality directly from the native nanodiscs.

Mapping light-driven conformational changes within the photosensory module of plant phytochrome B.

Organisms developed different photoreceptors to be able to adapt to changing environmental light conditions. Phytochromes are red/far-red (r/fr) photochromic photoreceptors that belong to the classical photoreceptors along with cryptochromes and phototropins. They convert absorbed light into a biological signal by switching between two states in a light-dependent manner therefore enabling the light control downstream signalling. Their Pfr conformation is the biological active form in plants, but until now only a structure of the ground state (Pr) was solved. Here, the authors provide information about structural changes occurring during photoconversion within phytochrome B and identify possible interaction sites for its N-terminal extension (NTE) utilising hydrogen/deuterium exchange rate analyses of its amide backbone. Especially, the newly identified light-dependency of two regions in the NTE are of particular interest for understanding the involvement of the phytochrome's NTE in the regulation of its downstream signalling.

UV-laser ablation of ionic liquid matrices.

Ionic liquid matrices are a new class of matrices used in MALDI mass spectrometry. The ablation process of several ionic liquid matrices was studied by determining the velocity distribution of ablated neutral matrix molecules. This was done by a postionization approach, where the neutrals were ionized in the ablation plume by a second laser pulse. It was found that a second, time-delayed ablation event occurs consisting completely of neutral molecules. To explain this, the reflected-shockwave model is used, which assumes that the shockwave emerging from the laser ablation is reflected at the sample holder surface. When the shockwave arrives at the sample surface it causes a second ablation.