Artificial Biomimetic Electrochemical Assemblies

Rapid, selective, and cost-effective detection and determination of clinically relevant biomolecule analytes for a better understanding of biological and physiological functions are becoming increasingly prominent. In this regard, biosensors represent a powerful tool to meet these requirements. Recent decades have seen biosensors gaining popularity due to their ability to design sensor platforms that are selective to determine target analytes. Naturally generated receptor units have a high affinity for their targets, which provides the selectivity of a device. However, such receptors are subject to instability under harsh environmental conditions and have consequently low durability. By applying principles of supramolecular chemistry, molecularly imprinted polymers (MIPs) can successfully replace natural receptors to circumvent these shortcomings. This review summarizes the recent achievements and analytical applications of electrosynthesized MIPs, in particular, for the detection of protein-based biomarkers. The scope of this review also includes the background behind electrochemical readouts and the origin of the gate effect in MIP-based biosensors.

Theranostic PSMA ligands with optimized backbones for intraoperative multimodal imaging and photodynamic therapy of prostate cancer

Introduction The first generation ligands for prostate-specific membrane antigen (PSMA)–targeted radio- and fluorescence-guided surgery followed by adjuvant photodynamic therapy (PDT) have already shown the potential of this approach. Here, we developed three new photosensitizer-based dual-labeled PSMA ligands by crucial modification of existing PSMA ligand backbone structures (PSMA-1007/PSMA-617) for multimodal imaging and targeted PDT of PCa. Methods Various new PSMA ligands were synthesized using solid-phase chemistry and provided with a DOTA chelator for 111In labeling and the fluorophore/photosensitizer IRDye700DX. The performance of three new dual-labeled ligands was compared with a previously published first-generation ligand (PSMA-N064) and a control ligand with an incomplete PSMA-binding motif. PSMA specificity, affinity, and PDT efficacy of these ligands were determined in LS174T-PSMA cells and control LS174T wildtype cells. Tumor targeting properties were evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies after dissection. Results In order to synthesize the new dual-labeled ligands, we modified the PSMA peptide linker by substitution of a glutamic acid into a lysine residue, providing a handle for conjugation of multiple functional moieties. Ligand optimization showed that the new backbone structure leads to high-affinity PSMA ligands (all IC50 < 50 nM). Moreover, ligand-mediated PDT led to a PSMA-specific decrease in cell viability in vitro (P < 0.001). Linker modification significantly improved tumor targeting compared to the previously developed PSMA-N064 ligand (≥ 20 ± 3%ID/g vs 14 ± 2%ID/g, P < 0.01) and enabled specific visualization of PMSA-positive tumors using both radionuclide and fluorescence imaging in mice. Conclusion The new high-affinity dual-labeled PSMA-targeting ligands with optimized backbone compositions showed increased tumor targeting and enabled multimodal image-guided PCa surgery combined with targeted photodynamic therapy.

Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

Regulation of Trafficking and Signaling of the High Affinity IgE Receptor by FcεRIβ and the Potential Impact of FcεRIβ Splicing in Allergic Inflammation

Mast cells are tissue-resident immune cells that function in both innate and adaptive immunity through the release of both preformed granule-stored mediators, and newly generated proinflammatory mediators that contribute to the generation of both the early and late phases of the allergic inflammatory response. Although mast cells can be activated by a vast array of mediators to contribute to homeostasis and pathophysiology in diverse settings and contexts, in this review, we will focus on the canonical setting of IgE-mediated activation and allergic inflammation. IgE-dependent activation of mast cells occurs through the high affinity IgE receptor, FcεRI, which is a multimeric receptor complex that, once crosslinked by antigen, triggers a cascade of signaling to generate a robust response in mast cells. Here, we discuss FcεRI structure and function, and describe established and emerging roles of the β subunit of FcεRI (FcεRIβ) in regulating mast cell function and FcεRI trafficking and signaling. We discuss current approaches to target IgE and FcεRI signaling and emerging approaches that could target FcεRIβ specifically. We examine how alternative splicing of FcεRIβ alters protein function and how manipulation of splicing could be employed as a therapeutic approach. Targeting FcεRI directly and/or IgE binding to FcεRI are promising approaches to therapeutics for allergic inflammation. The characteristic role of FcεRIβ in both trafficking and signaling of the FcεRI receptor complex, the specificity to IgE-mediated activation pathways, and the preferential expression in mast cells and basophils, makes FcεRIβ an excellent, but challenging, candidate for therapeutic strategies in allergy and asthma, if targeting can be realized.

SPR Analysis of SUMO-Murine Rap1-Interacting Factor 1 C-Terminal Domain Interaction with G4

One of the advantages of surface plasmon resonance is its sensitivity and real-time analyses performed by this method. These characteristics allow us to further investigate the interactions of challenging proteins like Rap1-interacting factor 1 (Rif1). Rif1 is a crucial protein responsible for regulating different cellular processes including DNA replication, repair, and transcription. Mammalian Rif1 is yet to be fully characterized, partly because it is predicted to be intrinsically disordered for a large portion of its polypeptide. This protein has recently been the target of research as a potential biomarker in many cancers. Therefore, finding its most potent interacting partner is of utmost importance. Previous studies showed Rif1’s affinity towards structured DNAs and amongst them, T6G24 was superior. Recent studies have shown mouse Rif1 (muRif1) C-terminal domain’s (CTD) role in binding to G-quadruplexes (G4). There were many concerns in investigating the Rif1 and G4 interaction, which can be minimized using SPR. Therefore, for the first time, we have assessed its binding with G4 at nano-molar concentrations with SPR which seems to be crucial for its binding analyses. Our results indicate that muRif1-CTD has a high affinity for this G4 sequence as it shows a very low KD (6 ± 1 nM).

Structural basis of BAK activation in mitochondrial apoptosis initiation

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Rapid video-based deep learning of cognate versus non-cognate T cell-dendritic cell interactions

Identification of cognate interactions between antigen-specific T cells and dendritic cells (DCs) is essential to understanding immunity and tolerance, and for developing therapies for cancer and autoimmune diseases. Conventional techniques for selecting antigen-specific T cells are time-consuming and limited to pre-defined antigenic peptide sequences. Here, we demonstrate the ability to use deep learning to rapidly classify videos of antigen-specific CD8+ T cells. The trained model distinguishes distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and DCs over 20 to 80 min. The model classified high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalized well to other types of high and low affinity CD8+ T cells. The classification accuracy achieved by the model was consistently higher than simple image analysis techniques, and conventional metrics used to differentiate between cognate and non-cognate T cells, such as speed. Also, we demonstrated that experimental addition of anti-CD40 antibodies improved model prediction. Overall, this method demonstrates the potential of video-based deep learning to rapidly classify cognate T cell-DC interactions, which may also be potentially integrated into high-throughput methods for selecting antigen-specific T cells in the future.

Atypical antipsychotic Lumateperone Beyond Schizophrenia: Seeking clarity in a time of Uncertainty

Lumateperone (ITI-007) is a serotonin 5HT2A tosylate salt with high affinity for dopamine D2 and D1 receptors and the serotonin transporter. It is unusual in that it controls serotonin, dopamine, and glutamate neurotransmission concurrently, all of which have been implicated in severe mental illness. Consider it a multi-targeted ligand and multifunctional modulator of the serotoninergic system with possible precognitive, antipsychotic, antidepressant, and anxiolytic properties. While lumateperone has been explored as a new agent for schizophrenia therapy, it also provides a unique therapeutic option for a range of other psychiatric and neurological diseases, including behavioural signs of dementia or Alzheimer's disease, sleep problems, and bipolar depression. Additionally, it had a better safety profile than placebo, with no significant extrapyramidal side effects, hyperprolactinemia, or changes in cardiometabolic or endocrine characteristics. Additional study is needed to validate and analyse lumateperone's effectiveness, as well as to identify prospective therapeutic targets. This article gives a comprehensive overview of the most notable results and potential future applications of this chemical in personalised medicine, particularly for neurodegenerative diseases.