Current Trends in Targeted Radionuclide Therapy Development

Introduction 1. Features of Targeted Delivery of Therapeutic Radionuclides 2. Design of Pharmaceuticals for Targeted Radionuclide Therapy (TRT) 2.1. Radionuclides 2.2. Synthesis of Radioconjugates 2.3. Targeting Carriers 4. Subcellular Targeting of Radionuclides 5. TRT Dosimetry Conclusion

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.

Regulation of spatially restricted gene expression: linking RNA localization and phase separation

Subcellular restriction of gene expression is crucial to the functioning of a wide variety of cell types. The cellular machinery driving spatially restricted gene expression has been studied for many years, but recent advances have highlighted novel mechanisms by which cells can generate subcellular microenvironments with specialized gene expression profiles. Particularly intriguing are recent findings that phase separation plays a role in certain RNA localization pathways. The burgeoning field of phase separation has revolutionized how we view cellular compartmentalization, revealing that, in addition to membrane-bound organelles, phase-separated cytoplasmic microenvironments — termed biomolecular condensates — are compositionally and functionally distinct from the surrounding cytoplasm, without the need for a lipid membrane. The coupling of phase separation and RNA localization allows for precise subcellular targeting, robust translational repression and dynamic recruitment of accessory proteins. Despite the growing interest in the intersection between RNA localization and phase separation, it remains to be seen how exactly components of the localization machinery, particularly motor proteins, are able to associate with these biomolecular condensates. Further studies of the formation, function, and transport of biomolecular condensates promise to provide a new mechanistic understanding of how cells restrict gene expression at a subcellular level.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7–11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

Semi-automated analysis of an optical ATP indicator in neural synapses

Since its discovery in fireflies, bioluminescence has been used in a wide range of biological assays, from reporter gene assays to in vivo imaging. Bioluminescent light is produced from the enzymatic action of luciferase on its substrate luciferin using ATP as a cofactor. Recently, subcellular targeting of luciferase to neural synaptic vesicles has led to the development of the sensor Syn-ATP that allows for quantitative measurement of presynaptic ATP levels. Manual analysis of presynaptic bioluminescence signals from Syn-ATP is challenging due to signal heterogeneity and cellular motion in long imaging sessions. Here, we present a pipeline for semi-automated image analysis of Syn-ATP signals in the nerve terminals of hippocampal neurons. Our method streamlines data analysis and reduces user-introduced bias, thus enhancing the reproducibility and reliability of quantitative ATP imaging.

Imaging the electrical activity of organelles in living cells

Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.

H-shaped miktobrush copolymer nanoassembly facilitates ultrafast nucleus delivery for multi-stimuli-cooperative tumour suppression

The potencies of antitumour compounds are often compromised by their restricted subcellular delivery to nucleus, although a variety of smart vehicles have been extensively designed to release drug in endocytic organelles. A major challenge remains in exploring the nanocarriers with robust and spatiotemporal responsiveness for yielding efficient subcellular targeting due to tumour heterogeneity. Herein, we show an H-shaped miktobrush copolymer nanoassembly (NAs) with ultrafast nucleus delivery for multi-stimuli-cooperative suppression against primary and metastatic triple-negative breast cancer (TNBC) models. The micellar NAs display acidity- and glutathione-responsive drug releases through the protonation and disulfide-bridge cleavage, which are further amplified by irreversible photo-controlled oxidation and phase transition in a ratiometric manner, leading to rapid morphological destruction and ultrafast cytoplasmic translocation into the nucleus from the lysosomes in a few minutes. These micellar NAs thus show a distinctly enhanced cooperativity of photochemotherapeutic efficacy through considerable apoptotic behavior, potently suppressing subcutaneous and orthotopic TNBC models, together with enhanced survival rates. Moreover, these NAs yield preferable anti-metastatic efficacy through the inhibition of metastasis-relevant proteins as compared to chemotherapy and surgical resection. These results provide the insight into multi-stimuli-responsive polymers for ultrafast nucleus delivery against aggressive tumours.