Preparation, characterization and cell labelling of strong pH-controlled bicolor fluorescence carbonized polymer dots

Multicolor fluorescence N-doped CPDs from dextrin water solution in strong acidic and alkaline environments were synthesized and characterized, which revealed that pH value plays a vital role in the process of CPD growth.

Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Donor-Acceptor Pyridinium Salts for Photo-Induced Electron Transfer Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes using Visible Light

Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling re-mains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship enabling optical triggering with visible light whilst simultaneously attenuating the probe’s photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of probe and short irradiation times (10-60 min) with violet light enabled efficient reactivity towards surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescent quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 450 nm LED irradiation. Peptide level enrichment from live cell labelling experiments identified 290 Trp modifications, with an 82% selectivity for Trp modification over other π-amino acids; demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.

NaNuTrap, a technique for in vivo cell nucleus labelling using nanobodies

In vivo cell labelling is challenging in fast developmental processes because many cell types differentiate more quickly than the maturation time of fluorescent proteins making visualization of these tissues impossible with standard techniques. Here we present a nanobody-based method, Nanobody Nuclear Trap (NaNuTrap), which works with the existing Gal4/UAS system in Drosophila and allows for early in vivo cell nuclei labelling independent of the fluorescent protein's maturation time. This restores the utility of fluorescent proteins that have longer maturation times, such as those used in two-photon imaging, for live imaging of fast or very early developmental processes. We also present a more general application of this system, whereby NaNuTrap can convert cytoplasmic GFP expressed in any existing transgenic fly line into a nuclear label. This nuclear re-localization of the fluorescent signal can improve the utility of the GFP label, for example in cell counting, as well as resulting in a general increase in intensity of the live fluorescent signal. We demonstrate these capabilities of NaNuTrap by effectively tracking subsets of cells during the fast movements associated with gastrulation.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

Nanotechnology and Biomedical Applications of Nanotechnology

Solid colloidal particles of size from 10 to 1000 Nanometre are known as Nanoparticles. Nanoparticles contribute many benefits to bigger particles such as enhanced surface-to-volume ratio and enhanced magnetic properties. Over the last few years, there has been an undeviating growing interest in using nanoparticles in different biomedical applications such as targeted drug delivery, hyperthermia, photo ablation therapy, bio imaging, and biosensors. Iron oxide nanoparticles have dominated applications, such as drug delivery, hyperthermia, bio imaging, cell labelling, and gene delivery, because of their superior properties such as chemical stability, non-toxicity, biocompatibility, high saturation magnetization, and high magnetic susceptibility. In this paper, biomedical applications of two different types of nanoparticles metal oxide nanoparticles and carbon nanotubes are discussed.

Nanotoxicity of multifunctional unlike cobalt nanoparticles (UCoNPs) with repercussions towards apoptosis and necrosis at nanobio-interfaces

: The development of multifunction nanoparticles proved their worth in the field of the discovery of drug/gene delivery, nanotheranostics (in-vivo imaging, coinciding diagnostics), in external healing intercessions, designing a nano-bio interface, and to do desired alterations in nanotherapeutic. Every so often, the cellular uptake of multifunctional unlike cobalt [Co, CoO, Co2(CO)8 and Co3O4] nanoparticles (UCoNPs) influenced cellular mechanics and initiated numerous repercussions (oxidative stress, tempted DNA damages, cyto-genotoxicity, and chromosomal damages), in pathways, routes and generate dysregulating factors in the biochemical transformations exceedingly. Unlike dimensions of UCoNPs-cell interfaces, their physical features (size, shape, shell structure, and surface chemistry), possessions on cell proliferation and differentiation are the vital whys and wherefores, which are hereby, specifically identified as the key causes responsible for nanotoxicity. In this review, the UCoNPs intricacies (cyto-genotoxicity, clastogenicity, and immunomodulatory), nanotoxicity, and associated repercussions have been highlighted and discussed. The interpretation of quantitative structure-activity relationships, chemical transformations, biological, and toxicological analysis are discussed. The concerns and influences of multifunctional UCoNPs on different cell mechanisms (mitochondria impermeability, hydrolysis of ATP, the concentration of Ca2+, impaired calcium clearance, defective autophagy, apoptosis, and necrosis), and interlinked properties (adhesion, motility, and internalisation dynamics, role in toxicity, surface hydrophilic and hydrophobicity, biokinetics and biomimetic behaviors of biochemical reactions) have been summarised. Various applications i.e. bio-imaging, cell labelling, gene delivery, enhanced chemical stability, and increased bio-compatibility are highlighted concerning apoptosis, necrosis, and nanobio-interfaces with suitable examples.

Immune cell labelling and tracking: implications for adoptive cell transfer therapies

Background The understanding of the role of different immune cell subsets that infiltrate tumors can help researchers in developing new targeted immunotherapies to reactivate or reprogram them against cancer. In addition to conventional drugs, new cell-based therapies, like adoptive cell transfer, proved to be successful in humans. Indeed, after the approval of anti-CD19 CAR-T cell therapy, researchers are trying to extend this approach to other cancer or cell types. Main body This review focuses on the different approaches to non-invasively monitor the biodistribution, trafficking and fate of immune therapeutic cells, evaluating their efficacy at preclinical and clinical stages. PubMed and Scopus databases were searched for published articles on the imaging of cell tracking in humans and preclinical models. Conclusion Labelling specific immune cell subtypes with specific radiopharmaceuticals, contrast agents or optical probes can elucidate new biological mechanisms or predict therapeutic outcome of adoptive cell transfer therapies. To date, no technique is considered the gold standard to image immune cells in adoptive cell transfer therapies.