Functionalized Nanomaterials as Tailored Theranostic Agents in Brain Imaging

Functionalized nanomaterials of various categories are essential for developing cancer nano-theranostics for brain diseases; however, some limitations exist in their effectiveness and clinical translation, such as toxicity, limited tumor penetration, and inability to cross blood–brain and blood-tumor barriers. Metal nanomaterials with functional fluorescent tags possess unique properties in improving their functional properties, including surface plasmon resonance (SPR), superparamagnetism, and photo/bioluminescence, which facilitates imaging applications in addition to their deliveries. Moreover, these multifunctional nanomaterials could be synthesized through various chemical modifications on their physical surfaces via attaching targeting peptides, fluorophores, and quantum dots (QD), which could improve the application of these nanomaterials by facilitating theranostic modalities. In addition to their inherent CT (Computed Tomography), MRI (Magnetic Resonance Imaging), PAI (Photo-acoustic imaging), and X-ray contrast imaging, various multifunctional nanoparticles with imaging probes serve as brain-targeted imaging candidates in several imaging modalities. The primary criteria of these functional nanomaterials for translational application to the brain must be zero toxicity. Moreover, the beneficial aspects of nano-theranostics of nanoparticles are their multifunctional systems proportioned towards personalized disease management via comprising diagnostic and therapeutic abilities in a single biodegradable nanomaterial. This review highlights the emerging aspects of engineered nanomaterials to reach and deliver therapeutics to the brain and how to improve this by adopting the imaging modalities for theranostic applications.

An Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-linked Glycosylation

The biopharmaceutical industry is transitioning towards adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Regulatory agencies such as the Food and Drug Administration (FDA) are further urging use of advanced PAT to analyze the design space to increase process knowledge and enable high quality biologics production. Post-translational modification of proteins, such as N-linked glycosylation are often critical quality attributes known to affect biologics safety and efficacy hence requiring close monitoring during manufacturing. Here, we developed an online sequential-injection based PAT system, called N-GLYcanyzer, that can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes design of an integrated mAb sampling and derivation system for antibody titer and glycoform analysis in under 2 hours. The N-GLYcanyzer process includes mAb capture, deglycosylation, fluorescent glycan labeling, and glycan enrichment for direct injection and analysis on an integrated high performance liquid chromatography (HPLC) system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was studied for optimum glycan recovery and enrichment. We find that 2-AB labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, gives higher glycan labeling efficiency under aqueous conditions leading to upwards of a 5-fold increase in fluorescent products intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at/on-line to an upstream bioreactor for automated and near real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese Hamster Ovary (CHO) cells.

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters

Antibiotic persistence is a phenomenon in which rare cells of a clonal bacterial population can survive antibiotic doses that kill their kin, even though the entire population is genetically susceptible. With antibiotic treatment failure on the rise, there is growing interest in understanding the molecular mechanisms underlying bacterial phenotypic heterogeneity and antibiotic persistence. However, elucidating these rare cell states can be technically challenging. The advent of single-cell techniques has enabled us to observe and quantitatively investigate individual cells in complex, phenotypically heterogeneous populations. In this review, we will discuss current technologies for studying persister phenotypes, including fluorescent tags and biosensors used to elucidate cellular processes; advances in flow cytometry, mass spectrometry, Raman spectroscopy, and microfluidics that contribute high-throughput and high-content information; and next-generation sequencing for powerful insights into genetic and transcriptomic programs. We will further discuss existing knowledge gaps, cutting-edge technologies that can address them, and how advances in single-cell microbiology can potentially improve infectious disease treatment outcomes.

Rhizostomins: A Novel Pigment Family From Rhizostome Jellyfish (Cnidaria, Scyphozoa)

Many pigments, such as melanins, are widely distributed throughout the animal kingdom. Others have arisen as novelties in particular lineages, for example, the Green Fluorescent Protein (GFP) found in cnidarians. While GFPs, widely used as fluorescent tags in biomedical research, are the most famous cnidarian example, other novel proteins have also been identified within this phylum. A blue protein that contains a Kringle (KR) domain inserted within a Frizzled cysteine-rich domain (Fz-CRD) was previously described from the jellyfish Rhizostoma pulmo (named rpulFKz1), however little is known about this pigment’s evolution or distribution among cnidarians. We performed a systematic search for homologs of this protein in published genomes and transcriptomes of 93 cnidarians. Phylogenetic analyses revealed eight predicted proteins that possess both domains in the same arrangement and that fall within the same clade as rpulFKz1. The sequence of one of these proteins contains motifs that match sequenced peptides of Cassio Blue, the blue pigment from Cassiopea xamachana. Another one of these proteins belongs to Stomolophus meleagris, and chemical studies on blue pigments that may occur in this genus have shown similarities to rpulFKz1 and Cassio Blue. Therefore, we hypothesize that the eight rpulFKz1 homologs identified are also pigment precursors. All precursors identified were exclusive to jellyfish in the order Rhizostomeae, so we herein name this new pigment family “rhizostomins.” Not all rhizostomes analyzed are blue, however, so these rhizostomin proteins may also be responsible for other colors, or perform other biochemical and biophysical roles. Previous studies have hypothesized that cnidarian pigments are photoprotective, and this study serves as basis for future investigations not only on the function of rhizostomins, but also on potential biotechnological applications for these proteins.

Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity

Vaccination is an essential public health measure for infectious disease prevention. The exposure of the immune system to vaccine formulations with the appropriate kinetics is critical for inducing protective immunity. In this work, faceted microneedle arrays were designed and fabricated utilizing a three-dimensional (3D)-printing technique called continuous liquid interface production (CLIP). The faceted microneedle design resulted in increased surface area as compared with the smooth square pyramidal design, ultimately leading to enhanced surface coating of model vaccine components (ovalbumin and CpG). Utilizing fluorescent tags and live-animal imaging, we evaluated in vivo cargo retention and bioavailability in mice as a function of route of delivery. Compared with subcutaneous bolus injection of the soluble components, microneedle transdermal delivery not only resulted in enhanced cargo retention in the skin but also improved immune cell activation in the draining lymph nodes. Furthermore, the microneedle vaccine induced a potent humoral immune response, with higher total IgG (Immunoglobulin G) and a more balanced IgG1/IgG2a repertoire and achieved dose sparing. Furthermore, it elicited T cell responses as characterized by functional cytotoxic CD8+ T cells and CD4+ T cells secreting Th1 (T helper type 1)-cytokines. Taken together, CLIP 3D–printed microneedles coated with vaccine components provide a useful platform for a noninvasive, self-applicable vaccination.

Degrading intestinal DAF-2 nearly doubles Caenorhabditis elegans lifespan without affecting development or reproduction

Twenty-eight years following the breakthrough discovery that a single-gene mutation of daf-2 can double the lifespan of Caenorhabditis elegans, it remains unclear where this gene, which encodes an insulin/IGF-1 receptor, is expressed and where it acts to regulate aging. Here, by inserting DNA sequences of fluorescent tags into the genomic locus of daf-2 and that of its downstream transcription factor daf-16, we determined that both genes are expressed in most or all tissues from embryos through adulthood, in line with their diverse functions. Using tissue-specific auxin-induced protein degradation, we determined that both DAF-2 and DAF-16 act in the intestine to regulate organismal aging. Strikingly, loss of DAF-2 in the intestine nearly doubled C. elegans lifespan but did not produce the adverse developmental or reproductive phenotypes associated with genetic daf-2 mutants. These findings unify the mechanism of lifespan regulation by genes and that by dietary restriction, and begin to focus anti-aging research on nutrient supply.

Rapid generation of homozygous fluorescent knock-in human cells using CRISPR/Cas9 genome editing and validation by automated imaging and digital PCR screening

We have previously described a protocol for genome engineering of mammalian cultured cancer cells with CRISPR/Cas9 to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here, we are updating this protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high efficiency electroporation of optimized CRISPR/Cas9 reagents, screening of single cell derived clones by automated bright field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital PCR (dPCR) and automated data analysis. Compared to the original protocol, our current procedure (i) significantly increases the efficiency of tag integration, (ii) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (iii) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with dPCR. The increased efficiency of the new procedure reduces the number of clones that need to be analysed in-depth by more than ten-fold, and yields up to 20% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 days to 10 days during the overall ~10 weeks procedure, allowing a single person to process up to 5 genes in parallel, assuming that validated reagents - e.g. PCR-primers, dPCR-assays, Western Blot antibodies - are available.

Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

Label-free super-resolution imaging below 90-nm using photon-reassignment

Background: Achieving resolutions below 100 nm is key for many fields, including biology and nanomaterial characterization. Although nearfield and electron microscopy are the gold standards for studying the nanoscale, optical microscopy has seen its resolution drastically improve in the last decades. So-called super-resolution microscopy is generally based on fluorescence photophysics and requires modification of the sample at least by adding fluorescent tags, an inevitably invasive step. Therefore, it remains very challenging and rewarding to achieve optical resolutions beyond the diffraction limit in label-free samples. Methods: Here, we present a breakthrough to unlock label-free 3D super-resolution imaging of any object including living biological samples. It is based on optical photon-reassignment in confocal reflectance imaging mode. Results: We demonstrate that we surpass the resolution of all fluorescence-based confocal systems by a factor ~1.5. We have obtained images with a 3D (x,y,z) optical resolution of (86x86x248) nm3 using a visible wavelength (445 nm) and a regular microscope objective (NA=1.3). The results are presented on nanoparticles as well as on (living) biological samples. Conclusions: This cost-effective approach double the resolution of reflectance confocal microscope with minimal modifications. It is therefore compatible with any microscope and sample, works in real-time, and does not require any signal processing.