Theranostics - a sure cure for cancer after 100 years?

Cancer has remained a formidable challenge in medicine and has claimed an enormous number of lives worldwide. Theranostics, combining diagnostic methods with personalized therapeutic approaches, shows huge potential to advance the battle against cancer. This review aims to provide an overview of theranostics in oncology: exploring its history, current advances, challenges, and prospects. We present the fundamental evolution of theranostics from radiotherapeutics, cellular therapeutics, and nanotherapeutics, showcasing critical milestones in the last decade. From the early concept of targeted drug delivery to the emergence of personalized medicine, theranostics has benefited from advances in imaging technologies, molecular biology, and nanomedicine. Furthermore, we emphasize pertinent illustrations showcasing that revolutionary strategies in cancer management enhance diagnostic accuracy and provide targeted therapies customized for individual patients, thereby facilitating the implementation of personalized medicine. Finally, we describe future perspectives on current challenges, emerging topics, and advances in the field.

A conserved phenylalanine motif among Teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared to the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among Teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 Teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time, and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homolog of Brachyhypopomus gauderio (B.g.) is inserted into EPG - EPG(B.g.) - the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation.

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

Computational peptide discovery with a genetic programming approach.

The development of peptides for therapeutic targets or biomarkers for disease diagnosis is a challenging task in protein engineering. Current approaches are tedious, often time-consuming and require complex laboratory data due to the vast search spaces that need to be considered. In silico methods can accelerate research and substantially reduce costs. Evolutionary algorithms are a promising approach for exploring large search spaces and can facilitate the discovery of new peptides. This study presents the development and use of a new variant of the genetic-programming-based POET algorithm, called POET Regex , where individuals are represented by a list of regular expressions. This algorithm was trained on a small curated dataset and employed to generate new peptides improving the sensitivity of peptides in magnetic resonance imaging with chemical exchange saturation transfer (CEST). The resulting model achieves a performance gain of 20% over the initial POET models and is able to predict a candidate peptide with a 58% performance increase compared to the gold-standard peptide. By combining the power of genetic programming with the flexibility of regular expressions, new peptide targets were identified that improve the sensitivity of detection by CEST. This approach provides a promising research direction for the efficient identification of peptides with therapeutic or diagnostic potential.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

Computational Peptide Discovery with a Genetic Programming Approach.

Background: The development of peptides for therapeutic targets or biomarkers for disease diagnosis is a challenging task in protein engineering. Current approaches are tedious, often time-consuming and require complex laboratory data due to the vast search space. In silico methods can accelerate research and substantially reduce costs. Evolutionary algorithms are a promising approach for exploring large search spaces and facilitating the discovery of new peptides. Results: This study presents the development and use of a variant of the initial POET algorithm, called POETRegex, which is based on genetic programming, where individuals are represented by a list of regular expressions. The program was trained on a small curated dataset and employed to predict new peptides that can improve the problem of sensitivity in detecting peptides through magnetic resonance imaging using chemical exchange saturation transfer (CEST). The resulting model achieves a performance gain of 20% over the initial POET variant and is able to predict a candidate peptide with a 58% performance increase compared to the gold-standard peptide. Conclusions: By combining the power of genetic programming with the flexibility of regular expressions, new potential peptide targets were identified to improve the sensitivity of detection by CEST. This approach provides a promising research direction for the efficient identification of peptides with therapeutic or diagnostic potential.

Closed-loop neurostimulation via expression of magnetogenetics-sensitive protein in inhibitory neurons leads to reduction of seizure activity in a rat model of epilepsy.

On-demand neurostimulation has shown success in epilepsy patients with pharmacoresistant seizures. Seizures produce magnetic fields that can be recorded using magnetoencephalography. We developed a new closed-loop approach to control seizure activity based on magnetogenetics using the electromagnetic perceptive gene (EPG) that encodes a protein that responds to magnetic fields. The EPG transgene was expressed in inhibitory interneurons under the hDlx promoter and kainic acid was used to induce acute seizures. In vivo electrophysiological signals were recorded. We found that hDlx EPG rats exhibited a significant delay in the onset of first seizure (1142.72 ± 186.35 s) compared to controls (644.03 ± 15.06 s) and significantly less seizures (4.11 ± 1.03) compared to controls (8.33 ± 1.58). These preliminary findings suggest that on-demand activation of EPG expressed in inhibitory interneurons suppresses seizure activity, and magnetogenetics via EPG may be an effective strategy to alleviate seizure severity in a closed-loop, and cell-specific fashion.

Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity.

Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor, thus allowing deeper brain regions to be imaged noninvasively. Here, we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes were optimized by high-throughput screening of linker libraries. The selected probes exhibit robust changes in light output in response to the extracellular presence of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

Development of a synthetic biosensor for chemical exchange MRI utilizing in silico optimized peptides.

Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed in E. coli and purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

Development of a Synthetic Biosensor for Chemical Exchange MRI Utilizing In Silico Optimized Peptides.

Chemical Exchange Saturation Transfer (CEST) magnetic resonance imaging (MRI) has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in-silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide 2.0, was designed to be 198 amino acids. SuperCESTide 2.0 was expressed in E. coli and purified with size-exclusion chromatography. The magnetic transfer ratio asymmetry (MTR asym ) generated by superCESTide 2.0 was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine, SP), Poly-L-Lysine (PLL), and human protamine (hPRM1). This data shows that novel peptides with sequences optimized in silico for CEST contrast that utilizes a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

A Genetic Programming Approach to Engineering MRI Reporter Genes.

Here we develop a mechanism of protein optimization using a computational approach known as "genetic programming". We developed an algorithm called Protein Optimization Engineering Tool (POET). Starting from a small library of literature values, the use of this tool allowed us to develop proteins that produce four times more MRI contrast than what was previously state-of-the-art. Interestingly, many of the peptides produced using POET were dramatically different with respect to their sequence and chemical environment than existing CEST producing peptides, and challenge prior understandings of how those peptides function. While existing algorithms for protein engineering rely on divergent evolution, POET relies on convergent evolution and consequently allows discovery of peptides with completely different sequences that perform the same function with as good or even better efficiency. Thus, this novel approach can be expanded beyond developing imaging agents and can be used widely in protein engineering.

A Novel Protein for the Bioremediation of Gadolinium Waste.

Several hundreds of tons of gadolinium-based contrast agents (GBCAs) are being dumped into the environment every year. Although macrocyclic GBCAs exhibit superior stability compared to their linear counterparts, we have found that the structural integrity of chelates are susceptible to ultraviolet light, regardless of configuration. In this study, we present a synthetic protein termed GLamouR that binds and reports gadolinium in an intensiometric manner. We then explore the extraction of gadolinium from GBCA-spiked artificial urine samples and investigate if the low picomolar concentrations reported in gadolinium-contaminated water sources pose a barrier for bioremediation. Based on promising results, we anticipate GLamouR can be used for detecting and mining REEs beyond gadolinium as well and hope to expand the biological toolbox for such applications.

Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology.

At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio-organic molecules. However, later, metal-based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal-free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein-based CEST agents, review the rationalization of enzyme-substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein-based CEST contrast agents.

Hyperaccumulation of Gadolinium by Methylorubrum extorquens AM1 Reveals Impacts of Lanthanides on Cellular Processes Beyond Methylotrophy.

Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57-62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo-HLn, for "evolved for Heavy Lanthanides." A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the "lanthasome." Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo-HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo-HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.

A Dual-Reporter Platform for Screening Tumor-Targeted Extracellular Vesicles.

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

Bioelectromagnetic Platform for Cell, Tissue, and In Vivo Stimulation.

Magnetogenetics is a new field that utilizes electromagnetic fields to remotely control cellular activity. In addition to the development of the biological genetic tools, this approach requires designing hardware with a specific set of demands for the electromagnets used to provide the desired stimulation for electrophysiology and imaging experiments. Here, we present a universal stimulus delivery system comprising four magnet designs compatible with electrophysiology, fluorescence and luminescence imaging, microscopy, and freely behaving animal experiments. The overall system includes a low-cost stimulation controller that enables rapid switching between active and sham stimulation trials as well as precise control of stimulation delivery thereby enabling repeatable and reproducible measurements.

Bioengineering of Genetically Encoded Gene Promoter Repressed by the Flavonoid Apigenin for Constructing Intracellular Sensor for Molecular Events.

In recent years, Synthetic Biology has emerged as a new discipline where functions that were traditionally performed by electronic devices are replaced by "cellular devices"; genetically encoded circuits constructed of DNA that are built from biological parts (aka bio-parts). The cellular devices can be used for sensing and responding to natural and artificial signals. However, a major challenge in the field is that the crosstalk between many cellular signaling pathways use the same signaling endogenous molecules that can result in undesired activation. To overcome this problem, we utilized a specific promoter that can activate genes with a natural, non-toxic ligand at a highly-induced transcription level with low background or undesirable off-target expression. Here we used the orphan aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that upon activation binds to specific AHR response elements (AHRE) of the Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) promoter. Flavonoids have been identified as AHR ligands. Data presented here show the successful creation of a synthetic gene "off" switch that can be monitored directly using an optical reporter gene. This is the first step towards bioengineering of a synthetic, nanoscale bio-part for constructing a sensor for molecular events.

Swimming direction of the glass catfish is responsive to magnetic stimulation.

Several marine species have developed a magnetic perception that is essential for navigation and detection of prey and predators. One of these species is the transparent glass catfish that contains an ampullary organ dedicated to sense magnetic fields. Here we examine the behavior of the glass catfish in response to static magnetic fields which will provide valuable insight on function of this magnetic response. By utilizing state of the art animal tracking software and artificial intelligence approaches, we quantified the effects of magnetic fields on the swimming direction of glass catfish. The results demonstrate that glass catfish placed in a radial arm maze, consistently swim away from magnetic fields over 20 µT and show adaptability to changing magnetic field direction and location.

Redesigned reporter gene for improved proton exchange-based molecular MRI contrast.

Reporter gene imaging allows for non-invasive monitoring of molecular processes in living cells, providing insights on the mechanisms underlying pathology and therapy. A lysine-rich protein (LRP) chemical exchange saturation transfer (CEST) MRI reporter gene has previously been developed and used to image tumor cells, cardiac viral gene transfer, and oncolytic virotherapy. However, the highly repetitive nature of the LRP reporter gene sequence leads to DNA recombination events and the expression of a range of truncated LRP protein fragments, thereby greatly limiting the CEST sensitivity. Here we report the use of a redesigned LRP reporter (rdLRP), aimed to provide excellent stability and CEST sensitivity. The rdLRP contains no DNA repeats or GC rich regions and 30% less positively charged amino-acids. RT-PCR of cell lysates transfected with rdLRP demonstrated a stable reporter gene with a single distinct band corresponding to full-length DNA. A distinct increase in CEST-MRI contrast was obtained in cell lysates of rdLRP transfected cells and in in vivo LRP expressing mouse brain tumors ([Formula: see text], n = 10).

Non-invasive neuromodulation using rTMS and the electromagnetic-perceptive gene (EPG) facilitates plasticity after nerve injury.

BACKGROUND: Twenty million Americans suffer from peripheral nerve injury. These patients often develop chronic pain and sensory dysfunctions. In the past decade, neuroimaging studies showed that these changes are associated with altered cortical excitation-inhibition balance and maladaptive plasticity. We tested if neuromodulation of the deprived sensory cortex could restore the cortical balance, and whether it would be effective in alleviating sensory complications. OBJECTIVE: We tested if non-invasive repetitive transcranial magnetic stimulation (rTMS) which induces neuronal excitability, and cell-specific magnetic activation via the Electromagnetic-perceptive gene (EPG) which is a novel gene that was identified and cloned from glass catfish and demonstrated to evoke neural responses when magnetically stimulated, can restore cortical excitability. METHODS: A rat model of forepaw denervation was used. rTMS was delivered every other day for 30 days, starting at the acute or at the chronic post-injury phase. A minimally-invasive neuromodulation via EPG was performed every day for 30 days starting at the chronic phase. A battery of behavioral tests was performed in the days and weeks following limb denervation in EPG-treated rats, and behavioral tests, fMRI and immunochemistry were performed in rTMS-treated rats. RESULTS: The results demonstrate that neuromodulation significantly improved long-term mobility, decreased anxiety and enhanced neuroplasticity. The results identify that both acute and delayed rTMS intervention facilitated rehabilitation. Moreover, the results implicate EPG as an effective cell-specific neuromodulation approach. CONCLUSION: Together, these results reinforce the growing amount of evidence from human and animal studies that are establishing neuromodulation as an effective strategy to promote plasticity and rehabilitation.