Ultra-high Throughput Research Articles

Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering.

Classical directed evolution is a powerful approach for engineering biomolecules with improved or novel functions. However, it traditionally relies on labour- and time-intensive iterative cycles, due in part to the need for multiple molecular biology steps, including DNA transformation, and limited screening throughput. In this study, we present an ultrahigh throughput in vivo continuous directed evolution system with thermosensitive inducible tunability, which is based on error-prone DNA polymerase expression modulated by engineered thermal-responsive repressor cI857, and genomic MutS mutant with temperature-sensitive defect for fixation of mutations in Escherichia coli. We demonstrated the success of the in vivo evolution platform with β-lactamase as a model, with an approximately 600-fold increase in the targeted mutation rate. Furthermore, the platform was combined with ultrahigh-throughput screening methods and employed to evolve α-amylase and the resveratrol biosynthetic pathway. After iterative rounds of enrichment, a mutant with a 48.3% improvement in α-amylase activity was identified via microfluidic droplet screening. In addition, when coupled with an in vivo biosensor in the resveratrol biosynthetic pathway, a variant with 1.7-fold higher resveratrol production was selected by fluorescence-activated cell sorting. In this study, thermal-responsive targeted mutagenesis coupled with ultrahigh-throughput screening was developed for the rapid evolution of enzymes and biosynthetic pathways.

Photoannealing of Microtissues Creates High-Density Capillary Network Containing Living Matter in a Volumetric-Independent Manner.

The vascular tree is crucial for the survival and function of large living tissues. Despite breakthroughs in 3D bioprinting to endow engineered tissues with large blood vessels, there is currently no approach to engineer high-density capillary networks into living tissues in a scalable manner. Here, photoannealing of living microtissue (PALM) is presented as a scalable strategy to engineer capillary-rich tissues. Specifically, in-air microfluidics is used to produce living microtissues composed of cell-laden microgels in ultrahigh throughput, which can be photoannealed into a monolithic living matter. Annealed microtissues inherently give rise to an open and interconnected pore network within the resulting living matter. Interestingly, utilizing soft microgels enables microgel deformation, which leads to the uniform formation of capillary-sized pores. Importantly, the ultrahigh throughput nature underlying the microtissue formation uniquely facilitates scalable production of living tissues of clinically relevant sizes (>1 cm3) with an integrated high-density capillary network. In short, PALM generates monolithic, microporous, modular tissues that meet the previously unsolved need for large engineered tissues containing high-density vascular networks, which is anticipated to advance the fields of engineered organs, regenerative medicine, and drug screening.

Protocol for high throughput 3D drug screening of patient derived melanoma and renal cell carcinoma

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.

On synergy between ultrahigh throughput screening and machine learning in biocatalyst engineering.

Protein design and directed evolution have separately contributed enormously to protein engineering. Without being mutually exclusive, the former relies on computation from first principles, while the latter is a combinatorial approach based on chance. Advances in ultrahigh throughput (uHT) screening, next generation sequencing and machine learning may create alternative routes to engineered proteins, where functional information linked to specific sequences is interpreted and extrapolated in silico. In particular, the miniaturisation of functional tests in water-in-oil emulsion droplets with picoliter volumes and their rapid generation and analysis (>1 kHz) allows screening of >107-membered libraries in a day. Subsequently, decoding the selected clones by short or long-read sequencing methods leads to large sequence-function datasets that may allow extrapolation from experimental directed evolution to further improved mutants beyond the observed hits. In this work, we explore experimental strategies for how to draw up 'fitness landscapes' in sequence space with uHT droplet microfluidics, review the current state of AI/ML in enzyme engineering and discuss how uHT datasets may be combined with AI/ML to make meaningful predictions and accelerate biocatalyst engineering.

Random mutagenesis of Phaeodactylum tricornutum using ultraviolet, chemical, and X-radiation demonstrates the need for temporal analysis of phenotype stability

We investigated two non-ionising mutagens in the form of ultraviolet radiation (UV) and ethyl methanosulfonate (EMS) and an ionising mutagen (X-ray) as methods to increase fucoxanthin content in the model diatom Phaeodactylum tricornutum. We implemented an ultra-high throughput method using fluorescence-activated cell sorting (FACS) and live culture spectral deconvolution for isolation and screening of potential pigment mutants, and assessed phenotype stability by measuring pigment content over 6 months using high-performance liquid chromatography (HPLC) to investigate the viability of long-term mutants. Both UV and EMS resulted in significantly higher fucoxanthin within the 6 month period after treatment, likely as a result of phenotype instability. A maximum fucoxanthin content of 135 ± 10% wild-type found in the EMS strain, a 35% increase. We found mutants generated using all methods underwent reversion to the wild-type phenotype within a 6 month time period. X-ray treatments produced a consistently unstable phenotype even at the maximum treatment of 1000 Grays, while a UV mutant and an EMS mutant reverted to wild-type after 4 months and 6 months, respectively, despite showing previously higher fucoxanthin than wild-type. This work provides new insights into key areas of microalgal biotechnology, by (i) demonstrating the use of an ionising mutagen (X-ray) on a biotechnologically relevant microalga, and by (ii) introducing temporal analysis of mutants which has substantial implications for strain creation and utility for industrial applications.

Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Machine Learning for Ultra High Throughput Screening of Organic Solar Cells: Solving the Needle in the Haystack Problem

AbstractOver the last two decades the organic solar cell community has synthesized tens of thousands of novel polymers and small molecules in the search for an optimum light harvesting material. These materials are often crudely evaluated simply by measuring the current–voltage (JV) curves in the light to obtain power conversion efficiencies (PCEs). Materials with low PCEs are quickly disregarded in the search for higher efficiencies. More complex measurements such as frequency/time domain characterization that could explain why the material performed as it is often not performed as they are too time consuming/complex. This limited feedback forced the field to advance using a more or less random walk of material development and has significantly slowed progress. Herein, a simple technique based on machine learning that can quickly and accurately extract recombination time constants and charge carrier mobilities as a function of light intensity simply from light/dark JV curves alone. This technique reduces the time to fully analyze a working cell from weeks to seconds and opens up the possibility of not only fully characterizing new devices as they are fabricated, but also data mining historical data sets for promising materials the community has overlooked.

Single-Step Biofabrication of In Situ Spheroid-Forming Compartmentalized Hydrogel for Clinical-Sized Cartilage Tissue Formation.

3D cellular spheroids offer more biomimetic microenvironments than conventional 2D cell culture technologies, which has proven value for many tissue engineering applications. Despite beneficiary effects of 3D cell culture, clinical translation of spheroid tissue engineering is challenged by limited scalability of current spheroid formation methods. Although recent adoption of droplet microfluidics can provide a continuous production process, use of oils and surfactants, generally low throughput, and requirement of additional biofabrication steps hinder clinical translation of spheroid culture. Here, the use of clean (e.g., oil-free and surfactant-free), ultra-high throughput (e.g., 8.5mL min-1 , 10 000 spheroids s-1 ), single-step, in-air microfluidic biofabrication of spheroid forming compartmentalized hydrogels is reported. This novel technique can reliably produce 1D fibers, 2D planes, and 3D volumescompartmentalized hydrogel constructs, which each allows for distinct (an)isotropic orientation of hollow spheroid-forming compartments. Spheroids produced within ink-jet bioprinted compartmentalized hydrogels outperform 2D cell cultures in terms of chondrogenic behavior. Moreover, the cellular spheroids can be harvested from compartmentalized hydrogels and used to build shape-stable centimeter-sized biomaterial-free living tissues in a bottom-up manner. Consequently, it is anticipated that in-air microfluidic production of spheroid-forming compartmentalized hydrogels can advance production and use of cellular spheroids for various biomedical applications.

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries.

Discovering new bioactive molecules is crucial for drug development. Finding a hit compound for a new drug target usually requires screening of millions of molecules. Affinity selection based technologies have revolutionized early hit discovery by enabling the rapid screening of libraries with millions or billions of compounds in short timeframes. In this Perspective, we describe recent technology breakthroughs that enable the screening of ultralarge synthetic peptidomimetic libraries with a barcode-free tandem mass spectrometry decoding strategy. A combination of combinatorial synthesis, affinity selection, automated de novo peptide sequencing algorithms, and advances in mass spectrometry instrumentation now enables hit discovery from synthetic libraries with over 100 million members. We provide a perspective on this powerful technology and showcase success stories featuring the discovery of high affinity binders for a number of drug targets including proteins, nucleic acids, and specific cell types. Further, we show the usage of the technology to discover synthetic peptidomimetics with specific functions and reactivity. We predict that affinity selection coupled with tandem mass spectrometry and automated de novo decoding will rapidly evolve further and become a broadly used drug discovery technology.

Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes

The genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 days. We apply the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS offers a promising solution for single-cell methylomic studies requiring examination of a large cell population.

Strategies for MALDI-MS method development to investigate different pharmaceutical drug modalities

High-throughput screening (HTS) is an ultra-high throughput tool that allows the identification of diverse, pharmaceutically active compounds in the early stages of drug discovery, by enabling chemists to screen reactions at micromole/nanomole scales. To eliminate the bottleneck arising from analysis of large libraries, Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) can be utilized as a compatible, ultra-fast, label-free, analysis tool. Upon integrating this technique with suitable liquid handling robots that can accurately spot matrices and analytes on a MALDI plate, chemical reactions can be screened at a sampling rate of <1 s per sample. While earlier efforts to improve MALDI-HTS applications were geared towards the development of automation and data processing tools, a systematic method development study across different drug modalities was needed to understand the contribution of different parameters such as MALDI matrices, choice of matrices, additives, and solvent composition, on MALDI-MS signal intensities of analytes. The current study will demonstrate the importance of initial MALDI-MS method development on small molecules, peptides, antibodies, and antibody drug conjugates. This study is designed as a starting point for MALDI-MS signal optimization, which can eventually lead to the development of effective MALDI-HTS methods for various modalities, thus paving the way for a wide range of applications, including but not limited to conformational analysis.

Wafer-Scale Monolithic Integration of Blue Micro-Light-Emitting Diodes and Green/Red Quantum Dots for Full-Color Displays

We demonstrated a simple method for achieving green and red colors by patterning quantum dots (QDs) on GaN-based blue micro-light-emitting diodes (μLEDs) using standard photolithography and dry etching process. The QDs could be patterned as small as 2 μm in size on a 4-inch wafer, revealing the ultra-high resolution and throughput capability of this method. The individual color-converted green and red μLEDs in a size of 10 μm × 10 μm exhibited a peak external quantum efficiency (EQE) of 9.6% and 14.7%, respectively. We also achieved the monolithic integration of red, green, and blue (RGB) μLEDs as a single pixel by sequentially patterning red and green QDs. The color gamut of the RGB μLEDs covered 83.4% of the Rec. 2020 color space in the CIE 1931 diagram. This method is very effective for patterning color-converted QDs with down to 2μm-size-resolution and achieving high performance for red/green μLEDs, while enabling scalable fabrication of full-color μLED displays.

Investigator-initiated phase 1 clinical trial of synthetic TIL: Autologous peripheral T cells engineered with polyclonal TIL-derived TCRs.

e14525 Background: The efficacy of tumor-infiltrating lymphocyte (TIL) therapy is limited by the rarity of anti-tumor TCRs and exhausted status of the product. Its improvement should focus on a high frequency of diverse anti-tumor TCRs with stem-like phenotype, which can be achieved synthetically by identifying multiple anti-tumor TCRs from the patient's intra-tumor TCR repertoire and subsequently engineering them into the patient’s peripheral T cells. Such a ‘synthetic TIL’ is cost-prohibitive using existing technologies. To enable a synthetic TIL product, we developed ultra-high throughput TCR gene synthesis and screening methods to synthesize and screen &gt; 50,000 TCRs from scRNA-Seq data from &gt; 30 patients and identified &gt; 100 tumor/NeoAg-reactive TCRs. The gene expression patterns of the T cells carrying such experimentally validated tumor/NeoAg-reactive TCRs gave rise to an algorithm called Interpreter to predict potentially anti-tumor TCRs from scRNA-Seq data. Methods: To translate synthetic TIL to the clinic, we developed a method to generate up to 100 sequence-verified TCR-encoding plasmids (selected by Interpreter) from fresh tumor biopsy in just 3 weeks. Our GMP-grade production uses a collective knock-in approach, where a mixture of up to 100 plasmids (each encoding a distinct TCR) at equal proportion are packaged into AAV which serve as template to precisely insert the TCR genes into a primarily monoallelic locus and simultaneously inhibit the endogenous TCR. This gene editing is characterized by a modified scRNA-Seq method called EntrySeq. In first-in-human, N-of-1 trials, 109 to 6x109 synthetic TIL cells were administered to solid tumor patients with or without lymphodepletion and IL2. An investigator-initiated Phase 1 trial was then opened to evaluate safety, preliminary efficacy, and cellular activity of synthetic TIL and to determine RP2D and MTD. Subjects with relapsed, unresectable locally advanced solid tumors who received at least 1 standard systemic therapy are allowed to enroll in this study. Those patients with no standard treatment regimens available, or those who are unable to obtain effective treatment due to economic conditions are also permitted. Results: EntrySeq confirmed that &gt; 80% of the T cells express only 1 TCR, avoiding mispairing of different TCRs. The frequencies of different TCRs were largely uniform and were stable through plasmid, AAV and T-cell mixtures. Up to 1.5x1010 synthetic TIL cells were produced from a patient’s leukopak. 40 to 60% of the T cells in the product for infusion express an exogenous TCR. In first-in-human trials, the administered T cells showed favorable persistence and safety profile. The Phase 1 trial is ongoing. Conclusions: We established a fast, affordable, and GMP-compliant process to manufacture synthetic TIL products, enabling a Phase 1 trial.

Ultra-high throughput dual channel liquid chromatography with tandem mass spectrometry for quantification of four immunosuppressants in whole blood for therapeutic drug monitoring

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is the golden standard for immunosuppressants analyses, where optimising throughput by parallel chromatography can reduce costs and turnaround time. We aimed to double our system throughput using a dual LC-MS/MS setup. Therefore, two independent UPLC systems were hyphenated to one triple quadrupole MS, with staggered injections from one autosampler on alternating columns. The method simultaneously measured the analytes tacrolimus, sirolimus, everolimus, and cyclosporin A in whole blood using isotope dilution, with a run time of 1.5 min. Using the dual LC-MS/MS system, net run-to-run time improved from 2.3 to 0.98 min per injection, where throughput increased from 26 to 61 injections per hour. For Performance Qualification, 1101 clinical samples were measured on the dual LC-MS/MS system in addition to the standard system, during a period of one month, and the results were compared using Passing Bablok regression and Bland Altman analysis. There was excellent agreement for all four analytes, with regression slopes of 0.98–1.02x and intercepts of -0.11–0.88 µg/L. Minor bias was demonstrated between the systems with mean differences from -0.93 to 1.43%. In conclusion, the throughput was doubled and idle MS time was reduced with good agreement to the standard system. Currently, the method is applied for clinical routine with frequent peak intensities of >180 injections per day.

Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop‐Based Microfluidics

AbstractEnzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor‐ and capital‐intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop‐based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5‐fold catalytic efficiency.

Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics.

Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

Fast Preparation of Peptide Tools to address new emerging Viruses: Case example SARS-CoV-2

Abstract The emergence of new virus species or variants poses a major challenge for healthcare systems. Countermeasures, including vaccine development, need to be developed without delay to ensure success. Vaccine and antibody development depend on highly efficient tools for antigen discovery, epitope mapping, diagnostics and (clinical) immune monitoring. Here we present a workflow for peptide tool development that can be rapidly adapted to emerging viruses. It combines bioinformatics algorithms and an ultra-high throughput peptide synthesis method alongside high-content assay formats. Data from different SARS-CoV-2 studies are presented addressing elucidation of both cellular and humoral immune responses. Robust spike-specific cellular immune responses were measured using SARS-CoV-2 Spike PepMix ™peptide pools during infection and/or vaccine response monitoring [ 1]. Using epitope mapping (matrix) pools an immunodominant T-cell epitope was identified that may contribute to the optimization of existing vaccines [ 2]. In addition, the immune response of Covid-19 convalescents against all major SARS-CoV-2 proteins was analyzed [ 2]. Humoral immune response analysis by high-density peptide microarrays identified immunodominant B cell epitopes as starting points for the development of alternative vaccines and diagnostic tests [ 3]. In conclusion, we show that readily available and tailored peptide formats, as exemplified for SARS-CoV-2, are important tools that contribute to the fast development and optimization of vaccines and diagnostic tests for emerging viruses of concern.

Detection speed optimization of the OI-RD microscope for ultra-high throughput screening.

The oblique-incidence reflectivity difference (OI-RD) microscope is a label-free detection system for microarrays that has many successful applications in high throughput drug screening. The increase and optimization of the detection speed of the OI-RD microscope will enable it to be a potential ultra-high throughput screening tool. This work presents a series of optimization methods that can significantly reduce the time to scan an OI-RD image. The wait time for the lock-in amplifier was decreased by the proper selection of the time constant and development of a new electronic amplifier. In addition, the time for the software to acquire data and for translation stage movement was also minimized. As a result, the detection speed of the OI-RD microscope is 10 times faster than before, making the OI-RD microscope suitable for ultra-high throughput screening applications.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Swarm of UAVs for Network Management in 6G: A Technical Review

Fifth-generation (5G) cellular networks have led to the implementation of beyond 5G (B5G) networks, which are capable of incorporating autonomous services to swarm of unmanned aerial vehicles (UAVs). They provide capacity expansion strategies to address massive connectivity issues and guarantee ultra-high throughput and low latency, especially in extreme or emergency situations where network density, bandwidth, and traffic patterns fluctuate. On the one hand, 6G technology integrates AI/ML, IoT, and blockchain to establish ultra-reliable, intelligent, secure, and ubiquitous UAV networks. 6G networks, on the other hand, rely on new enabling technologies such as air interface and transmission technologies, as well as a unique network design, posing new challenges for the swarm of UAVs.Keeping these challenges in mind, this article focuses on the security and privacy, intelligence, and energy-efficiency issues faced by swarms of UAVs operating in 6G mobile network. In this state-of-the-art review, we integrated blockchain and AI/ML with UAV networks utilizing the 6G ecosystem. The key findings are then presented, and potential research challenges are identified. We conclude the review by shedding light on future research in this emerging field of research.