High-Throughput DNA-Encoded Libraries Affinity Selection Platform for Binder Identification with Solid Support Protein Immobilization.

DNA-encoded libraries (DELs) have demonstrated to be one of the most powerful technologies within the ligand identification toolbox, widely used either in academia or biotech and pharma companies. DEL methodology utilizes affinity selection (AS) as the approach to interrogate the protein of interest for the identification of binders. Here we present a high-throughput, fully automated AS platform developed to fulfill industrial standards and compatible with different assay formats to improve the reproducibility of the AS process for DEL binders identification. This platform is flexible enough to virtually set aside all kinds of DELs and AS methods and conditions using immobilized proteins. It bears the two main immobilization methods to support of the proteins of interest: magnetic beads or resin tip columns. A combination of a broad variety of protocol options with a wide range of different experimental conditions can be set up with a throughput of 96 samples at the same time. In addition, small modifications of the protocols provide the platform with the versatility to run not only the routine DEL screens, but also test covalent libraries, the successful immobilization of the proteins of interest, and many other experiments that may be required. This versatile AS platform for DEL can be a powerful instrument for direct application of the technology in academic and industry settings.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

Corrigendum: Intrathecal drug delivery: Advances and applications in the management of chronic pain patient.

[This corrects the article DOI: 10.3389/fpain.2022.900566.].

Intrathecal Drug Delivery: Advances and Applications in the Management of Chronic Pain Patient.

Advances in our understanding of the biology of spinal systems in organizing and defining the content of exteroceptive information upon which higher centers define the state of the organism and its role in the regulation of somatic and automatic output, defining the motor response of the organism, along with the unique biology and spatial organization of this space, have resulted in an increased focus on therapeutics targeted at this extracranial neuraxial space. Intrathecal (IT) drug delivery systems (IDDS) are well-established as an effective therapeutic approach to patients with chronic non-malignant or malignant pain and as a tool for management of patients with severe spasticity and to deliver therapeutics that address a myriad of spinal pathologies. The risk to benefit ratio of IDD makes it a useful interventional approach. While not without risks, this approach has a significant therapeutic safety margin when employed using drugs with a validated safety profile and by skilled practioners. The present review addresses current advances in our understanding of the biology and dynamics of the intrathecal space, therapeutic platforms, novel therapeutics, delivery technology, issues of safety and rational implementation of its therapy, with a particular emphasis upon the management of pain.

A new strategy in lung/lobe isolation in patients with a lung abscess or a previous lung resection using double lumen tubes combined with bronchial blockers.

The combined use of a double-lumen tube and a bronchial blocker can be very helpful in two different clinical scenarios: (1) in isolating not only the contralateral lung, but also the lobe/s of the same lung in which the infected lobe must be resected, (2) in preventing/treating hypoxemia because of the presence of a contralateral lobectomy. A cardiothoracic anesthesiologist must expertise this technique to avoid complications during surgery.

Adjacent segment syndrome after failed back surgery: biomechanics, diagnosis, and treatment.

The adjacent segment syndrome is defined as the changes in the adjacent structures of an operated spinal level that produce symptoms of pain and disability, which worsen the quality of life of a patient. Pain management specialists must be aware of these biomechanical changes brought by spinal surgeries, as well as of the symptoms associated with pain after surgery, to reach an appropriate diagnosis and provide an adequate treatment. Specialized pain literature contains few reports on specific management of patients using the terms "adjacent segment syndrome, degeneration or disease;" most of the literature comes from surgical journals. It is necessary to perform studies with a population sample comprising patients with adjacent segment syndrome after spinal surgery, since almost all treatments applied in this group are extrapolated from those used in patients with pain originating in the same area but who have not previously undergone spine surgery. Therefore, we consider necessary for pain physicians to understand the underlying biomechanics, promote the diagnosis of this condition, and analyze possible treatments in patients with adjacent segment disease to alleviate their pain and improve their quality of life.

Updated management of occipital nerve stimulator lead migration: case report of a technical challenge.

Electrode migration is a challenge, even with adequate anchoring techniques, due to the high mechanical stress on components of occipital nerve stimulation (ONS) for headache disorders. When a lead displacement of an ONS implant is diagnosed, there are currently different approaches described for its management. Nevertheless current neuromodulation devices are designed like a continuum of components without any intermediate connector, and if a lead displacement is diagnosed, the solution is the complete removal of the electrode from its placement, and its repositioning through an ex-novo procedure. The described technique can allow ONS leads to be revised while minimizing the need to reopen incisions over the IPG, thus improving patients' intraoperative and postoperative discomfort, shortening surgical time and medical costs, reasonably reducing the incidence of infective postoperative complications.

Intrathecal Drug Delivery.

Targeted intrathecal (IT) drug delivery systems (IDDS) are well established as an effective treatment of patients with chronic nonmalignant or malignant pain, and as a tool for management of patients with severe spasticity. The risk to benefit ratio of IDD makes it a relatively safe therapy for both cancer- and noncancer-related pain, but it is not free of risks, so it should be managed at specific centers. Recent technological advances, new therapeutic applications, reported complications, and the costs as well as maintenance required for this therapy require the need to stay up to date about new recommendations that may improve outcomes. This chapter reviews all technological issues regarding IDDS implantation with follow-up and pharmacological recommendations published during recent years that provide evidence-based decision-making process in the management of chronic pain and spasticity in patients.

Microanatomy Relevant to Intrathecal Drug Delivery.

This chapter describes the microanatomy of the spinal cord that is relevant to intrathecal drug delivery started with covering of the spinal cord that are pierced to enter the intrathecal space. The dural sac is mostly constituted by the outer layer of dura and the inner layer called arachnoid membrane, which regulates diffusion of drugs into the intrathecal space. The pia matter surrounding the spinal cord is a permeable structure allowing the passage of drugs through intercellular spaces. The relationship between nerve roots, CSF, and subarachnoid catheters determines the passage of an intrathecal catheter which can cause damage to nerve roots and spinal cord. Multiple factors may be involved in the mechanisms of drug diffusion across the membranes of the spinal cord, as well as in their dilution with the CSF, which will lead to the final drug distribution and availability at nerve roots and the spinal cord.

A Decoupled Automation Platform for Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.

Regional analgesia techniques for pain management in patients admitted to the intensive care unit.

Controlling pain should be a priority in the clinical practice of intensive care units (ICUs). Monomodal analgesic approaches, such as the administration of opioids, are widely employed; however, the widespread use of opioids has catastrophic consequences, given their multiple side effects and the development of dependence. Regional analgesia (RA), with single or continuous dosing using neuraxial and peripheral catheters, can play an important role in multimodal analgesia for management of pain in critical care patients. RA provides superior pain control, as compared to systemic treatments, and is associated with a lower rate of side effects. Nevertheless, RA remains underused in ICUs. Many critically ill, post-surgical or traumatically injured patients would benefit from these techniques. For these reasons, we aim to establish a set of potential indications integrating the use of RA in analgesia protocols routinely used in ICUs. We performed a review of literature sources with contrasted evidence levels to present RA techniques and their potential applications in ICU patients.

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures.

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

Automated liquid-liquid extraction workstation for library synthesis and its use in the parallel and chromatography-free synthesis of 2-alkyl-3-alkyl-4-(3H)-quinazolinones.

An automated liquid-liquid extraction workstation has been developed. This module processes up to 96 samples in an automated and parallel mode avoiding the time-consuming and intensive sample manipulation during the workup process. To validate the workstation, a highly automated and chromatography-free synthesis of differentially substituted quinazolin-4(3H)-ones with two diversity points has been carried out using isatoic anhydride as starting material.