De novo gene synthesis and protein expression are established technologies that can give access to nearly any target DNA or protein sequence, allowing for engineering of biologics. In this presentation we demonstrate how considering more than codon usage in gene optimization can boost protein expression. Advanced mammalian expression systems can further enhance antibody yields by getting more product from the same culture volumes. Customer examples demonstrate the benefits of a continuous workflow, from gene to protein, to accelerate discovery and early development.

Learning Objectives:

1. Explain how Enhancing Protein Expression: optimize gene synthesis techniques beyond codon usage can significantly improve protein expression rates, facilitating the engineering of biologics.

2. Summarize how Maximizing Antibody Yields: utilize advanced mammalian expression systems can increase antibody yields, extracting more product from identical culture volumes to accelerate discovery and early-stage development processes.

Surface plasmon resonance is a real-time and label-free technique that offers low sample consumption and high throughput when compared to other biophysical techniques and has been widely adopted in pharmaceutical and biotechnology industries as the gold standard for determining accurate kinetics and affinity values.

The field of real-time, label-free SPR protein-binding analysis has historically been marked with steep learning curves and can often seem daunting to new or inexperienced users, being viewed as an ‘art’ instead of a ‘science’ due in part to the lack of exposure to the technique. The next generation SPR approach challenges this notion through user-friendly technology.

One the of the main roadblocks to determining accurate kinetics and affinity values is optimization of the analyte concentration series for use in an assay, which often stems from users lacking basic knowledge required to design, execute, and analyze their assays

The Octet® SF3 SPR system contains a unique injection method called OneStep® that simplifies the process of choosing the correct analyte concentration. It allows users to generate accurate kinetics and affinity values from a single analyte concentration, saving a substantial amount of time on assay development and assay performance compared to standard SPR methods.

Learning Objectives: 

1. Learn how to speed up fragment-based drug discovery by weeks.

2. Learn how to hit the sweet spot for binding kinetic experiments.

3. Discover new possibilities to conduct competition assays label-free and in real-time.

mRNA‑based products are rapidly growing in the biotherapeutics space. Important considerations need to be made to develop a process for mRNA synthesis that consistently generates high‑yield and high‑quality products. This 15-minute webinar discusses the material quality attributes and the process considerations that are required to successfully synthesize mRNA using in vitro transcription. It also covers the important factors that go into scaling‑up the production of mRNA‑based therapies.

Learning objectives:

1. Recognize the importance of raw materials in mRNA production.

2. Discuss quality attributes of mRNA raw materials.

3. Review considerations for mRNA synthesis and scale‑up.

Traditional liver perfusion procedures – used to isolate hepatocytes – are difficult to perform and require extensive training, steady hands and long-standing experience.

In this webinar, a new, automated method of isolating viable hepatocytes from resected liver tissue will be presented, making liver perfusion easier. Our expert speaker will also discuss how to optimize yield and viability of the isolated hepatocytes, as well as non-parenchymal liver cells and provide insights into cell cultivation of hepatocytes and their characterization.

Learning Objectives:

1. Explain how the new perfusion technology works and how simple it is to introduce into your lab.

2. Review what the benefits are compared to complex traditional perfusion procedures.

3. Review how automated liver perfusion creates a smooth transition to your downstream applications.

Background and aim:
G protein-coupled receptors (GPCRs) are vital membrane proteins involved in cellular and physiological processes, including sensory perception, signaling, neurotransmission, and immune response. Consequently, understanding their function and regulation is particularly relevant for developing targeted therapies for human diseases. A family of three single-transmembrane proteins, called receptor activity-modifying proteins (RAMPs), has been shown to co-evolve and act as regulators of GPCR function. Still, system-wide GPCR-RAMP interactions have remained underexplored (Kotliar et al., 2023, PharmaRev).

Methods:
We developed a multiplexed system for a multi-modal analysis of the membranebound receptors. The versatile system is based on combinatorial epitope tags in a co-expression library of > 200 GPCRs and RAMPs. Following solubilization into micelles, we apply bead-based immuno-capture assays to determine the receptors' expression, epitope presentation, and RAMP interactions. The data is then implemented into interactive web interfaces to simplify the evaluation.

Results:
At first, we evaluated our concept on a set of 25 secretine-like GPCRs and their interactions with RAMPs (Lorenzen et al., 2019, SciAdv). We then expanded the versatility of our platform to > 200 GPCR and validated the specificity of > 400 anti-GPCR antibodies (Dahl et al., 2023, SciAdv). To demonstrate the system's utility, we studied the downstream effects of GPCR-RAMP binding for the Itch receptor (Kotliar et al., 2023, JBC). More recently, we used the full library to map the interactome of GPCRs and RAMPs system-wide (Kotliar et al., submitted).

Conclusion:
The developed multiplexed system has allowed us to study the analytically challenging membrane receptors and reveal interactions with system-wide implications for GPCR regulation.

Interoceptive signals dynamically interact with the environment to shape appropriate defensive behaviors. Neurons in the hypothalamus regulate internal states, such as thirst and circadian rhythmicity, and send projections to the extended amygdala to influence defensive responses, including fear and anxiety-like behaviors. Two hypothalamic hormones and neuromodulators, oxytocin (OT) and arginine-vasopressin (AVP) modulate parturition and lactation, water and electrolyte balance and as well as circadian rhythmicity. Neurons of the bed nucleus of the stria terminalis (BNST) express both oxytocin (OTR) and vasopressin receptors and mediates defensive behaviors, including fear- and anxiety behaviors. Here we show that both OT and AVP directly excite BNST neurons via oxytocin receptor (OTR) transmission. These excitatory effects are also confirmed by specifically recording excitability of fluorescent OTR-neurons from OTR-Cre transgenic male rats after application of AVP or selective OTR agonist, TGOT. Considering the well-established role of BNST in avoidance and fear-related behaviors, we used chemogenetics in OTR-Cre rats and demonstrate that silencing of OTR-BNST neurons significantly reduces exploration of open arms of the elevated plus-maze and increases cue-induced vigilance measured in the fear-potentiated startle. We demonstrate how OTR-BNST neurons exited by hypothalamic OT and AVP play a major role in regulating BNST excitability, overcoming threat avoidance, and reducing fear responses to ambiguous threats. Using neuronal tracing studies and peptide optogenetics we demonstrate that distinct populations of hypothalamic OT and AVP neurons send projections to the BNST. Therefore, changes in the activity of internal state-sensitive hypothalamic nuclei will directly impact OTR neuron excitability in the BNST via hypothalamic inputs to shape appropriate, physiologically relevant levels of defensive behaviors.

Learning Objectives:

1. Describe the impact of hypothalamic hormones (oxytocin, vasopressin) on the extended amygdala activity and defensive behaviors. 

2. Explain how chemogenetic manipulations of BNST neurons affect fear- and anxiety-like behaviors.

3. Explain which hypothalamic neurohypophysial neurons innervate the bed nucleus of the stria terminalis (BNST).

This presentation explores the pharmacokinetics (PK) and pharmacodynamics (PD) of β-lactam antibiotic dosing, particularly in ICU patients. To optimize PK/PD targets, two strategies are possible and include population level and individual Precision Dosing approaches. This presentation emphasizes the critical need for precise dosing strategies to optimize β-lactam exposures to improve efficacy. Variability in PK, driven by high inter-individual variability in clearance and volume, translates to poor target attainment and underscores the importance of tailored Precision Dosing approaches. The presentation also touches on Bayesian probability concepts, showcasing how conditional probabilities can aid in decision-making for Precision antibiotic dosing strategies. Challenges related to therapeutic drug monitoring and its role in refining antibiotic administration are addressed. Alternate population level approaches such as prolonged infusion methods are discussed for the ability to increase achievement of therapeutic targets in sepsis.

Learning Objectives:

1. Review the PK/PD for β-lactams.

2. Describe variability in β-lactam exposures.

3. List strategies to achieve targets.

4. Review recent clinical literature on strategy outcomes.

Multiplex immunofluorescence is a powerful tool for phenotyping cells from a single tissue slide, while preserving spatial context. Multiplex immunofluorescence can be used to profile dozens of biomarkers from a single four-micron tissue section, enabling high-throughput biomarker discovery without exhausting precious tissue or disrupting tissue architecture. Accurate and reproducible image analysis is a key aspect to generating multiplex IF datasets. This talk will cover some image analysis strategies for generating data from multiplex IF images, as well as some challenges associated with multiplex IF. In particular, the use of deep-learning based pipelines to perform the segmentation and classification tasks needed for reliable single-cell phenotyping.  Finally, spatial analytics build upon single-cell phenotyping to reveal the complex cell interactions that define the tumor microenvironment. We illustrate two case studies using multiplex IF: characterizing tertiary lymphoid structures in colorectal carcinomas; and detecting cell-cell interactions in the perivascular niche of triple-negative breast cancers. Multiplex IF, when combined with accurate digital image analysis, yields high-quality multiplex immunofluorescence datasets and facilitates deep interrogation of the tumor microenvironment.

Learning Objectives:

1. Summarize the key steps for image analysis of multiplex IF.

2. Review the challenges of multiplex IF analysis and ways to mitigate them.

3. Explain several use cases of multiplex IF technology.

Advancements in the field of drug discovery and chemical development have necessitated the integration of computer-assisted technologies to expedite processes, uncover efficiencies, and foster innovation in synthesis workflows. SYNTHIA™ retrosynthesis software has emerged as a powerful tool, utilizing sophisticated algorithms, advanced chemical logic, and an expert-coded chemistry rule database to efficiently identify pathways for known and novel synthetic targets. By applying user-guided customizations to search parameters, tailored results can be obtained to meet specific project requirements. The seamless customizability of SYNTHIA™ facilitates its integration into diverse technology workflows. Although significant progress has been made, synthetic chemistry remains a highly complex and time-consuming field. The synthesis of novel molecules with unique properties is crucial for drug discovery but often poses challenges and causes bottlenecks in the overall process. To address this issue, the application of customized search parameters within SYNTHIA™ enables the efficient planning of routes to target molecules. This optimized approach eliminates the need for extensive time spent on manual route planning, searching for appropriate starting materials, and executing bench experiments. Consequently, the drug discovery process is accelerated, enabling researchers to focus on crucial aspects of molecule development and property optimization.

The cancer drug discovery landscape is shifting. While research continues to grow in cost and complexity, the pace of development has never been faster. Biopharmaceutical companies can’t afford to waste time and resources pursuing ineffective or unsafe ideas.

In this talk, our experts discuss how biopharmaceutical companies can leverage key genomic, biomedical, and clinical trial databases to improve and accelerate cancer drug discovery while avoiding potential pitfalls. Through a series of use cases, attendees will learn about expert-curated knowledge from the Wellcome Sanger Institute and QIAGEN, and how to use these resources to better predict cancer-driving effects of mutations, identify available drugs that target specific variants, and accelerate indication expansion and repurposing of existing cancer therapies.

Learning objectives:  

1. Examine use-cases of how to leverage expert-curated databases across multiple phases of cancer drug development.

2. Discover key applications of these databases, including determining the function, frequency, and actionability of specific mutations.

3. Discuss potential pitfalls and clinical consequences, and how to avoid them early with data-driven drug target and biomarker qualification.

Current cancer research is hindered by the limitations of existing experimental systems, which fall short in demonstrating concordance with human studies. In this way, we aim to develop models for studying mechanisms that initiate and progress carcinogenesis, focusing initially on Small Cell Lung Cancer (SCLC), the most aggressive type of lung cancer. My currently developed cell culture models based on directed differentiation of human pluripotent stem cells (hPSC) reveal why certain constellations of genetic changes drive carcinogenesis in specialized human cell lineages. These tractable experimental systems enable studying cancer in great depth, using genetically defined human cells that can be characterized at the single cell level. I will also describe our recent work in studying lung adenocarcinoma using the cells derived from hPSCs.

Learning Objectives:

1. Explain the advance of human pluripotent stem cell technology, and its application for disease modeling.

2. Review the technology to generate pulmonary neuroendocrine cells by directed differentiation of human pluripotent stem cells.

3. Review how to establish the model for various stages of small cell lung cancer using the human cells derived from pluripotent stem cells.

NAFLD (non-alcoholic fatty liver disease) is a global epidemic that affects 25% of the world’s population, with its epicentre in the West. Twenty percent of NAFLD patients go on to develop the most severe form of the disease, NASH (metabolic-associated steatohepatitis) which is characterized by lipid accumulation, infiltration of immune cells, and liver fibrosis. While NASH is well-characterized, there is currently no therapeutic cure for these diseases. One of the main reasons preclinical models for testing efficacy fail to adequately predict clinical outcomes is that current preclinical models mostly comprise animal models that fail to recreate the full spectrum of the disease and often lead to poor translation to humans. There is an urgent need to improve current preclinical NASH models to better predict drug efficacy. Microphysiological systems (MPS), also known as organ-on-a-chip (OOC), have been developed as a new alternative method (NAM) to bridge the gap between preclinical predictions and clinical outcomes. MPS models of the liver can capture the complexity of the liver’s microenvironment and express a wide range of human-relevant translational biomarkers. In this NASH MPS model, multiple primary cells (hepatocytes, hepatic stellate cells, and Kupffer cells) are co-cultured on a perfused scaffold to form a 3D microtissue. Exposure of this microtissue to fatty media generates a clinical-like NASH phenotype, making this model a good fit for drug efficacy testing. Here, we show the model’s potential to assess the efficacy of four anti-NASH compounds, currently in late-stage clinical trials, using a panel of inflammatory, fibrotic, and steatosis biomarkers. We also discuss its ability to investigate the mechanism of drug action.

Learning Objectives: 

1. Identify preclinical drug development challenges for NASH studies.

2. Introduce a liver MPS model for investigating NASH.

3. Demonstrate how this NASH liver MPS provide translatable insights into drug efficacy for NASH therapeutics.

Central Nervous System (CNS) therapies have significantly progressed in the last decade, propelled by emerging treatment modalities including gene therapies. Neurological indications remain amongst the most challenging to address with longer development timelines and a lower success rate when compared to other therapeutic areas. This session will discuss CNS delivery considerations when using non-clinical models with a focus on best practices. Translational challenges related to biodistribution, species relevance and overall study design will be discussed. The session will also review the status of innovative CNS dosing approaches such as convection enhanced delivery (CED) and focused ultrasound (FUS) in non-clinical models.

Learning Objectives:

1. Understand the range of strategies available when planning central nervous system delivery in preclinical models.

2. Familiarize participants with success factors and sources of variation when conducting central nervous system delivery studies in non-clinical models.

3. Review basic scientific endpoints that are included in preclinical safety studies with central nervous system treatment.

Absorption, Distribution, Metabolism, and Excretion (ADME) are important parameters to determine the pharmacokinetic profile of a drug, and therefore a crucial measurement for drug efficacy. Animal models and immortalized cell lines have long been considered the standard preclinical tools for estimating a drug’s ADME profile for humans. Yet, 90% of newly developed therapeutic agents still fail to gain market entry, predominantly because the current preclinical methods – 2D cell culture and in vivo testing, fail to adequately predict a drug’s safety and efficacy in humans.

With recent advancements in science, a growing number of regulatory bodies are investigating and integrating the use of new approach methodologies (NAMs) into Drug Discovery workflows. In particular, microphysiological systems (MPS), also known as organ-on-a-chip (OOC), have elevated the preclinical toolbox. By combining primary cells with a fluidic flow, MPS models allow for more physiological relevance, as well as improved translatability of preclinical studies. We have established an interconnected Gut/Liver MPS model using our bespoke Multi-Organ OOC system. The model’s inter-organ crosstalk capabilities facilitate ADME studies to better predict oral bioavailability of a drug compound.

To improve on current in vitro gut models, that mainly use Caco-2 cells, we integrated a primary gut model (RepliGut®) into our MPS model. By developing a specialized co-culture media, we successfully preserved the functionality markers of both the gut and liver for at least 48 hours. Combined with primary liver cells, the model shows greater genotypic and phenotypic relevance to humans, when compared to immortalized cell lines. Finally, the model was dosed with drug compounds- temocapril and midazolam, which demonstrated improved predictive capacity of their ADME profiles.

To conclude, this primary Gut/Liver-on-a-chip model enables us to study the combined effects of intestinal absorption and hepatic metabolism. This demonstrates the potential of MPS as an alternative to standard preclinical methods to better estimate ADME profiles of compounds. With the use of these more predictive models, drug developers can gain earlier insights, thereby promoting a more cost effective and successful pipeline.

Learning Objectives: 

1. Summarize the benefits and challenges of current preclinical methods in drug discovery and development.

2. Review new alternative methodologies (NAMs) and microphysiological systems (MPS) and their application areas.

3. Examine the potential of MPS to improve on in vitro to in vivo translatability of preclinical studies for oral bioavailability assessment.

Diosgenin is a botanical steroidal saponin with anti-inflammatory, antioxidant, anti-thrombotic, anti- apoptotic, anti-depressant, and anti-nociceptive properties. However, it's unknown how diosgenin affects anti-nociception. TRPV1, a transient receptor potential vanilloid, is crucial for nociception. Therefore, we investigated whether TRPV1 antagonism mediates the anti-nociceptive effects of diosgenin. In vivo mouse experiments were performed to examine nociception-related behavior, while in vitro experiments were per-formed to examine calcium currents in dorsal root ganglion (DRG) and Chinese hamster ovary (CHO) cells. The duration of capsaicin-induced licking (pain behavior) was significantly reduced following oral and intraplantar administration of diosgenin, approaching levels observed in mice treated with the TRPV1 antagonist BCTC. Additionally, oral administration of diosgenin reduced capsaicin-induced thermal hyperalgesia. Further, diosgenin reduced capsaicin- induced Ca2+ currents in a dose-dependent manner in both DRG and CHO cells. Oral administration of diosgenin also improved thermal and mechanical hyperalgesia in the sciatic nerve constriction injury- induced chronic pain model by reducing the expression of TRPV1 and inflammatory cytokines in DRG cells. Overall, our findings imply that diosgenin has analgesic benefits in a mouse model of neuropathic pain by inhibiting TRPV1 and reducing inflammation in the DRG.

Across many cancers, mortality is associated with metastasis and acquired drug resistance, both of which often manifest in recurrent disease. Central to these phenomena is epithelial to mesenchymal transition (EMT), a developmental process reactivated during tumor progression. During EMT, transcriptional regulation of cell adhesion and cytoskeletal genes is altered, and cells become motile. For many years, EMT has been closely tied to metastasis, but in recent years its roles in drug resistance and stem cell phenotype have also been uncovered. While EMT is thus an attractive therapeutic target, progress in drug development for EMT has been limited, as the transcription factors that mediate the process have been dubbed “undruggable”. Novel approaches are therefore needed.

Our recent work in ovarian cancer has revealed that mesenchymal cells may be vulnerable to therapies targeting DNA repair and VEGF signaling, but in unexpected ways. We present here our ongoing work exploring the use of olaparib, cediranib, and various marine alkaloids as a supplement to conventional chemotherapy to more effectively kill cancer cells, particularly those that have undergone EMT. We also present our comprehensive research paradigm, where we use live cell imaging in conjunction with drug screening and gene expression assays to paint a complete picture of EMT biomarkers, cell migration, and drug action across ovarian, colorectal, and oral cancers.

Learning Objectives: 

1. Describe the various effects of EMT in the course of cancer progression.

2. Explain the known and putative mechanisms of action of olaparib and cediranib.

3. Propose experimental designs involving complementary assay techniques.

The study of a single cell, the fundamental unit of our complex bodies, has become an important field in cellular biology, and researchers are using single-cell analysis to shed new light on basic biological questions and understand disease mechanisms. There is thus an urgent requirement for technologies enabling high-throughput assay and isolation of single cells for further characterization.

Using microfluidics, single cells can be encapsulated in picodroplets with a volume that is 100,000s times lower than a typical bulk volume method, Picodroplet-based methods to analyse single cells provide important benefits in research, including rapid generation for use as miniaturized independent “test tubes” for high-throughput bioassays, encapsulation of single cells (and their corresponding secreted molecules) to extend applicable assay types, and a low-shear stress environment to protect cells. This facilitates large-scale experiments at the single cell level; not only to enable the rapid detection and high-throughput sample processing, but also time- and cost-effective screening compared to conventional methods.

Using this technology, a range of novel bioassays can be developed for several applications, including biotherapeutic discovery and development and cell therapy research. In this webinar, we will describe these droplet-based approaches and how they can be harnessed to advance single-cell analysis, enabling the discovery and development of novel therapeutics.

Learning objectives:

1. Clarify the main advantages of studying single cell as opposed to cell bulk analysis.

2. Explain picodroplets microfluidics.

3. Describe how they can be applied to therapeutic research: CAR-T therapy’s case study will be shared to help illustrate the benefit of functional assays in picodroplets for single-cell analysis.

Proliferation biomarkers are proteins that are up-regulated in growing cells. Immunological staining of tumor tissue can provide an indication of the aggressivity of the tumor and provide information as to the likely therapeutic response. Most such biomarkers can only be measured immunohistologically requiring biopsy and making repeat sampling difficult. Thymidine Kinase 1 (TK1) is up-regulated in cells synthesising DNA and many studies have shown it to be found in increased concentrations in tumor tissue. However, uniquely TK1 is also measurable in body fluids making it a convenient biomarker for following cell proliferation. However, previous assays based on enzymatic activity have under-estimated TK1, particularly in samples from subjects with solid tumors, such as prostate and breast. Immunoassays using antibodies specific for the TK210 antigen increase the clinical sensitivity of TK1 and make the assay more robust. This presentation will demonstrate the value of TK1 immunoassays in the study of anti-tumor therapies in in-vitro, xenograft and pre-clinical studies. Examples will be taken from several tumor types, particularly recent data from studies on prostate cancer.

Learning Objectives:

1. Review how proliferation biomarkers can add value to studies with other tumor biomarkers.

2. Summarize Thymidine Kinase 1 as a mechanistic biomarker.

3. Review the use of Thymidine Kinase 1 immunoassay as a valuable translational biomarker applicable from drug discovery through to clinical trials.

Globally there has been a constant rise in the population suffering from cardiovascular diseases mostly due to metabolic complications. We have made use of two high throughput omics-based approaches, transcriptomics and metabolomics to understand the pharmacological and toxicological properties of apigenin and chrysin. Both apigenin and chrysin are naturally occurring compounds belonging to the flavonoid family and present in fruits and vegetables, which are a part of our daily dietary regimen. Our work revealed that apigenin and chrysin were able to able to down regulate cholesterol biosynthesis pathway by inhibiting transcripts encoding for enzymes. Additionally, based on our recent study involving metabolomic analysis, we affirmed the role of both apigenin and chrysin as hypocholesterolemic agents. Interestingly, in metabolomic studies, we also found that apigenin and chrysin were able to downregulate major metabolites involved in uric acid metabolism including xanthosine, hypoxanthine and xanthine. It is mandatory to conduct in vivo studies, then at least we will be able to confirm whether these sister flavonoids could suppress cholesterol and uric acid excess and promote ketogenic pathway. The results from in vivo studies will confirm whether apigenin and chrysin are ideal protective agents against cardiovascular and systemic complications and whether we can recommend their potential use in humans.

Learning Objectives:

1. Summarize the overall therapeutic properties of apigenin and chrysin based on Omics based approaches.

2. Explain the potential adverse effects associated with apigenin and chrysin based on omics based interpretation.

3. Review transcriptional targets that are regulated by apigenin and chrysin.

4. Demonstrate knowledge of metabolites that are regulated by apigenin and chrysin.

5. Review confirmation of the hypocholesterolemic property of both apigenin and chrysin in vitro.

Spatial analysis of protein or gene expression is vital to understanding the distribution, phenotypes, and interactions between cells within tumor microenvironment (TME). Traditionally, multiplexed spatial analysis has been performed using methods to detect either protein or RNA separately. Combining spatial analysis of protein-RNA on a single specimen is a powerful method to identify the cellular source of secreted proteins, characterize the cytokine signature, study gene expression in specific cell types as defined by protein biomarkers, or map the distribution of CAR-T+ cells within the TME. Here we present a validation study of a combined immunofluorescent (IF) and in situ hybridization (ISH) assay for co-detection of multiple protein and RNA markers on a single formalin fixed-paraffin embedded (FFPE) sample. The validation study was performed on 3 colorectal cancer samples using a 2plx-ISH and 17plx-IF integrated assay for detection of multiple RNA and protein biomarkers. Expression of each RNA and protein biomarker was quantified and inter/intra run coefficient of variation (CV) were calculated for the precision assessment. The results demonstrate robustness of all RNA and protein markers analyzed as well as the ability to spatially correlate RNA expression in different populations of the TME.

Learning Objectives:

1. Identify the advantages of a multiplexed spatial biology approach for immune-oncology research.

2. Demonstrate understanding of a multiplexed IF-ISH spatial assay workflow and the associated challenges.

3. Describe assay validation design and understand methods to analyze and assess results.