2024 Webinars

 Environmental Focus 

Sources, Emission Inventory and Environmental Occurrences of New Pollutants in China

Guorui Liu, PhD | Professor | Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (China)

Abstract: Persistent toxic pollutants (PTS) have properties such as high toxicity, bioaccumulation, and long-range transport. PTS is ubiquitous in the global environment. This presentation mainly focuses on new pollutants of global concern, such as hexachlorobutadiene, halogenated carbazoles, and so on. Their sources, emission inventory and environmental behaviors will be discussed, which are helpful to protect human health and promote sustainable development goals.

Introducing EPA Method 1628: A New Paradigm for Clean Water Act Compliance Monitoring of PCBs

Chip McCarty, PhD | Senior Scientist | General Dynamics Information Technology (USA)

Abstract: The USEPA Office of Water currently requires monitoring of PCBs in wastewater discharges using techniques such as EPA Method 608, a dual-column GC/ECD procedure for organochlorine pesticides and seven Aroclor mixtures, which was the most practical approach available in the late 1970s when the first wastewater methods were proposed at 40 CFR Part 136. In the 40 years since those regulations were finalized, the environmental monitoring landscape has changed dramatically.

 NMR Focus 

Synthesis of ¹³C-Methyl-Labeled Amino Acids and Their Incorporation into Proteins in Mammalian Cells

Andrew Hinck, PhD | Professor and Deputy Chair | Department of Structural Biology, University of Pittsburgh (USA)

Abstract: Isotopic labeling of methyl-substituted proteinogenic amino acids with ¹³C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with ¹⁵N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of ¹³C-methyl-labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable ¹³C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet ¹³C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to ¹³C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, ¹³C-g2-Ile and ¹³C-g1,g2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2–3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as ¹³C-d1-Ile and ¹³C-d1,d2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of ¹³C methyl-labeled amino acids generated de novo by a versatile synthetic route.

New Way for Quantitatively Imaging Brain Energy Metabolism Using Deuterium (²H) MRS Imaging and Isotope-Labeled Glucose at Ultrahigh Field

Wei Chen, PhD | Professor | Radiology Department, Center for Magnetic Resonance Research (CMRR), University of Minnesota (USA)

Abstract: Decades ago, Ackerman et al. demonstrated the ability to measure cerebral blood flow in vivo using deuterium (²H) MRS or imaging combined with exogenous deuterated water (D₂O) as a freely diffusible tracer.¹ In 2011, Mateescu et al. reported the feasibility of measuring deuterium-labeled glucose and metabolically produced deuterated water (HDO) in the mouse head using ²H MRS and uniformly deuterated glucose as an substrate.² In 2014, we presented the first rat brain ²H MRS study at 16.4T that measured the deuterium-labeled glucose (Glc), mixed glutamate and glutamine (Glx), and lactate (Lac) with excellent SNR and temporal resolution after an IV administration of deuterated D-Glucose-6,6-d₂ (d66), and demonstrated the feasibility of simultaneously determining the cerebral metabolic rates of glucose consumption (CMRGlc) and TCA cycle (VTAC) using a kinetic model and the dynamic changes of [Glx] and [Glc] in the rat brain.^3,4 In 2016, we reported the first 3D ²H MRS imaging (DMRSI) study of rat brain tumors, which used the [Lac]/[Glx] ratio as a sensitive marker of the “Warburg effect” associated with cancer biology, showing excellent contrast between brain tumors and normal appearing brain tissues.^5,6 Since then the ²H MRS/DMRSI or DMI technique has been applied to study various tumors in preclinical models or human brains.^7-10 Recently, we have employed the advanced subspace-based denoising and machine learning methods to largely improve the SNR and spatial-temporal resolution of the DMRSI, and made it possible to map intra-tumor heterogeneity.^11,12 Furthermore, we reported for the first time the possibility of mapping three key metabolic rates of CMRGlc, VTAC, and lactate production rate (CMRLac) using high-resolution dynamic DMRSI covering the entire human brain at 7T.¹³ In summary, DMRSI is emerging as an important metabolic imaging modality with significant merits compared to other imaging methods. It is becoming an important tool for studying metabolic reprograming between glycolytic and oxidative metabolism in healthy brain, brain aging and many brain disorders including cancer, stroke and neurodegeneration, and it has a potential for translation.

RNA in Action: Bring RNA Structure to Life

Qi Zhang, PhD | Professor and Associate Chair | Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill (USA)

Abstract: The ongoing discoveries of regulatory RNAs with diverse activities in gene expression and regulation have transformed our view of RNA’s functions in cellular physiology and disease. Despite this progress, a critical gap remains in elucidating the mechanisms underlying RNA activities, where these highly dynamic molecules constantly morph between alternative conformations, each triggered by specific cellular signals. These conformational transitions can occur across a wide range of timescales, from picoseconds to seconds and beyond; yet, conventional static structures convey little of this dynamic nature of RNA, which is crucial for orchestrating their cellular activities. Hence, to fully understand RNA biology, we need to reimagine RNA structural biology, where RNAs are viewed as dynamic ensembles characterized by probability distributions of fluctuating conformations, each with a distinct lifetime. In this presentation, I will discuss our recent progress in uncovering such an ensemble perspective of RNA molecules toward a quantitative and predictive framework for understanding how RNAs harness conformational dynamics to drive cellular activities.

Feeding Spiders Isotope-Enriched Amino Acids to Label Their Silks for NMR Investigation

Greg Holland, PhD | Professor | College of Sciences – Chemistry and Biochemistry, San Diego State University (USA)

Abstract: Over 300 million years spiders have evolved to produce seven different types of silk. The silks are comprised almost entirely of protein and are used for a diverse range of applications including web construction, egg case production and wrapping prey. The silks vary dramatically in their mechanical and physical properties with the major ampullate silk (dragline) exhibiting a strength that exceeds steel by weight and a toughness greater than Kevlar while, flagelliform silk has an elasticity comparable to rubber. Our lab is focused on understanding the molecular structure and dynamics of the proteins that comprise the various spider silk fibers with MAS solid-state NMR. It is the folded structures and hierarchical organization of these proteins that imparts spider silks their impressive yet, diverse mechanical properties. Our research team has been developing and applying SSNMR to probe secondary structure, hydrogen-bonding, side chain dynamics, and oligomeric protein assembly all of which are crucial to understanding spider silk formation and the resulting fiber properties. Recently, we have focused on using solution NMR to understand the protein-rich fluid within the various silk producing glands to investigate the conformational structure and dynamics prior to fiber formation and determine the important biochemical triggers responsible for converting this hydrogel-like protein solution to fibers with unparalleled properties. It is our belief that a better fundamental understanding of spider silk protein structure and assembly process will accelerate the ability to mimic and reproduce similar biologically inspired materials in the lab. These NMR approaches all require isotopic enrichment (¹³C/¹⁵N) that are administered to the spider in their water supply. I will discuss how we do it, the types of NMR experiments it enables and the molecular information gained in the context of silk formation.

A transcript of this presentation in English is available here.

Quantitative Benchtop NMR for Vaccine Development

Adam Sutton, PhD | Associate Principal Scientist  | Merck Research Labs (USA)

Abstract: Several different types of vaccines are regularly examined in the pharmaceutical industry. Vaccines can contain large molecules such as proteins and polysaccharides or larger particles such as attenuated viruses, lipid nanoparticles or virus like particles. All these vaccine types contain a complex mixture of small and macromolecules, therefore several analytical tools often need to be assessed in order to assist in the various stages of vaccine process development. Benchtop NMR is an advancing technology that can be a versatile analytical tool that provides quantitative information about the multiple components, using a single internal standard, that arise in the development of a vaccine. Benchtop NMR often involves minimal sample preparation since macromolecular species are usually not detectable or can be removed by T2 filters. The software for benchtop NMR is easy to use and provides automatic data processing further making benchtop NMR an attractive analytical tool that can be quickly assessed when comparing different analytical approaches. In this presentation the use of benchtop NMR as an alternative to chromatographic methods will be discussed. Its use for real-time monitoring and fast method development will be demonstrated. Examples of using benchtop NMR to quickly determine solvent compositions, to confirm excipients and study alum sedimentation in vaccine-related samples are some of the examples that will be covered, with some comparison to chromatography-based methods. Furthermore, we will discuss how benchtop NMR method development is possible even from those with no NMR experience, which are clear advantages for the implementation of benchtop NMR in the pharmaceutical industry.

A transcript of the presentation in English is available here.

 Mass Spec Focus 

The Use of Stable Isotopes to Understand Metabolic Disease in Pediatrics

Melanie Cree, MD, PhD | Associate Professor Pediatrics Endocrinology | University of Colorado Anschutz and Children’s Hospital Colorado (USA)

Abstract: Pediatric metabolic disease is increasing at an astronomical rate, in line with the obesity epidemic. Understanding the underlying pathology, in particular, tissue specific insulin resistance and substrate metabolism is critical for the development of new therapeutics. Intravenous and oral tracers can be utilized in different settings: fasting, with hyperinsulinemic-euglycemic clamps or oral glucose tolerance tests to understand metabolism under different metabolic states. Combining studies in youth with different methodologies and tracers, and with clinical interventions is leading towards new therapeutic targets.

A transcript of this presentation in English is available here.

A Targeted LC-MS/MS Method for Routine Monitoring of Cell Culture Media Components in Biotherapeutic Processes

Jared Kress, BSc | Scientist | Merck (USA)

Abstract: Cell culture media (CCM) optimization is a critical step during the development and scale up of biotherapeutic production. In particular, the emphasis on quality by design has made it necessary to understand how the components of CCM change during production and how these changes relate to product quality. There is a vital need to develop analytical assays that can provide comprehensive, yet accurate, CCM profiling for a wide range of biotherapeutic types produced from, or are themselves, living cells. Herein, we present a robust method that allows commendable retention and separation of an excess of 110 compounds spanning a multitude of metabolic classes. By using the MSK-QReSS (Quantification, Retention, and System Suitability) kit containing isotope-enriched ¹³C and ¹⁵N metabolite mixes, the developed method enabled the level of key metabolites in culture, as well as spent media, to be relatively quantified. Through statistical analysis, metabolite levels are visualized to understand similarities and differences throughout various manufacturing stages and provide clear indication of CCM components behaviors during biotherapeutic production.

A transcript of this presentation in English is available here.

Investigation of the Dystrophin Associated Protein Complex Using a SILAC Strategy

Emily Canessa, BSc | PhD Student | School of Pharmacy and Pharmaceutical Sciences, Binghamton University (USA)

Abstract: The dystrophin associated protein complex (DAPC) is an important glycoprotein complex that helps to maintain the membrane stability of muscle fiber sarcolemma. Central to this complex is the protein dystrophin which anchors the DAPC to the actin cytoskeleton of the cell. In Duchenne muscular dystrophy (DMD) this protein is absent, leading to fiber degradation, muscle atrophy, and loss of ambulation by age 12. Current FDA-approved therapies aim to restore dystrophin expression in patient tissue, but the amount and function of restored dystrophins are not well characterized. To address this, we used two different SILAC strategies to improve our current understanding of the interaction between dystrophin and the DAPC. In one study we used a pulse-chase SILAC labeling strategy to study the DAPC protein turnover in mdx mice treated with an exon-skipping therapy to restore dystrophin expression. In a second study we spiked human muscle lysate with SILAC-labeled myotubes in order to quantify the DAPC in patients with a milder form of muscular dystrophy where dystrophin is present in varying decreased amounts. By using these two different SILAC strategies we were able to better characterize the role dystrophin amount plays in the stability of the DAPC. This greater understanding of the complex will help to explain the efficacy of current dystrophin replacement therapies, as well as aid the designing of newer ones.

A transcript of this presentation in English is available here.

Utilization of Stable Isotope-Labeled Metabolites for Automation in Data Processing in Microchip CE-MS Metabolomics

Will Thompson, PhD | Principal Scientist | 908 Devices (USA)

Abstract: Metabolomics has demonstrated the ability to measure hundreds of metabolites in diverse sample types. Nonetheless, data analysis remains a key bottleneck in targeted and nontargeted approaches. For example, system suitability testing (SST) has been widely adopted as common practice in metabolomics but human intervention is often required to accept or reject system suitability. After experiments have been run, data quality assessment is often made by somewhat arbitrary and manual approaches, resulting in inconsistent results and wasted time. Finally, errors in metabolite/peak assignment require arduous manual curation. Heavily utilizing stable isotope-labeled internal standards (SIL-IS), we have developed a novel Windows application which automates instrument orchestration, SST interrogation, raw data QC, and the quantitative data analysis pipeline for microchip CE-MS metabolomics. This presentation will focus on the use of SIL-IS for migration time indexing, rapid data quality checking, and use in automated correction of common peak-selection errors made by metabolomics software.

A transcript of this presentation in English is available here.