T cells exquisitely discriminate between antigenic and self-peptide ligands presented on major histocompatibility complexes (pMHCs) despite subtle differences in affinities. This aspect of adaptive immunity is required for robust, specific, and highly selective T cell activation and to avoid autoimmunity in the background of more abundant self pMHCs. We recently introduced a reconstitution-based molecular impulse-response assay that demonstrated how T cell receptors (TCR) execute this single molecule precision in antigenic pMHC detection. Interestingly, T cells effect a spatiotemporally-correlated antigen detection mechanism that translates a series of input ligand:TCR binding events into activation responses. Cell-based immunotherapies reprogram T cells to eliminate cancer cells through the engineering of tumor-associated antigen-specific (TAA) chimeric antigen receptors (CARs). Many TAAs are epitopes of highly overexpressed cell surface proteins but are rarely tumor-exclusive. CARs have been highly effective in eliminating cancers of blood cells but continue to be minimally effective against solid tumors. The hyperactivation of CAR T cells, as well as off-target toxicity, have plagued the translation of mobilizing T cells as a therapeutic. We propose that a mechanistic, single molecule understanding of the CAR T cell input:response function is needed to optimally harness anti-tumor responses and minimize side effects. I will discuss our efforts to measure molecular activation thresholds in primary, anti-tumor CAR T cells.

Learning Objectives:

1. Define single molecule triggering of T cells

2. Explain an impulse-response function

3. Identify at least 2 parameters that may control CAR T cell triggering and activation.

The glomerular filtration barrier, generated through the interaction between podocytes and glomerular endothelial cells (GECs) and separated by a thin layer of glomerular basement membrane (GBM), is structurally responsible for filtering the blood and preventing the loss of proteins. The mature GBM is composed of laminin-521, collagen α3α4α5(IV), and agrin. Mutations in the alpha chains of collagen IV genes lead to a hereditary chronic kidney disease identified as Alport syndrome (AS). To study the cellular and molecular mechanisms responsible for glomerular injury and podocyte loss and design therapeutics strategies targeted to the glomerulus, we must develop human physiologically relevant models of the glomerulus. We designed a glomerulus-on-a-chip (GOAC) constituted by human amniotic fluid kidney progenitor-derived podocytes (hAKPC-P) and human glomerular endothelial cells (hGEC) seeded on OrganoplatesTM(MIMETAS). Our system is characterized by the absence of an artificial membrane separating the two monolayers. The GOAC system recapitulated the function and structure of the glomerulus, including permselectivity. In long-term cultures, seeded cells maintained morphology and formed capillary-like structures that expressed slit diaphragm proteins. Chips generated using these AS podocytes presented impaired permselectivity to albumin due to a dysfunctional assembly of the GBM, typical of AS. We show that miR-193a and serpins may also play key roles in influencing glomerular homeostasis, and modulating their expression may minimize disease progression. Our GOAC system offers a unique prospect for disease-modifying studies and can serve as a platform for personalized medicine studies.
 
Learning Objectives:

1. Explain the structure and function of the glomerular filtration barrier, the pathophysiology of Alport syndrome, and the role of miR193.

2. Explore the glomerulus-on-a-chip model and how it may be used to model disease and in studies of personalized medicine. 

The explosion of interest in extracellular vesicles has largely coincided with the development of tools which allow their rapid and routine characterization, in this presentation we will identify and discuss the fundamental principles behind several common tools used for quantifying individual EVs and consider what limitations and strengths should be recognized in each technique. With this understanding we will walk through example data comparing bulk and single particle measurements to develop a better understanding of the real composition of EVs. Lastly we will consider which techniques should be selected for a given investigation based on the research question at hand. 

Learning Objectives:

1. Identify the subtle differences in techniques for detecting single nanoparticles.

2. Review how the choice of a particular technique can influence the collection of the data.

3. Explain how to choose the appropriate technique for their work based on the research question under investigation. 

Bladder cancer is the 4th most prevalent cancer in men, 11th in women and presents one of the highest rates of post-treatment recurrence (70%) among malignancies. Despite its impact on human health, therapeutic approaches against bladder cancer are of limited efficacy. 

We previously developed a strategy based on receptor micro-clustering (µC) to induce agent fast internalization by bladder tumor cells.  Importantly, µC effects can be elicited by different multivalent agents including anthrax toxin that clusters as heptamers at the plasma membrane inducing its own uptake. Further, and considering that bladder tumor cells express high levels of Epidermal Growth Factor (EGF) Receptor (EGFR), we used EGF to target this lethal toxin against cancer cells. 

Here we show that the EGF-toxin induces its own internalization and delivery into the cytosol of targeted cells within min of exposure. This agent was up to 1000 times more efficient that current anti-bladder cancer agents such as mitomycin C and Gemcitabine at eliminating bladder cancer cells. Further we also found that this agent was capable of eliminating Cancer Stem Cells of bladder and prostate origin. We established that under bladder instillation conditions, the toxin eliminated human, canine and mouse bladder cancer cells, including Her2-positive cells, with a LC50<1nM (<<intoxication dose ~1μM) and exposure time<3min.

Since µC enhances internalization of cargoes independently of receptor dimer formation, we found that this strategy was insensitive to known efficacy-decreasing factors to EGF-based approaches such as internalization interference by presence of Her2 or by endocytosis-limiting mutations. Further, dogs with spontaneous, treatment-resistant Her2-positive bladder cancer (closely mimicking the human condition) showed marked reduction in tumor mass when treated with EGF-toxin. Safety studies indicated no deleterious effects in normal and patient dogs.

These studies provide the foundation for an innovative and transformative anti-bladder cancer therapy. The EGF-toxin is a superior agent due to its high efficacy, fast action (minutes vs. current treatments requiring more than 2h retention of therapeutics in the bladder) and enhanced safety, but also due to its ability to target cancer stem cells and Her2-positive tumors. 
 

Learning Objectives:

1. Review the current impact of bladder cancer and the limitations of available treatment options.

2. Review the novel paradigms for the developments of more efficient and specific anti-bladder therapeutics.

Actin cytoskeleton is essential for eukaryotic cells. To exert its countless cellular functions, actin undergoes dynamic remodeling orchestrated by the large number of actin-interacting factors. Cellular conditions favor actin assembly (polymerization) as opposed to actin disassembly (depolymerization), and our understanding of actin disassembly mechanisms is still incomplete. In this talk I will discuss a unique mechanism of actin disassembly that relies on site- and stereo-specific oxidation/reduction of actin. This enzymatic oxidation is introduced by the Mical family of enzymes, and it primes actin filaments for disassembly. I will discuss how this posttranslational modification synergizes with some of the key actin regulators – cofilin and profilin – to orchestrate actin disassembly independently of its nucleotide-bound state and to suppress its repolymerization. 

Learning objectives:

1. Recognize that oxidation of proteins can be a regulatory mechanism as opposed to oxidative damage.

2. Explain how site-specific oxidation introduced by Mical enzymes synergizes with other regulatory proteins to promote disassembly of actin filaments.
 

Microtubule plus-end tracking proteins (+TIPs) play key roles in the regulation of microtubule dynamics in vivo. These proteins bind to and regulate microtubule “plus‐ends”, which are the dynamic ends that explore and interact with the cellular periphery. Despite their importance, it is still unclear how various +TIPs interact with each other and with plus‐ends to control microtubule behaviors, particularly in vivo. My lab studies +TIP function by quantitative analysis of microtubule plus-end behaviors using high‐resolution live‐imaging data of cultured embryonic Xenopus laevis cells. Previously, we demonstrated that the transforming acidic coiled‐coil (TACC) domain family member, TACC3, can function as a +TIP to promote microtubule polymerization dynamics, in collaboration with the microtubule polymerase, XMAP215. In this talk, I will discuss recent work on how TACC3 may be mediating axon guidance, as well as how its binding partner, XMAP215, appears to promote MT-F-actin interaction in embryonic neuronal growth cones.

Learning Objectives:

1. Describe the roles of different cytoskeletal elements in cell migration

2. Identify what a microtubule plus-end-tracking protein is and its importance

3. Explain how XMAP215 may have a novel function in aligning different cytoskeletal elements

Stem cells respond to both physical and biochemical changes in their stem cell niche. An ideal scaffold for tissue engineering application should mimic the microenvironment for natural tissue development and present the appropriate biochemical and topographical cues in a spatially controlled manner. Studies have shown that physical forces from the substrate topography play a role in stem cell proliferation, migration and cell fate determination. Our research group is interested in studying the interactions of adult and pluripotent stem cells with nanotopography, the mechanism of the topography-induced cell behavior and how to apply this knowledge to direct stem cell differentiation for tissue engineering applications.  In this presentation, nanotopography-regulation on adult stem cells and pluripotent stem cells (PSCs) will be presented as examples of applying nanotopography in stem cell regulation.

In attempt to understand the sensing mechanisms for nanotopography, we investigated the roles of focal adhesion signaling and cytoskeletal contractility in topography-induced differentiation. We showed that the nanotopography-induced differentiation mesenchymal stem cells (hMSCs) is modulated through cell mechanotransduction by the integrin-activated focal adhesion kinase (FAK). In addition, our mechanistic study in hMSCs and hPSCs confirmed that the regulation was dependent upon actomyosin contractility, suggesting a direct force-dependent mechanism. Using a 3D traction force system to study the cell contractility on anisotropic grating topography, focal adhesions were found to be aligned parallel to the grating direction as compared to cells on flat substrates. The 3D traction stress measurements revealed that elongated cells on grating substrates exert anisotropic traction stresses, in the direction parallel to the grating direction. Focal adhesion alignment along the grating direction may result in increased actin stress fiber formation in the direction parallel to the grating, leading to polarized traction stresses which drive cell elongation.  The temporal presentation of topography also plays a significant role in the differentiation.

Learning Objectives:

1. Recognize how micro- and nanostructures in the cell microenvironment can affect cell responses.

2. Explain the potential mechanism of how cells sense these structures.

Neutrophils are the most abundant immune cells in human blood and are crucial for host defense against bacterial and fungal infections. Neutrophils are made mainly in the bone marrow from where they travel to different tissues with the blood circulation. Neutrophils are recruited from the blood stream into the tissue through a cascade of events that entail neutrophil rolling and arrest on the endothelium and migration through the vessel wall. Neutrophils roll at a velocity about 1000-fold less compared to the blood flow velocity, which enables the neutrophils to screen the vessel wall for inflammatory mediators and facilitates arrest. The importance of neutrophil rolling is demonstrated by the increased risk of severe bacterial infection in patients harboring genetic deficiency in selectin mediated neutrophil interaction with the endothelium, that is crucial for rolling. Neutrophil rolling is further stabilized by the formation of long thin membrane tethers from the endothelial bound microvilli of the rolling neutrophils. We found that tethers can fail and detach from the neutrophil. This will result in unstable neutrophil rolling and often detachment of the rolling neutrophil from the vessel wall. The separated tethers will form sliding, adhering or circulating elongated neutrophil-derived structures (ENDS) in the vessel lumen. We found ENDS to be devoid of mitochondria, endoplasmic reticulum, or DNA, but enriched for S100A8, S100A9. In in vitro experiments within hours of formation, ENDS round up and release S100A8–S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. We detected 10–100-fold elevated ENDS count in blood plasma of septic patients compared to healthy donors. Unlike neutrophil-derived extracellular vesicles, ENDS do not stain for CD9, CD63, and CD81. Thus, ENDS are a new class of bloodborne microparticles with a formation mechanism linked to neutrophil rolling on the vessel wall.

Learning objective: 

1. Explain neutrophils and neutrophil rolling.

2. Explain the consequences of tether detachment from rolling neutrophils.

3. Explain how ENDS compare to other neutrophil-derived extracellular particles.
 

While providing indispensable insight into immune processes, the live cell imaging of immune cells poses unique challenges due to their non-adherent nature. Suspension cells such as immune cells can easily move away from the FOV (field of view) during imaging by perturbation such as reagent introduction, often necessitating the use of surface coating to artificially immobilize cells. In addition, unlike adherent cells, interacting suspension cells form aggregates, making it very difficult to track and analyze individual cells during imaging. Here we describe the novel, microfluidic-based dynamic live cell imaging platform CellASIC® ONIX2, where microenvironmental parameters such as the perfusion of media/reagent, temperature, and gas compositions can be precisely controlled on-demand via software during the entire imaging experiment. Unlike most live imaging platforms merely aiming to maintain temperature and gas at certain levels, CellASIC® ONIX2 platform’s ability to control fluidics and perfusion offers true on-demand control of cellular microenvironment during live cell imaging, and its standard plate form factor can instantly upgrade any existing inverted microscope into a fully capable live cell imaging platform. Overview of applications covering hypoxia, apoptosis, migration, and suspension immune cell imaging will be presented. Specific emphasis will be given to microfluidic designs targeted for use with different cell types as well as fluorescent probes for live cell imaging. Any scientists planning to start live cell imaging experiments, as well as experienced imaging scientists wanting to broaden their applications will benefit from this session. 

Learning Objectives:

1. Evaluate the advantages and limitations of current live cell imaging platforms

2. Discuss the emerging field of microfluidic based dynamic imaging

3. Review current state of the art regarding microfluidics based dynamic imaging

Cells in their natural environment are surrounded by a complex extracellular matrix that provides structure and functional cues to the surrounding cells. Hydrogels can be used to approximate this environment for 3D cell culture, but require preparative work including hydrogel assembly, hydrogel optimization, and cell encapsulation. Learn how our fully ready-to-use TrueGel3D™ HTS hydrogel plates help you to quickly and easily establish 3D cell cultures for high-throughput screening and high content analysis. Data will be presented to show how these automation-compatible plates can be used for imaging 3D cultures in immuno-oncology, angiogenesis, neuroscience, and ADME/tox studies.

Learning Objectives:

1. Explain how TrueGel3D® HTS plates solve current limitations of 3D cell cultures for drug discovery.

2. Identify how to easily establish cocultures of tumor spheroids and T-cells in 3D using TrueGel3D® HTS plates

3. Evaluate performing high-throughput drug screen using TrueGel3D® HTS plates

Cells of the liver and pancreas are highly polarized and well differentiated, contributing to food digestion through the secretion of lipid emulsifying bile, and proteolytic juice into the gut.  Both have sophisticated cytoskeletal membrane trafficking machineries that aid in this secretory process while ensuring the maintenance of apical and basolateral membrane domains.  Unfortunately, these organs can become steatotic (fatty), leading to a variety of different diseases.  Further, as the liver is a central blood filtering organ, it also can become infected by a substantial variety of different pathogens.  Both steatosis and chronic infections are known to lead to cancers of the liver and pancreas that are among the most lethal of all cancers with exceptionally poor prognoses. 

This presentation is divided into three parts that are aimed at providing insights into the membrane trafficking processes that play a key role in diseases and neoplasia of the liver and pancreas.  The focus is the role of the actin cytoskeleton and its interaction with both small regulatory GTPases (Rabs) and the large mechanochemical GTPases (dynamins).  The presentation will cover:  first, cytoskeletal dynamics at the leading edge of invading tumor cells that support metastatic migration, second, identification of a new autophagic process (lipophagy) that acts to degrade lipid droplets in steatotic hepatocytes, and finally, an anti-viral dynamin family member that associates with hepatocyte mitochondria to alter the genome of this organelle.

Key references for this presentation include:
•    The cell biology of the hepatocyte: A membrane trafficking machine.  J. Cell Biol. 2019 Jul 1;218(7):2096-2112. 
•    Dynamin 2 interacts with α-actinin 4 to drive tumor cell invasion. Mol. Biol. Cell. 2020 Mar 15;31(6):439-451. 
•    A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets.  Sci. Adv. 2016 Dec 16;2(12):e1601470. 
•    The anti-viral dynamin family member MxB participates in mitochondrial integrity. Nat. Commun. 2020 Feb 26;11(1):1048.

Learning Objectives:

1. Analyze one cytoskeletal component of tumor cell metastasis.

2. Defining the basis of hepatocellular steatosis and “lipophagy”.

3. Defining a role for an anti-viral dynamin in mitochondrial integrity.

Research focused on SARS-CoV-2 has become a primary focus in genomic and molecular biology fields. With a constantly changing field with new methods for screening and studying virus-host interactions, availability of accurate, efficient, and consistent tools is critical. In this webinar we will discuss the latest advancements in thermostable DNA polymerases enabling sensitive and fast amplification, even from samples with suboptimal purity. Field applications and publications will be explored to demonstrate how reverse transcription enzymes with increased levels of efficiency, sensitivity and inhibitor tolerance have been used in SARS-CoV-2 research, including in RT- qPCR, deep sequencing and cloning applications.

Learning Objectives:

1. Review Sars-Cov-2 research trends and focused areas.
2. Explain major technologies used for Sars-Cov-2 research.
3. Review important features and key considerations of DNA polymerase and reverse transcriptase used for Sars-Cov-2 research.

Extracellular vesicles provide a tool to measure tumour characteristics in blood, at various stages of cancer development.  We have extensively used multiplex RNA bead-based assays (Quantigene assays Thermo Fisher Scientific) to detect gene amplifications in colorectal cancer tissues in multiple sites within a primary tumour, showing intra-tumour heterogeneity and in matched lymph nodes and metastatic lesions. These studies provided the selection of a gene amplification panel to study tumour-derived exosomes in blood from various stages of colorectal cancer. The selected panel includes TPX2, TOP2A, CCND3 and MYC. Of interest, TPX2 is overexpressed in 86% of colorectal cancer (CRC) samples when compared to normal colon epithelium, 50% of lung metastatic lesions show the highest fold increase of TOP2A when compared to the primary CRC tissue, CCND3 was exclusively overexpressed in lug metastasis, and MYC is consistently gained in metastasis, irrelative to site of metastatic lesion. To ensure that the methodology used is sensitive enough to measure these amplifications in patient blood-derived exosomes, we used colorectal cancer cell lines and generated exosomes following serum starvation. The mean fluorescence intensity (xMAP® Technology, Luminex) of RNA representing the amplified genes, were measured for exosomes and respective matched originator cells. Correlation analysis show an coefficient of determination, r2 of 0.95, showing that MFI magnitude derived from exosomes reflect the RNA amount from originator cells, and that gene amplification in originator cells can be measured using RNA quantification in exosomes. This led us to isolate exosomes from patients’ plasma and run a 40-plex RNA bead-based assay to measure gene amplification in tumour-derived extracellular vesicles. The 40-plex assay included 18 epithelial-metastatic transition and metastasis associated gene amplifications.  TPX2, MET and TOP2A were detected in the exosomes and matched tissue samples from the same patient. This shows the versatility of the Quantigene® assay (Thermo Fisher Scientific) and the sensitivity of the xMAP® Technology (Luminex) to detect tumour characteristics in blood. This augers well to develop protocols for early detection of disease, and early detection of metastatic disease during therapy or tumour recurrence.

Learning Objectives:

1. Assess the use of multiplex assays to measure panels of biomarkers simultaneously provides a solution to the complexity of cellular biology.

2. Examine how RNA bead-based assays provide the sensitivity required to measure mRNA in exosomes that reflect gene amplification/s in originator cellular models. 

3. Interpret how optimising methodologies through the integration of technologies, enhance end-point measurement sensitivity for tumor-derived exosome detection in patients.