RNA-Seq has been used for the past decade to gain significant insights into gene expression. Using bulk methods however only allow for an understanding of the average gene expression within a tissue. Single cell RNA-seq gives a much more insightful picture of what is happening at the cellular level, but by dissociating tissues into their cellular components valuable information about tissue organization and cell to cell interactions are lost. The Visium Spatial Gene Expression Solution provides researchers with the ability to analyze gene expression across histological tissue sections using unbiased, spatially barcoded, mRNA capture probes. This discovery platform supports the analysis of all polyadenylated transcript and can be used to analyze thousands of genes and tens of thousands of transcripts within a few square microns of tissue. Here we demonstrate the platform’s capabilities to analyze a variety of human and mouse tissues giving deeper insight into the relationships between tissue morphology and gene expression.

The active shaping of biological membranes is essential for a variety of cellular functions including but not limited to cell migration, cell division, organelle morphology, and cell and membrane fusion. To this end, cells have evolved the ability to shape membranes through interactions between lipids and membrane shaping proteins or through direct modification of membrane lipids. Exactly how and when cells choose to employ these distinct membrane shaping tools remains a topic of intense study. Using the ciliate, Tetrahymena thermophila, our lab is trying to identify novel membrane shaping mechanisms and the signaling pathways by which they are regulated. 

Learning Objectives: 

1. Tetrahymena mating requires cell-cell fusion and selective autophagy 
2. Membrane shaping proteins, Reticulon and SNX4, may play a role in cell-cell fusion and selective autophagy respectively


 

Lowe syndrome (LS) is a lethal X-linked genetic disorder caused by caused by mutations in OCRL1 gene, which encodes a lipid phosphatase Ocrl1, important for many cellular processes. Our lab identified that lack of Ocrl1 function results in defects in cell spreading, cell migration and primary cilia assembly. Over 200 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. The phosphatase domain is a hotspot for disease-causing mutations harboring over 80 unique missense mutations. Our results indicate that different mutations within this domain have different effects on Ocrl1 distribution and on triggering cellular phenotypes. This is the first study to establish the link between genotype and phenotype in Lowe syndrome.

Learning Objectives:
1. Studying the relationship between of genotype and phenotype of Lowe Syndrome patients.
2. Learning the function of different domains of Ocrl1 in different cellular processes.

The ability to discern spatial gene expression differences in complex biological systems is critical to our understanding of developmental biology and the progression of disease. However, the complexity presented by heterogeneous tissue has been historically difficult to overcome. Immunohistochemistry, ISH, and H&E staining, foundational tools for understanding tissue architecture, are based on a combination of gene expression and cell morphology information. Though recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data, these experiments require dissociated cells and cannot preserve morphological context, until now.

The Visium Spatial Gene Expression Solution from 10x Genomics analyzes complete transcriptomes in intact tissue sections, allowing you to discover genes and markers relevant to your research without having to rely on known targets. Preserving spatial resolution offers critical information for understanding the relationships between cellular function, phenotype, and location in the tissue.

Environment drives bacterial functional diversification. Molecular functional abilities of individual microbes and microbiomes from different envrionment conditions, such as temperature or salinity or even the sick or healthy individual gut, are clearly different. Understanding the functions encoded in the (meta)genomes of microbiomes is thus vital for mapping their environmental preferences. The high-throughput (meta)genomic sequencing, coupled with the growing computational resources, has unlocked new horizons. However, making sense of this deluge of data requires efficient and accurate analytical techniques. The attendees are expected to leave the presentation with understandings the concept of how environment impacts on micro[biom]e functions and computational tools that we built to facilitate the analysis.  

The innate immune response requires continuous surveillance of the environment, and the ability to detect and react to pathogens and danger signals. Phagocytosis –the ingestion of particulate matter ≥0.5 µm in diameter– and macropinocytosis –the gulping of large volumes of extracellular fluid– are key components of innate immunity.  Both processes are complex and elegant, involving receptors and signal transduction, as well as cytoskeletal and membrane remodeling; a compendium of cell biology! These processes are often subverted by viruses, bacteria and fungi that take advantage of the host cells to establish a niche that favors their growth, replication and dissemination. My presentation will consist of a review of basic aspects of phagocytosis and macropinocytosis, followed by two sections describing recent advances in the field that have revealed the involvement of unique cytoskeletal structures (in the case of phagocytosis) and of ion channels (in macropinocytosis).

Learning objectives:

1. Understand the stages and functional roles of macropinocytosis and phagocytosis.

2. Appreciate the key role of phosphoinositides and inorganic ions in signaling and driving the formation and maturation of phagosomes and macropinosomes.

Spatial transcriptomics methods permit gene expression from focal areas within a tissue to be profiled while maintaining the morphologic context of the tissue microenvironment. This presentation will address considerations based on the needs of a study for selecting the best fit to what are new methods still in their infancy. These  important considerations start with; i) whether focal profiling will provide data that profiling of bulk tissue will not; ii) is it necessary to profile single cells or are larger focal areas such as single glands sufficient; iii) what is the size of such histologic regions and Is it necessary to have histologically discrete profiling without cross-contamination from adjacent histologic regions or cells; iv) What is the throughput required, just a few sections or lots of sections from 100+ tissues, animals, or subjects, and within each section, how many focal areas; v) Is it desirable to measure splice variants, snRNA, long non-coding RNA, histones - if so, then a 3’ biased assay method would be unsuited for the need vi) What tissue type does the method use, and how does that match up with what the investigator has available - Frfsh frozen, FFPE, H&E stained, antibody-stained; vii) What is the sensitivity – can all the genes measured from a bulk sample, including low expressed ones be measured; viii) What is the dynamic range; i.e. can a broad range of expressed genes from low to high be measured quantitatively at the same time. As methods continue in their development, the choice will also be whether the investigator wants to measure a panel of antigens at the same time as gene expression. If so, can this be from different serial sections, or does it have to be from the same section, same focal area. And of course, there is the question of cost. Different methods will be presented within the context of these questions, and spatial profiling data using TempO-Seq® will be provided to demonstrate several of these considerations. This data will also demonstrate the potential of spatial profiling in understanding the specificity and context of gene expression within the tissue microenvironment.

Neuroinflammation has been implicated as a factor in alcohol-induced neurodegeneration, but the role of the neuroimmune system in alcohol consumption has only recently come to the forefront. The drinking in the dark paradigm (DID) is an alcohol use disorder model that is uniquely suited to study the consummatory mechanisms of binge drinking. Our recent work has characterized the neuroimmune response following ethanol consumption in the DID. Our results indicate that a history of binge-like ethanol drinking promotes a proinflammatory cytokine response in the amygdala and hippocampus. Importantly, direct administration of anti-inflammatory cytokines can reduce ethanol consumption. These findings highlight the reciprocal relationship between alcohol abuse and the neuroimmune response. Alcohol misuse dysregulates cytokine concentrations but manipulating the neuroimmune response may curb excessive consumption.

Learning Objectives:

1. Understand that binge drinking can alter neuroimmune cells and function
2. Describe how manipulation of cytokines reduces binge drinking