In this tutorial, we introduce cytoNet, a cloud-based image analysis software designed to quantify the structure of cell communities from microscope images. Using principles of graph theory, cytoNet identifies spatial relationships between cells and evaluates the effect of cell-cell interactions on individual cell phenotypes. Example applications are provided to characterize endothelial cell structures and differentiation of neural stem cells.

In these tutorials, we introduce GAIN, a graphical method to automatically analyze neurite outgrowth from microscopy images. Given an input of paired neuronal nuclear and cytoskeletal microscopy images, the GAIN algorithm calculates neurite length statistics linked to individual cells or clusters of cells. As output, GAIN produces a table of neurite lengths from cell body to neurite tip per cell cluster in an image along with a count of cells per cluster. GAIN’s performance compares favorably with the popular ImageJ plugin NeuriteTracer for counting neurons, and provides the added benefit of assigning neurites to their respective cell bodies. In summary, GAIN provides a new tool to improve the robust assessment of neural cells by image-based analysis.

In this tutorial, we introduce the AML Clinical Outcomes & Proteomics dataset and illustrate how to explore the data using an interactive visualization tool. 199 acute myeloid leukemia patients’ protein expression profiles for 231 proteins are displayed in relation to the patients’ clinical outcomes (overall survival and remission duration) and demographics (e.g., gender, prior malignancy). The visualization enables rapid identification of patterns in the data and exploration of relationships between outcome, proteomics, and clinical covariates.

In these tutorials, we introduce GAIN, a graphical method to automatically analyze neurite outgrowth from microscopy images. Given an input of paired neuronal nuclear and cytoskeletal microscopy images, the GAIN algorithm calculates neurite length statistics linked to individual cells or clusters of cells. As output, GAIN produces a table of neurite lengths from cell body to neurite tip per cell cluster in an image along with a count of cells per cluster. GAIN’s performance compares favorably with the popular ImageJ plugin NeuriteTracer for counting neurons, and provides the added benefit of assigning neurites to their respective cell bodies. In summary, GAIN provides a new tool to improve the robust assessment of neural cells by image-based analysis.

This Keystone Symposia SciTalk was made possible by a grant from

Coronaviruses (CoVs) thrive in animal reservoirs and represent a constant threat to human health as most recently exemplified the 2012 emergence of MERS-CoV, which is responsible for 2229 reported cases and 791 deaths worldwide. In anticipation of the next CoV outbreak, there is an imminent need for a vaccine solution. CoV spike (S) proteins mediate cellular attachment and membrane fusion and are therefore the primary target of protective antibodies. Instability and low expression of full-length CoV S proteins has historically hindered their development as vaccine antigens. Stabilizing other enveloped viral class I fusion proteins (e.g. RSV fusion (F) glycoprotein) in the functional prefusion conformation has resulted in highly immunogenic protein subunit candidate vaccines. To that end, we sought to evaluate stabilized prefusion CoV S trimers as vaccine candidates. Using structure-guided protein engineering, stabilizing mutations were identified to maintain several CoV S proteins across genera as trimers in their prefusion conformation (pre-S). To date, we have stabilized S proteins of 5 CoVs that infect humans: MERS, SARS, HKU1, OC43, and 229E. This presentation details our efforts to characterize the immunogenicity of MERS pre-S in mice. We show pre-S elicits more robust neutralizing antibodies to multiple MERS strains than S1 monomer or wild-type versions of S trimers, and protects mice from lethal challenge at low dose. Dissection of MERS pre-S immune mouse serum reveals MERS pre-S vaccination induces neutralizing antibodies to multiple domains of the trimer including to conserved regions outside of the receptor-binding domain. Additionally, we have optimized our MERS pre-S design for mRNA vaccine delivery, which yields robust neutralizing antibody responses. Looking forward to the next CoV outbreak, we are developing antigen design and vaccination strategies to target diverse neutralization-sensitive sites on pre-S and identifying sites that can elicit broadly neutralizing antibody responses. Our findings suggest that it may be possible to identify a generalizable solution for designing vaccine antigens for newly-emerging coronaviruses.

This Keystone Symposia SciTalk was made possible by a grant from

Understanding how different cell types recognize and respond to danger-associated molecular patterns (DAMPs) could lead to a better understanding of basic mechanisms of pain plasticity and lead to new therapeutic insights. DAMPs are produced in a broad variety of tissue injury paradigms and are linked to the generation of pain and pain plasticity underlying chronic pain. High-mobility group-box1 (HMGB1) is a major DAMP that regulates pain states, it is produced after injury, and activates toll-like receptor-4 (TLR4). Here we have employed a novel transgenic model that allows for cre-mediated deletion of a floxed TLR4 allele, utilizing NaV1.8 and LysozymeM to drive cre expression in peripheral nociceptors or macrophages, respectively. We observed a robust sexual dimorphic behavioral effect in mechanical hypersensitivity and hyperalgesic priming when TLR4 is removed from peripheral macrophages in males versus nociceptors in females. After intraplantar administration of HMGB1, wild-type (WT) littermates of both sexes show a mechanical hypersensitivity that is blocked in males when TLR4 is removed from macrophages while it is blocked in females when TLR4 is removed from nociceptors.  Conversely, macrophage TLR4 KO females and nociceptor TLR4 KO males develop mechanical hypersensitivity similar to their WT littermates. Moreover, these cell-specific TLR4 KO’s were also associated with decreased hyperalgesic priming precipitated by PGE₂ injection in respective groups (macrophages in males and nociceptors in females). We then assessed whether intrathecal administration of LPS, another TLR4 agonist, is capable of likewise mediating a sex- and cell-specific behavioral response. These experiments show a similar trend toward cell-specific TLR4-mediated modulation of pain in a sex-dependent manner.  Collectively our work demonstrates a cell-specific effect of TLR4 in acute pain plasticity and in the transition to a chronic pain state, pointing to a clear sex-difference in how DAMPs promote pain.

This Keystone Symposia SciTalk was made possible by a grant from

Mutations in protein kinases have been associated with the development of cancer. Research efforts in developing inhibitor molecules against the active site of the kinase catalytic subunit have shown positive effects such as prolonging patient survival. Unfortunately, in most cases these treatments result in drug resistance and tumor relapse. An alternative and novel approach is to identify allosteric sites that control protein kinase activity. In the Maillard laboratory, we will develop new single molecule biophysical methods to study how protein kinases are allosterically regulated. This study will guide the development of vitally needed novel therapeutic measures like allosteric inhibitors.

This Keystone Symposia SciTalk was made possible by a grant from

This Keystone Symposia SciTalk was made possible by a grant from

This Keystone Symposia SciTalk was made possible by a grant from