In response to the limited research experiences for young scholars during the COVID-19 pandemic and community interest, we developed the Pre-College Rosetta Internship Opportunity (PCR-IO). The ...mission of PCR-IO was to offer a program to increase equitable access to computational biomolecular research. The PCR-IO program engaged rising senior high school students in a protein therapeutic design project in which they produced novel structural models using the PyRosetta and Foldit software packages. The program comprised a year-long series of activities, with an immersive summer internship that involved students in research as the cornerstone. These activities aimed to support the overarching goal of the program by expanding participating students’ social capital and technical skills, making them more likely to consider and succeed in STEM in their future endeavors. Here we describe the program’s components and rollout and discuss successes and challenges in implementing a remote computational research-based educational high school program. We observed considerable student skill development and conclude that the program created real added value to student participants’ education. We also uncovered issues associated with curriculum pace and found that the required mentorship effort exceeded our expectations. This perspective is intended to offer insight, share recommendations, and create dialog to increase propagation of research-based computational internships, and to shed light on how much novice students can accomplish with mentorship, structured curricula, and access to the research community.
The Rickettsiales-like prokaryote and causative agent of Withering Syndrome (WS)-
Xenohaliotis californiensis (
Xc)-decimated black abalone populations along the Pacific coast of North America. White ...abalone-
-are also susceptible to WS and have become nearly extinct in the wild due to overfishing in the 1970s.
Xenohaliotis californiensis proliferates within epithelial cells of the abalone gastrointestinal tract and causes clinical signs of starvation. In 2012, evidence of a putative bacteriophage associated with
Xc in red abalone-
-was described. Recently, histologic examination of animals with
Xc infection in California abalone populations universally appear to have the phage-containing inclusions. In this study, we investigated the current virulence of
Xc in red abalone and white abalone at different environmental temperatures. Using a comparative experimental design, we observed differences over time between the two abalone species in mortality, body condition, and bacterial load by quantitative real time PCR (qPCR). By day 251, all white abalone exposed to the current variant of
Xc held in the warm water (18.5 °C) treatment died, while red abalone exposed to the same conditions had a mortality rate of only 10%, despite a relatively heavy bacterial burden as determined by qPCR of posterior esophagus tissue and histological assessment at the termination of the experiment. These data support the current status of
. Xc as less virulent in red abalone, and may provide correlative evidence of a protective phage interaction. However, white abalone appear to remain highly susceptible to this disease. These findings have important implications for implementation of a white abalone recovery program, particularly with respect to the thermal regimes of locations where captively-reared individuals will be outplanted.
Including undergraduate research in STEM education is a well-supported and growing high-impact practice that has been made much more scalable through integrating these experiences into the classroom. ...Here we describe a new biochemistry Course-based Undergraduate Research Experience (CURE) that follows a design-to-data workflow with a strong connection to a worldwide community of protein modeling software developers. Analysis of psychosocial developments in association with participating in this CURE from the first set of students formally participating in the course suggest a beneficial effect on attributes associated with STEM persistence. To increase successful propagation, the design of the CURE’s curriculum, supporting learning materials, and instructor resources are provided to make it facile for faculty at any institution to join this network and implement the CURE. With this foundation, we expect student participation and the data set to continue to grow.
Rickettsiales-like prokaryote Candidatus Xenohaliotis californiensis (Ca. Xc), the causative agent of abalone withering syndrome (WS), has decimated abalone populations along the Pacific coast of ...North America. The federally endangered white abalone, (Haliotis sorenseni) are particularly susceptible to this agent and are now nearly extinct in the wild. Candidatus Xc proliferates in epithelial cells of the abalone gastrointestinal tract and causes clinical signs of starvation. The specific aims were to: (1) develop an axenic media to support the growth of Ca. Xc, (2) observe effects of reduced pathogenicity of Ca. Xc in the presence of bacteriophage in red (H. rufescens) and white abalone under conditions that previously have been shown to exacerbate withering syndrome, and (3) collect preliminary data on undergraduate student engagement with research on the abalone microbiota through a novel first-year seminar. Axenic culture of members of the Order Rickettsiales, including numerous human pathogens, has not been achieved for any species. In a related project by our collaborators, genetic analysis suggested that Ca. Xc is the most ancestral form of the Anaplasmataceae family of Rickettsiales studied to date. Accordingly, we hypothesized that Ca. Xc may require simpler culture conditions than other Rickettsiales. We attempted to culture Ca. Xc from Ca. Xc-infected red abalone gut tissue using a basal medium that supports growth of Piscirickettsia salmonis, a marine facultative intracellular pathogen of fish. Unfortunately, we were unable to observe Ca. Xc. growth even with supplementation of media with a wide variety of additives. In order to gain a better understanding on the pathogenesis of Ca. Xc in abalone, and to investigate the attenuation conferred by a recently described bacteriophage, infectious challenges were pursued at different temperatures. We hypothesized that the phage hyperparasite would reduce the pathogenicity of Ca. Xc infection in red and white abalone and in temperature regimes that have previously either slowed (cold water) or enhanced (warm water) disease progression. Red and white abalone were assigned to different seawater temperature regimens (14.0°C or 18.5°C) with and without Ca. Xc exposure all in the presence of phage. Animal health was assessed by weight and length measurements, Ca. Xc 16S rRNA gene copies in tank fecal and tissue samples by quantitative real-time PCR, histological data, and mortality rates. Using a comparative experimental design, we observed differences over time in mortality and growth rates of the two species and bacterial load by qPCR. By day 251, all of the white abalone exposed to Ca. Xc in the warm water treatment had died. A different response was observed in red abalone exposed to the same conditions, with a mortality rate of less than 10%. In comparison to historical experiments conducted prior to the presence of the phage, red abalone showed significantly improved (36%) survival rate in the presence of phage. Red abalone showed advanced infection as evidenced by high Ca. Xc gene copy numbers by tissue qPCR and large numbers of bacterial inclusions histologically but the pathogen burden seemed to have little effect on red abalone health and survival. White abalone had fewer organisms at either temperature treatment as determined by both qPCR and histology but had far greater negative health and survival outcomes. These data support the novel phage hyperparasite as a protective agent in red abalone, while white abalone remain susceptible to withering syndrome in its presence. The abalone microbiota has recently begun to be investigated in relation to WS. In an effort to engage young scientists, we undertook efforts to incorporate a microbiota study of abalone with WS into a course-based undergraduate research experience class (CURE). Course-based undergraduate research experiences provide students with the unique opportunity to participate in a research project that is of interest to the scientific community. To test the impact of CUREs on UC Davis undergraduates, we developed a course focused on identifying bacteria from diseased and heathy abalone. In this course, bacterial isolates were whole-genome sequenced and seven genomes were published with students as authors. A second course focused on isolation and characterization of tannin-tolerant bacteria isolated from koala fecal material. We evaluated the courses using surveys and student reflections to assess the immediate impact of these CUREs. The data collected supported the conclusions that the course was successfully delivered, allowed exposure of the students to research, and indicated a modest increase in student’s scientific self-efficacy in association with the course. Overall this project seeks to investigate Candidatus Xenohaliotis californiensis in vitro growth, changes in pathogenesis, and new ways to incorporated research into pedagogical practices.
Hands-on research provides insight into the process of science and has been linked to increased retention of students in STEM disciplines. While large research universities can provide valuable ...undergraduate research experiences in laboratories, most cannot accommodate all of the students seeking research apprenticeships. Course-based undergraduate research experiences (CUREs) offer a scalable solution to this problem by facilitating faculty-mentored student research on novel problems through the structure of unit-bearing classes. Yet, implementation of CUREs de novo in large-enrollment introductory courses can be challenging at both institutional and individual instructional levels. We investigated whether First-Year Seminars (FYS), small credit-bearing classes targeted at freshman and transfer students, which are common in large universities, could provide a venue for CUREs. We found that in association with taking these courses, students reported attitudinal gains linked to STEM persistence and that the FYSCURE participant body demographically represented the campus undergraduate population. Here, we describe the successful implementation of twenty-four CUREs spanning a diverse range of topics through the FYS program at a large research institution.
Abstract Background: Categorizing breast cancer HER2/ERBB2 expression as “positive” or “negative” is no longer sufficient, with evidence that treatment response and outcomes are associated with HER2 ...“low” status and HER2 intratumoral heterogeneity. We hypothesized that interrogating HER2 heterogeneity (HER2het) across multiple spatial resolutions would more accurately capture HER2 diversity and be associated with clinical outcomes. Methods: We interrogated tumor cell and microenvironmental features by profiling 1,113,204 single cells in tissue sections from 171 HER2+/HER2low cancers via custom 25-marker high dimensional multiplexed immunofluorescence (HDmIF) using NeoGenomics MultiOmyx, with adjacent section HER2 immunohistochemistry (IHC). We developed novel metrics to concurrently: 1) interrogate HER2 heterogeneity at four spatial resolutions; 2) use machine learning to translate HER2 IF to IHC, termed ‘HAIQu’ (HER2 Automated Immunofluorescence Quantification), scoring HER2 IF expression according to ASCO/CAP guidelines; 3) delineate HER2 signaling phenotypes at the tumor cell level based on six HER2-related proteins; 4) evaluate immunophenotype of 23 immune cell types. We evaluated the association of these novel HER2het metrics with patient clinicopathologic features, recurrence-free survival (RFS), overall survival (OS), and diverse antibody markers representing tumor cell intrinsic processes and tumor-immune microenvironment (TME). Results: 1166 regions of interest were analyzed from 208 unique tumors profiled. Median follow-up from diagnosis was 143 months and 98.9% (n=183/185) received HER2-directed therapy in the (neo)adjuvant or metastatic setting. Our HAIQu scoring system effectively translated adjacent section HER2 IF to IHC with 97.9% concordance between HAIQu and clinical IHC scoring. Single-cell phenotypic analyses of 392,984 HER2+/PanCK+ tumor cells’ concurrent expression of six HER2-positive breast cancer related proteins (HER2, HER3, EGFR, pAKT, ER, KI67) using an unsupervised neural network-based self-organizing map approach resulted in 7 HER2 signaling cell phenotypes. Most patient samples are dominated by a single cell cluster but, intriguingly, Cluster 1 cells (EGFR-low) predominate in tumors with high HER2 cell membrane heterogeneity (ANOVA p=0.003). Evaluation of immunophenotype in hormone receptor-negative, HER2+ tumors, demonstrated significant association with immune cluster and recurrence-free survival (RFS; log-rank p=0.024) with zero RFS events among immune-high versus median survival of only 53.5mo among immune low-PDL1 low tumors. A multivariable Cox proportional hazards model including single cell HER2-heterogeneity (only significant metric on univariate), receptor subtype, and immunophenotype cluster demonstrated significant association with RFS (overall model log-rank p=0.005) and each significantly contributed to the model (all p< 0.05). Conclusions: We present novel metrics of HER2 heterogeneity via HDmIF, which offer detailed characterization of the diversity of HER2 expression in a large, clinically-annotated cohort with long-term follow-up. Identification of a strong association between immunophenotype and RFS supports further investigation of the highly immune activated subsets of ER-/HER2+ breast cancer. Strong correspondence of HER2 IF and IHC and our HAIQu methodology offers a pathway to translation of HER2het metrics to clinical practice. Table 1. Association of HER2 Heterogeneity Metrics with Recurrence-Free Survival Citation Format: David Tallman, Anna Juncker-Jensen, Harry Nunns, Kevin Gallagher, Heather LeFebvre, Karen Yamamoto, Katharine Collier, Mark Vater, Ava Strahan, Mathew Cherian, Ashley Pariser, Preeti Sudheendra, Bhuvaneswari Ramaswamy, Margaret Gatti-Mays, Ainura Kyshtoobayeva, Zaibo Li, Daniel Stover, Kai Johnson. Novel Metrics of HER2 Heterogeneity in HER2-Positive and HER2-Low Breast Cancer via High Dimensional Multiplexed Immunofluorescence Spatial Profiling abstract. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-03.
Abstract
Background: Breast cancer is characterized by distinct molecular subtypes based on expression of estrogen and progesterone hormone receptors (ER and PR), and epidermal growth factor receptor ...2 (HER2). To investigate presence and location of distinct immune cell populations on single cell level we utilized the multiplexed immunofluorescence (mIF) platform MultiOmyx to investigate the tumor immune microenvironment (TME) in HER2+ breast cancer. We have built on growing data implicating distinct immunophenotypes in the breast cancer TME with breast cancer outcomes by profiling 1) prevalence; 2 location (e.g. intratumoral, stromal); 3) phenotype (e.g. activated, exhausted) of infiltrating immune cells.
Methods: We optimized a custom 26-marker MultiOmyx panel interrogating HER2 IF expression, HER2 signaling, and immune markers. This mIF platform leverages serial IF image capture to allow concurrent profiling of all 26 markers on a pathologic section at single cell resolution. We applied the 26-marker panel to a tissue microarray of 208 unique patients with matched tumor/normal tissue cores (1-4 cores/patient; total 333 tumor and 307 normal cores). HER2-positive was defined via ASCO/CAP guidelines; HER2-low was defined as HER2 immunohistochemistry (IHC) 1+/2+ but HER2 in-situ hybridization (ISH) negative.
Results: The 208 unique tumors profiled included 88.9% (185/208) HER2-positive and 11.1% (23/208) HER2-low; 62.5% (130/208) hormone receptor (HR) positive and 37.5% (78/208) HR negative. Median follow-up from diagnosis was 143 months. Among HER2-positive patients, 98.9% (n=183/185) received HER2-directed therapy in the (neo)adjuvant or metastatic setting or were diagnosed prior to FDA approval of trastuzumab for early stage disease. In sum, from 1166 regions of interest in 640 total cores, a total of 1,076,700 single cells were profiled via the 26-marker panel. Tumors categorized based on HR and HER2 reflected distinct immunophenotype. Relative to HR+HER2low, HR-HER2low tumors had a significant increase in the density of T cells (CD3) and tumor-associated macrophages (TAMs) (CD68). The increase in T cells was observed for T helper (CD3+CD4+), T regulatory (CD3+CD4+FoxP3+), as well as T cytotoxic cells (CD3+CD8+). Observed increases in pro-tumorigenic M2 TAM density (CD68+CD163+) were observed for both HR negative subtypes when compared to HR+HER2+ and HR+HER2low groups, indicating a negative correlation between M2 TAM infiltration and ER/PR status. HER2low tumors had significantly lower tumor PDL1+ than HER2+ tumors. Additionally, 18 of the 208 patients with equal distribution across the hormone-receptor subtypes underwent whole slide multiplexed analysis to perform a deeper spatial analysis.
Conclusions: In a large, clinically annotated cohort of breast cancers distinct immunophenotypes are evident among HR and HER2+ subsets.
Citation Format: Anna Juncker-Jensen, David Tallman, Harry Nunns, Heather Lefebvre, Karen Yamamoto, Katharine A. Collier, Mark Vater, Ava Strahan, Ava Willoughby, Olivia Bouchard, Madison Kingsbury, Mathew Cherian, Ashley C. Pariser, Preeti K. Sudheendra, Bhuvaneswari Ramaswamy, Margaret Gatti-Mays, Ainura Kyshtoobayeva, Zaibo Li, Daniel G. Stover. Single-cell immunoprofiling and spatial analysis of hormone receptor subtypes in HER2+ and HER2low breast tumors using multiplexed immunofluorescence. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4639.
PDF
