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We present a chromosome-length genome assembly and annotation of the Black Petaltail dragonfly (Tanypteryx hageni). This habitat specialist diverged from its sister species over 70 million years ago, and separated from the most closely related Odonata with a reference genome 150 million years ago. Using PacBio HiFi reads and Hi-C data for scaffolding we produce one of the most high quality Odonata genomes to date. A scaffold N50 of 206.6 Mb and a BUSCO score of 96.8% indicate high contiguity and completeness.SignificanceWe provide a chromosome-length assembly of the Black Petaltail dragonfly (Tanypteryx hageni), the first genome assembly for any non-libelluloid dragonfly. The Black Petaltail diverged from its sister species over 70 million years ago.T. hageni, like its confamilials, occupies fen habitats in its nymphal stage, a life history uncommon in the vast majority of dragonflies. We hope that the availability of this assembly will facilitate research onT. hageniand other petaltail species, to better understand their ecology and support conservation efforts.

Evaporative cooling is an energy efficient form of air conditioning in dry climates that functions by pulling hot, dry outdoor air across a wet evaporative pad. While evaporative coolers can help save energy, they also have the potential to influence human health. Studies have shown residential evaporative coolers may pull outdoor air pollutants into the home or contribute to elevated levels of indoor bioaerosols that may be harmful to health. There is also evidence that evaporative coolers can enable a diverse microbial environment that may confer early-life immunological protection against the development of allergies and asthma or exacerbate these same hypersensitivities. This review summarizes the current knowledge of bioaerosol and microbiological studies associated with evaporative coolers, focusing on harmful and potentially helpful outcomes from their use. We evaluate the effects of evaporative coolers on indoor bacterial endotoxins, fungal β-(1 → 3)-D-glucans, dust mite antigens, residential microbial communities, and Legionella pneumophila. To our knowledge, this is the first review to summarize and evaluate studies on the influence that evaporative coolers have on the bioaerosol and microbiological profile of homes. This brings to light a gap in the literature on evaporative coolers, which is the lack of data on health effects associated with their use.

Graves’ disease (GD) is a thyroid-specific autoimmune disease with a high prevalence worldwide. The disease is primarily mediated by B cells, which produce autoantibodies against the thyroid-stimulating hormone receptor (TSHR), chronically stimulating it and leading to high levels of thyroid hormones in the body. Interest in characterizing the immune response in GD has motivated many phenotyping studies. The immunophenotype of the cells involved and the interplay between them and their secreted factors are crucial to understanding disease progression and future treatment options. T cell populations are markedly distinct, including increased levels of Th17 and follicular helper T cells (Tfh), while Treg cells appear to be impaired. Some B cells subsets are autoreactive, and anti-TSHR antibodies are the key disease-causing outcome of this interplay. Though some consensus across phenotyping studies will be discussed here, there are also complexities that are yet to be resolved. A better understanding of the immunophenotype of Graves’ disease can lead to improved treatment strategies and novel drug targets.

Periodontitis is a chronic inflammatory oral disease that affects approximately 42% of adults 30 years of age or older in the United States. In response to microbial dysbiosis within the periodontal pockets surrounding teeth, the host immune system generates an inflammatory environment in which soft tissue and alveolar bone destruction occur. The objective of this study was to identify diagnostic biomarkers and the mechanistic drivers of inflammation in periodontitis to identify drugs that may be repurposed to treat chronic inflammation. A meta-analysis comprised of two independent RNA-seq datasets was performed. RNA-seq analysis, signal pathway impact analysis, protein-protein interaction analysis, and drug target analysis were performed to identify the critical pathways and key players that initiate inflammation in periodontitis as well as to predict potential drug targets. Seventy-eight differentially expressed genes, 10 significantly impacted signaling pathways, and 10 hub proteins in periodontal gingival tissue were identified. The top 10 drugs that may be repurposed for treating periodontitis were then predicted from the gene expression and pathway data. The efficacy of these drugs in treating periodontitis has yet to be investigated. However, this analysis indicates that these drugs may serve as potential therapeutics to treat inflammation in gingival tissue affected by periodontitis.

A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer’s disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A (ACKR2-V41A; rs2228467). High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation (β-Arrestin-dependent signaling pathway) upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD.

Obesity is an established risk factor for many cancers and has recently been found to alter the efficacy of T cell–based immunotherapies. Currently, however, the effects of obesity on immunometabolism remain unclear. Understanding these associations is critical, given the fact that T cell metabolism is tightly linked to effector function. Thus, any obesity-associated changes in T cell bioenergetics are likely to drive functional changes at the cellular level, alter the metabolome and cytokine/chemokine milieu, and impact cancer immunotherapy outcomes. Here, we provide a brief overview of T cell metabolism in the presence and absence of solid tumor growth and summarize current literature regarding obesity-associated changes in T cell function and bioenergetics. We also discuss recent findings related to the impact of host obesity on cancer immunotherapy outcomes and present potential mechanisms by which T cell metabolism may influence therapeutic efficacy. Finally, we describe promising pharmaceutical therapies that are being investigated for their ability to improve CD8 T cell metabolism and enhance cancer immunotherapy outcomes in patients, regardless of their obesity status.

Human pathogens belonging to theAlphavirusgenus, in theTogaviridaefamily, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.

We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes—all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.

Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined “trained immunity” and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The “exposome” is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

Background Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. Methods We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. Results Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73–66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. Conclusions The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

Since the completion of the Human Genome Project in 2003, genomic sequencing has become a prominent tool used by diverse disciplines in modern science. In the past 20 years, the cost of genomic sequencing has decreased exponentially, making it affordable and accessible. Bioinformatic and biological studies have produced significant scientific breakthroughs using the wealth of genomic information now available. Alongside the scientific benefit of genomics, companies offer direct-to-consumer genetic testing which provide health, trait, and ancestry information to the public. A key area that must be addressed is education about what conclusions can be made from this genomic information and integrating genomic education with foundational genetic principles already taught in academic settings. The promise of personal genomics providing disease treatment is exciting, but many challenges remain to validate genomic predictions and diagnostic correlations. Ethical and societal concerns must also be addressed regarding how personal genomic information is used. This genomics revolution provides a powerful opportunity to educate students, clinicians, and the public on scientific and ethical issues in a personal way to increase learning. In this review, we discuss the influence of personal genomics in society and focus on the importance and benefits of genomics education in the classroom, clinics, and the public and explore the potential consequences of personal genomic education.

Evita G Weagel,1 Wei Meng,1 Michelle H Townsend,1 Edwin J Velazquez,1 Rachel A Brog,1 Michael W Boyer,2 K Scott Weber,1 Richard A Robison,1 Kim L O’Neill1 1Department of Microbiology and Molecular Biology, Brigham Young University, Provo, 2Division of Hematology and Hematologic Malignancies, Department of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA Abstract: TK1 is an enzyme involved in DNA synthesis and repair. TK1 is usually found elevated in cancer patients’ serum, which makes it a useful tumor proliferation biomarker that strongly correlates with cancer stage, metastatic capabilities, and recurrence risk. In this study, we show that TK1 is upregulated and localizes on the plasma membrane of Burkitt’s lymphoma, acute promyelocytic leukemia, T cell leukemia, and acute lymphoblastic leukemia (ALL). Using flow cytometry, we confirmed that TK1 localizes on the surface of Raji, HL60, and Jurkat cell lines and on ALL clinical samples. Using fluorescent microscopy, we found a strong association of TK1 with the plasma membrane in Raji, HL60, and Jurkat cell lines. These findings were also confirmed by scanning electron microscopy. Our study also shows that this phenomenon does not occur on normal resting or proliferating lymphocytes. In addition, we show that membrane TK1 is found in all oligomeric forms ranging from monomer to tetramer and exhibits enzymatic activity. These findings suggest TK1 as a possible target for immunotherapy with the potential to be utilized in the treatment of hematological cancers. Keywords: Burkitt’s lymphoma, acute lymphoblastic leukemia, ALL, thymidine kinase 1, surface antigen

House dust mites are typically absent in homes in arid and semiarid climates due to low humidity. Evaporative "swamp" cooling significantly increases indoor humidity in dry climates and is suspected of promoting dust mite survival in these regions. We investigated the prevalence and concentration of mite allergens in dust from low-income homes (N = 22) with evaporative coolers in Utah County, Utah. Overall, 15 homes (68.2%) were positive for either Der p 1 or Der f 1 in at least 1 location. Geometric mean allergen levels in mattresses were 0.107 and 0.087 ug/g dust for Der p 1 and Der f 1, respectively. In furniture, levels were 0.143 and 0.165 ug/g dust for Der p1 and Der f 1, respectively. The percentage of positive homes in this study was much higher than previously reported in larger homes with swamp coolers in the same community. These results suggest socioeconomic factors may play a role in dust mite allergen prevalence in homes with evaporative coolers in dry climates.

Recent work suggests that evaporative coolers increase the level and diversity of bioaerosols, but this association remains understudied in low-income homes. We conducted a cross-sectional study of metropolitan, low-income homes in Utah with evaporative coolers (n = 20) and central air conditioners (n = 28). Dust samples (N = 147) were collected from four locations in each home and analyzed for dust-mite allergens Der p1 and Der f1, endotoxins, and β-(1 → 3)-d-glucans. In all sample locations combined, Der p1 or Der f1 was significantly higher in evaporative cooler versus central air conditioning homes (OR = 2.29, 95% CI = 1.05-4.98). Endotoxin concentration was significantly higher in evaporative cooler versus central air conditioning homes in furniture (geometric mean (GM) = 8.05 vs 2.85 EU/mg, P

Flow cytometry is a versatile and high throughput technique for rapid and efficient biological testing. It requires a high level of conceptual, technical, and analytical skills to properly design experiments, effectively operate flow cytometry machines, and analyze the data. A lack of training and development of any of these three skills can result in underutilization and improper use of flow cytometric machines that can impede research progress. Often students develop these conceptual, technical, and analysis skills from trial and error, but many students either do not use this powerful flow cytometry technology, use it improperly or ineffectively, or give up using it without proper training and support. Here we report on a course which teaches flow cytometry skills to undergraduate and graduate students. The design of this course is unique in that it teaches conceptual, technical, and analytical skills related to flow cytometry in a full semester format. Undergraduate and graduate students reported significant increases in their confidence levels over the course of the semester. Here we provide our findings and resources for others who may want to implement a similar course.

Flavonoids are dietary compounds with potential anti-diabetes activities. Many flavonoids have poor bioavailability and thus low circulating concentrations. Unabsorbed flavonoids are metabolized by the gut microbiota to smaller metabolites, which are more bioavailable than their precursors. The activities of these metabolites may be partly responsible for associations between flavonoids and health. However, these activities remain poorly understood. We investigated bioactivities of flavonoid microbial metabolites [hippuric acid (HA), homovanillic acid (HVA), and 5-phenylvaleric acid (5PVA)] in primary skeletal muscle and β-cells compared to a native flavonoid ([(−)-epicatechin, EC]. In muscle, EC was the most potent stimulator of glucose oxidation, while 5PVA and HA simulated glucose metabolism at 25 μM, and all compounds preserved mitochondrial function after insult. However, EC and the metabolites did not uncouple mitochonndrial respiration, with the exception of 5PVA at10 μM. In β-cells, all metabolites more potently enhanced glucose-stimulated insulin secretion (GSIS) compared to EC. Unlike EC, the metabolites appear to enhance GSIS without enhancing β-cell mitochondrial respiration or increasing expression of mitochondrial electron transport chain components, and with varying effects on β-cell insulin content. The present results demonstrate the activities of flavonoid microbial metabolites for preservation of β-cell function and glucose utilization. Additionally, our data suggest that metabolites and native compounds may act by distinct mechanisms, suggesting complementary and synergistic activities in vivo which warrant further investigation. This raises the intriguing prospect that bioavailability of native dietary flavonoids may not be as critical of a limiting factor to bioactivity as previously thought.

Calcium influx is critical for T cell effector function and fate. T cells are activated when T cell receptors (TCRs) engage peptides presented by antigen-presenting cells (APC), causing an increase of intracellular calcium (Ca2+) concentration. Co-receptors stabilize interactions between the TCR and its ligand, the peptide-major histocompatibility complex (pMHC), and enhance Ca2+ signaling and T cell activation. Conversely, some co-receptors can dampen Ca2+ signaling and inhibit T cell activation. Immune checkpoint therapies block inhibitory co-receptors, such as cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), to increase T cell Ca2+ signaling and promote T cell survival. Similar to CTLA-4 and PD-1, the co-receptor CD5 has been known to act as a negative regulator of T cell activation and to alter Ca2+ signaling and T cell function. Though much is known about the role of CD5 in B cells, recent research has expanded our understanding of CD5 function in T cells. Here we review these recent findings and discuss how our improved understanding of CD5 Ca2+ signaling regulation could be useful for basic and clinical research.

A critical area of emphasis for science educators is the identification of effective means of teaching and engaging undergraduate students. Personal microbiome analysis is a means of identifying the microbial communities found on or in our body. We hypothesized the use of personal microbiome analysis in the classroom could improve science education by making courses more applied and engaging for undergraduate students. We determined to test this prediction in three Brigham Young University undergraduate courses: Immunology, Advanced Molecular Biology Laboratory, and Genomics. These three courses have a two-week microbiome unit and students during the 2016 semester students could submit their own personal microbiome kit or use the demo data, whereas during the 2017 semester students were given access to microbiome data from an anonymous individual. The students were surveyed before, during, and after the human microbiome unit to determine whether analyzing their own personal microbiome data, compared to analyzing demo microbiome data, impacted student engagement and interest. We found that personal microbiome analysis significantly enhanced the engagement and interest of students while completing microbiome assignments, the self-reported time students spent researching the microbiome during the two week microbiome unit, and the attitudes of students regarding the course overall. Thus, we found that integrating personal microbiome analysis in the classroom was a powerful means of improving student engagement and interest in undergraduate science courses.

Education research within the STEM disciplines applies a scholarly approach to teaching and learning, with the intent of better understanding how people learn and of improving pedagogy at the undergraduate level. Most of the professionals practicing in this field have ‘crossed over’ from other disciplinary fields and thus have faced challenges in becoming experts in a new discipline. In this article, we offer a novel framework for approaching education research design called Backward Design in Education Research. It is patterned on backward curricular design and provides a three-step, systematic approach to designing education projects: 1) Define a research question that leads to a testable causal hypothesis based on a theoretical rationale; 2) Choose or design the assessment instruments to test the research hypothesis; and 3) Develop an experimental protocol that will be effective in testing the research hypothesis. This approach provides a systematic method to develop and carry out evidence-based research design.

Background Lung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1’s potential as a tumor biomarker and immunotherapeutic target and clinical relevance. Methods We assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1’s clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients. Results We found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1’s localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients. Conclusions Our results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer. Electronic supplementary material The online version of this article (10.1186/s12935-018-0633-9) contains supplementary material, which is available to authorized users.

The goal of an undergraduate laboratory course should be not only to introduce the students to biology methodologies and techniques, but also to teach them independent analytical thinking skills and proper experiment design. This is especially true for advanced biology laboratory courses that undergraduate students typically take as a junior or senior in college. Many courses achieve the goal of teaching techniques, but fail to approach the larger goal of teaching critical thinking, experimental design, and student independence. Here we describe a study examining the application of the scaffolding instructional philosophy in which students are taught molecular techniques with decreasing guidance to force the development of analytical thinking skills and prepare undergraduate students for independent laboratory research. This method was applied to our advanced molecular biology laboratory class and resulted in an increase of confidence among the undergraduate students in their abilities to perform independent research.

Michelle H Townsend, Michael D Anderson, Evita G Weagel, Edwin J Velazquez, K Scott Weber, Richard A Robison, Kim L O’Neill Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, USA Abstract: In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. Toexpand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines. Keywords: non-small-cell lung cancer, NCI-H460, A549, surface antigen, hypoxanthine guanine phosphoribosyltransferase

The adaptive immune response is orchestrated by T helper cells and their function is dependent upon interactions between the T cell receptor (TCR), peptide MHC (pMHC) and co-receptors. TCR-pMHC interactions initiate calcium signaling cascades which determine T cell activation, survival, proliferation and differentiation. CD5 is a co-receptor that plays an important role in regulating T cell signaling and fate during thymocyte education. CD5 surface expression on mature single positive thymocytes correlates with the TCR signal strength for positive selecting self-ligands. CD5 also plays a role in T cell function after thymic development is complete. Peripheral T cells with higher CD5 expression respond better to foreign antigen than those with lower CD5 expression and CD5-high T cells are enriched in memory populations. In our study, we examined the role of CD5 expression and calcium signaling in the primary response of T cells using two Listeria monocytogenes specific T helper cells (LLO118 and LLO56). These T cells recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have divergent primary and secondary responses and different levels of CD5 expression. We found that each T cell has unique calcium mobilization in response to in vitro stimulation with LLO190-205 and that CD5 expression levels in these cells changed over time following stimulation. LLO56 naïve T helper cells, which expresses higher levels of CD5, have higher calcium mobilization than naïve LLO118 T cells. Three days after in vitro stimulation, LLO118 T cells had more robust calcium mobilization than LLO56 and there were no differences in calcium mobilization 8 days after in vitro stimulation. To further evaluate the role of CD5, we measured calcium signaling in CD5 knockout LLO118 and LLO56 T cells at these three time points and found that CD5 plays a significant role in promoting the calcium signaling of naïve CD5-high LLO56 T cells.

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide–MHC complex. Deprivation of self peptide–MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide–MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic ‘education’ in determining the intrinsic functionality of CD4+ T cells. A r t i c l e s

The purpose of this study was to examine the possible effects that Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) may have on the immune response in the tumor microenvironment. HPRT is a salvage pathway enzyme responsible for recycling guanosine throughout the cell cycle, and we have shown that it is significantly upregulated in numerous solid malignancies. As a necessary component of the S phase in the cell cycle, guanosine has also been regarded as a potential neuroprotectant in the central nervous system (CNS). We thus hypothesized that there may be a correlation between the observed upregulation of HPRT and the relative downregulation of the immune system mediated by the differential regulation of guanosine. Using data from The Cancer Genome Atlas (TCGA), we analyzed over 200 cytokine genes and found a negative correlation between the level of HPRT expression in cancer and the general expression level of both pro-inflammatory and anti-inflammatory cytokine expression. We further analyzed data showing the relative infiltration level of individual immune cell subsets in solid tumors compared with elevated HPRT levels. We found the most significant negative correlations between HPRT expression and immune cell infiltration for B-cells (p < 0.001), CD8+ T-cells (p < 0.01), CD4+ T-cells (p < 0.001), macrophages (p < 0.001), neutrophils (p < 0.001), and dendritic cells (p < 0.001) in lung squamous cell carcinoma. Other significant negative correlations were found in kidney renal clear cell carcinoma, prostate adenocarcinoma, and breast invasive carcinoma. To further analyze this reduction in infiltration, we examined the relationship between HPRT expression and both costimulatory and coinhibitory molecules and found a significant negative correlation. Because of these observed associations between HPRT levels and cytokine expression, we hypothesized that the increased guanosine production was the mechanism by which HPRT was affecting the immune system in the tumor microenvironment. To test this, we examined the level of Ca2+ influx using Fluo-4 assays of B-cell (Raji), T-cell (Jurkat), and monocyte (THP-1) cell lines when treated with guanosine as a measure of overall cell activation. We then compared this activation to untreated cells and cells treated with adenosine, a known anti-inflammatory agent. When treated with guanosine, a significant reduction in activation was found in THP-1s (p < 0.01) and Rajis (p < 0.01), but not in Jurkats. However, under hypoxic and hypoglycemic conditions similar to those commonly found within the tumor microenvironment, Jurkat activation was significantly reduced (p < 0.05) when treated with guanosine. From this data, we conclude that guanosine levels significantly reduce immune cell activation in the tumor microenvironment and that the upregulation of HPRT in malignant tissue may be one of the contributing factors of the immunosuppressive nature of the microenvironment. Citation Format: Michelle H. Townsend, Zachary D. Ewell, Claudia M. Tellez Freitas, Dallas J. Larsen, Eliza L. Bitter, Kelsey B. Bennion, Stephen R. Piccolo, K Scott Weber, Richard A. Robison, Kim L. O'Neill. HPRT overexpression may contribute to the immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2369.

High affinity T cell receptors (TCRs) are absent from the native T cell repertoire. While current hypotheses propose that high affinity TCRs are more sensitive to antigen and have a competitive advantage in immune responses, emerging evidence suggests that higher affinity T cells may reach a threshold where they experience decreased functionality, loss of binding specificity, and become prone to anergy. High affinity TCRs are especially attractive to facilitate immunotherapies as native TCR binding affinity may be too low to effectively initiate activation. However, the effects of increased TCR affinity for CD4+ T cells specific for naturally occurring epitopes is poorly understood. To address this question, we engineered via yeast display several class II-restricted high affinity TCRs specific for a naturally occurring peptide from Listeria monocytogenes protein listeriolysin O. Our high affinity clones have KD values as low as 8.7 nM and half-lives as long as 174 minutes when measured by tetramer dissociation assays. This panel of class II restricted high affinity TCRs specific for a naturally occurring Listeria monocytogenes epitope provides a novel means of testing the role of affinity and CD4+ T cell activation responses in the context of an infection.

Changes in the peptide and MHC molecules have been extensively examined for how they alter T cell activation, but many fewer studies have examined the TCR. Structural studies of how TCR differences alter T cell specificity have focused on broad variation in the CDR3 loops. However, changes in the CDR1 and 2 loops can also alter TCR recognition of pMHC. In this study we focus on two mutations in the CDR1α loop of the TCR that increased the affinity of a TCR for agonist Hb(64-76)/I-E(k) by increasing the on-rate of the reaction. These same mutations also conferred broader recognition of altered peptide ligands. TCR transgenic mice expressing the CDR1α mutations had altered thymic selection, as most of the T cells were negatively selected compared to T cells expressing the wildtype TCR. The few T cells that escaped negative selection and were found in the periphery were rendered anergic, thereby avoiding autoimmunity. T cells with the CDR1α mutations were completely deleted in the presence of Hb(64-76) as an endogenous peptide. Interestingly, the wildtype T cells were not eliminated, identifying a threshold affinity for negative selection where a 3-fold increase in affinity is the difference between incomplete and complete deletion. Overall, these studies highlight how small changes in the TCR can increase the affinity of TCR:pMHC but with the consequences of skewing selection and producing an unresponsive T cell.

Helper T cells are critical for protective immunity, CD8 + T-cell memory, and CD4 + recall responses, but whether the same or distinct CD4 + T cells are involved in these responses has not been established. Here we describe two CD4 + T cells, LLO118 and LLO56, specific for an immunodominant Listeria monocytogenes epitope, with dramatically different responses to primary and secondary infection. Comparing in vivo responses, LLO118 T cells proliferate more strongly to primary infection, whereas surprisingly, LLO56 has a superior CD4 + recall response to secondary infection. LLO118 T cells provide more robust help for CD8 + T-cell responses to secondary infection than LLO56. We found no detectable differences in antigen sensitivity, but naive LLO118 T cells have much lower levels of CD5 and their T-cell receptor levels are dramatically down-regulated after their strong primary response. Thus, distinct CD4 + helper T cells are specialized to help either in primary or secondary responses to infection.

This study aims to evaluate the expression of the salvage pathway enzymes DCK, APRT, and HPRT in colorectal cancer cells to investigate their potential as biomarkers for diagnosis and treatment. As the third most common cancer diagnosed in the United States, there remains a need to identify biomarkers capable of detecting and characterizing colorectal cancers. Because of their involvement in nucleotide synthesis and their role in cell cycle regulation and proliferation, we hypothesized an increase in these enzymes within malignancy. In order to quantify expression, we utilized two colorectal cancer cell lines (HT29 and SW620) as well as healthy and malignant patient tissue samples. Expression in tissues was evaluated using immunohistochemistry (IHC), and surface presence of the enzymes was assessed via confocal microscopy, flow cytometry, and scanning electron microscopy. Staining of malignant tissue samples using IHC showed upregulation of HPRT in 59% of patients when compared to control tissues derived from healthy patients (p=0.001). Surface analysis was performed utilizing flow cytometry, confocal microscopy, and scanning electron microscopy. Upon flow cytometry evaluation no significant presence of either DCK or APRT was found on the membranes of SW620 and HT29 cells (p=0.234 and 0.93, respectively), while HPRT expression was significantly increased on the surface of both SW620 and HT29 cells (p=0.013). Cell population fluorescence increased 58% and 28% in SW620 and HT29 cells, respectively, compared to controls. Confocal microscopy images showed direct overlap between SW620 cells stained with membrane-specific dye and anti-HPRT antibody, suggesting localization on the membrane. To further confirm the surface presence of HPRT, cells were treated with gold-labelled HPRT antibody and visualized under a scanning electron microscope to determine if surface expression was randomly dispersed across the cell surface. When treated with anti-HPRT antibodies, there was an increase in gold weight percentage on both SW620 (p=8.14x10 -6) and HT29 (p=1.74x10 -4) cells compared to controls, confirming HPRT presence and showing random surface localization patterns. Surface analysis was also conducted on malignant samples of patients with colorectal cancer. Of three samples, one of them exhibited significant HPRT surface localization. This supports the variation found within the tissues, as HPRT overexpression is not found in every patient. One patient did exhibit significant levels of HPRT on the surface of their malignant cells; this indicates HPRT as a potential target in patients that exhibit upregulation of the enzyme. These results suggest a relationship between colorectal cancers and HPRT, and indicate HPRT as a potential target for the detection and treatment of colorectal cancers. Citation Format: Michelle H. Townsend, Eric C. Olsen, Evita G. Weagel, Edwin J. Velasquez, Abigail M. Felsted, Weston Burrup, K Scott Weber, Richard Robison, Kim L. O'Neill. HPRT: A biomarker and potential target for detection and treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 771.

T cells are key players in the adaptive immune response and undergo metabolic changes upon activation. CD5 is a co-receptor found on T cells and plays a significant role in regulating T cell thymic development, intracellular signaling and cytokine production. Previous studies have found that naïve T cells with high CD5 expression (CD5hi) have increased TCR signal strength and enhances immune response to foreign peptide in the periphery. Additionally, we have reported that CD5hi naïve T cells have higher calcium mobilization and improved T cell activation compared to CD5lo T cells. Calcium influx levels can modulate and influence metabolic changes in T cells. Thus, we hypothesized that CD5hi, CD5lo and CD5 deficient T cells have different bioenergetic demands that affect metabolic pathways and T cell activation. We evaluated the effects of CD5 levels on metabolism using CD5 deficient mice vs wild type controls and found CD5 deficient T cells had significant differences in metabolic function. Recently published work has described a connection between increased T cell metabolism and altered cognitive function in PD-1 deficient mice. We have also found significant differences between CD5 deficient and wild type mice in marble burying rates, elevated plus and water maze behavior and open field activity. These behavioral test results suggest CD5 deficient mice have altered cognitive function and higher levels of anxiety. Thus, CD5 deficiency alters T cell metabolic and cognitive function.

Protein kinase C-θ (PKCθ) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCθ in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCθ in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCθ in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCθ−/− mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCθ−/− thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCθ−/− mice. Inefficient positive selection of n3.L2 PKCθ−/− CD4 single-positive cells resulted from “weaker” signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCθ−/− mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCθ−/− mice. These findings now place PKCθ as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.