Characterizing control of memory CD8 T cell differentiation by BTB-ZF transcription factor Zbtb20.

Members of the BTB-ZF transcription factor family regulate the immune system. Our laboratory identified that family member Zbtb20 contributes to the differentiation, recall responses, and metabolism of CD8 T cells. Here, we report a characterization of the transcriptional and epigenetic signatures controlled by Zbtb20 at single-cell resolution during the effector and memory phases of the CD8 T cell response. Without Zbtb20, transcriptional programs associated with memory CD8 T cell formation were up-regulated throughout the CD8 T response. A signature of open chromatin was associated with genes controlling T cell activation, consistent with the known impact on differentiation. In addition, memory CD8 T cells lacking Zbtb20 were characterized by open chromatin regions with overrepresentation of AP-1 transcription factor motifs and elevated RNA- and protein-level expressions of the corresponding AP-1 components. Finally, we describe motifs and genomic annotations from the DNA targets of Zbtb20 in CD8 T cells identified by cleavage under targets and release under nuclease (CUT&RUN). Together, these data establish the transcriptional and epigenetic networks contributing to the control of CD8 T cell responses by Zbtb20.

Science Scholars: Integrating Scientific Research Into Undergraduate Medical Education Through a Comprehensive Student-Led Preclinical Elective.

Introduction: One of the goals of evidence-based medical education is to familiarize future health care practitioners with the scientific method so they can interpret scholarly literature and communicate appropriately with patients. However, many students lack the skills necessary to conduct research themselves. We describe a preclinical elective course designed to equip students with these skills through workshops, mentorship, and research experience. Methods: Through an application process, we selected first-year medical (M1) students who expressed interest in conducting basic, translational, or clinical research. Throughout the yearlong curriculum, students attended a series of 10 1-hour workshops to learn the skills necessary to engage in research. Additionally, each student was paired with a peer mentor. As their final project, students completed a specific aims page based on their projected research study. Results: Over the course of 3 years, 96% of students secured a research position for the summer following M1, and 36% secured positions at external institutions with nationally competitive funding, compared to 10% of their peers who did not participate in the elective. Of students, 80% indicated that this elective helped them find and secure these research positions, and 75% of students reported that they learned valuable skills not taught in their medical curriculum. Discussion: Participation in a preclinical research elective can provide immediate value in the form of research skills with the prospect of stimulating a lifelong interest in scientific inquiry. Our curriculum was delivered in a medical school setting, however it is applicable to any health care professional school.

Tumor immune infiltration estimated from gene expression profiles predicts colorectal cancer relapse.

A substantial fraction of patients with stage I-III colorectal adenocarcinoma (CRC) experience disease relapse after surgery with curative intent. However, biomarkers for predicting the likelihood of CRC relapse have not been fully explored. Therefore, we assessed the association between tumor infiltration by a broad array of innate and adaptive immune cell types and CRC relapse risk. We implemented a discovery-validation design including a discovery dataset from Moffitt Cancer Center (MCC; Tampa, FL) and three independent validation datasets: (1) GSE41258 (2) the Molecular Epidemiology of Colorectal Cancer (MECC) study, and (3) GSE39582. Infiltration by 22 immune cell types was inferred from tumor gene expression data, and the association between immune infiltration by each cell type and relapse-free survival was assessed using Cox proportional hazards regression. Within each of the four independent cohorts, CD4+ memory activated T cell (HR: 0.93, 95% CI: 0.90-0.96; FDR = 0.0001) infiltration was associated with longer time to disease relapse, independent of stage, microsatellite instability, and adjuvant therapy. Based on our meta-analysis across the four datasets, 10 innate and adaptive immune cell types associated with disease relapse of which 2 were internally validated using multiplex immunofluorescence. Moreover, immune cell type infiltration was a better predictors of disease relapse than Consensus Molecular Subtype (CMS) and other expression-based biomarkers (Immune-AICMCC:238.1-238.9; CMS-AICMCC: 241.0). These data suggest that transcriptome-derived immune profiles are prognostic indicators of CRC relapse and quantification of both innate and adaptive immune cell types may serve as candidate biomarkers for predicting prognosis and guiding frequency and modality of disease surveillance.

Zbtb20 Restrains CD8 T Cell Immunometabolism and Restricts Memory Differentiation and Antitumor Immunity.

CD8 T cell differentiation is orchestrated by dynamic metabolic changes that direct activation, proliferation, cytotoxic function, and epigenetic changes. We report that the BTB-ZF family transcriptional repressor Zbtb20 negatively regulates CD8 T cell metabolism and memory differentiation in mice. Effector and memory CD8 T cells with conditional Zbtb20 deficiency displayed enhanced mitochondrial and glycolytic metabolism, and memory CD8 T cells had enhanced spare respiratory capacity. Furthermore, Zbtb20-deficient CD8 T cells displayed increased flexibility in the use of mitochondrial fuel sources. Phenotypic and transcriptional skewing toward the memory fate was observed during the CD8 T cell response to Listeria monocytogenes Memory cells mounted larger secondary responses and conferred better protection following tumor challenge. These data suggest that inactivation of Zbtb20 may offer an approach to enhance metabolic activity and flexibility and improve memory CD8 T cell differentiation, useful attributes for T cells used in adoptive immunotherapy.

The tumor microenvironment of colorectal cancer metastases: opportunities in cancer immunotherapy.

About a fifth of individuals with colorectal cancer (CRC) present with disease metastasis at the time of diagnosis. While the role of the tumor microenvironment (TME) in governing CRC progression is undeniable, the role of the TME in either establishing or suppressing the formation of distant metastases of CRC is less well established. Despite advances in immunotherapy, many individuals with metastatic CRC do not respond to standard-of-care therapy. Therefore, understanding the role of the TME in establishing distant metastases is essential for developing new immunological agents. Here, we summarize our current understanding of the TME of CRC metastases, describe differences between the TME of primary tumors and their distant metastases, and discuss advances in the design and combinations of immunotherapeutic agents.

Could aflatoxin B1 production by Aspergillus flavus affect the severity of keratitis: an experience in two tertiary health care centers, Egypt.

To investigate the expression of AFB1 gene in isolates obtained from corneal scrapping samples from keratitis patients and to correlate the quantity of AFB1 to the severity of keratitis. An observational study was undertaken in Medical Microbiology and Immunology department, Mansoura University, Egypt, over corneal scrapping samples that were cultured aiming to isolate fungal causes of infective keratitis followed by AFB1 gene detection in Aspergillus flavus isolates by nested PCR then quantitation of the toxin by TLC. Out of 843 corneal scrapping samples collected from patients with infective keratitis, positive fungal growth was identified in 277 cases (32.9%). A. flavus was the commonest fungal agent isolated in 93 cases (33.6%). The AFB1 toxin-encoding gene was detected in 63.4% of A. flavus isolates. There was a positive correlation between the quantity of produced AFB1 toxin and the degree of severity of keratitis (P value < 0.0001*). Aspergillus flavus was the most common cause of fungal keratitis, with the AFB1 toxin-encoding gene detected in more than half of the isolates. A significant correlation between the degree of severity of keratitis and the quantity of produced AFB1 toxin was detected. Therefore, exploring presence or absence of AFB1 toxin is important for the clinicians in their diagnostic assessment and selection of proper treatment choices.

Transcriptomic Differences between Primary Colorectal Adenocarcinomas and Distant Metastases Reveal Metastatic Colorectal Cancer Subtypes.

Approximately 20% of colorectal cancer patients with colorectal adenocarcinomas present with metastases at the time of diagnosis, and therapies that specially target these metastases are lacking. We present a novel approach for investigating transcriptomic differences between primary colorectal adenocarcinoma and distant metastases, which may help to identify primary tumors with high risk for future dissemination and to inform the development of metastasis-targeted therapies. To effectively compare the transcriptomes of primary colorectal adenocarcinoma and metastatic lesions at both the gene and pathway levels, we eliminated tissue specificity of the "host" organs where tumors are located and adjusted for confounders such as exposure to chemotherapy and radiation, and identified that metastases were characterized by reduced epithelial-mesenchymal transition (EMT) but increased MYC target and DNA-repair pathway activities. FBN2 and MMP3 were the most differentially expressed genes between primary tumors and metastases. The two subtypes of colorectal adenocarcinoma metastases that were identified, EMT inflammatory and proliferative, were distinct from the consensus molecular subtype (CMS) 3, suggesting subtype exclusivity. In summary, this study highlights transcriptomic differences between primary tumors and colorectal adenocarcinoma metastases and delineates pathways that are activated in metastases that could be targeted in colorectal adenocarcinoma patients with metastatic disease. SIGNIFICANCE: These findings identify a colorectal adenocarcinoma metastasis-specific gene-expression signature that is free from potentially confounding background signals coming from treatment exposure and the normal host tissue that the metastasis is now situated within.

Quantification of Subdural Electrode Shift Between Initial Implantation, Postimplantation Computed Tomography, and Subsequent Resection Surgery.

BACKGROUND: Subdural electrodes are often implanted for localization of epileptic regions. Postoperative computed tomography (CT) is typically acquired to locate electrode positions for planning any subsequent surgical resection. Electrodes are assumed to remain stationary between CT acquisition and resection surgery. OBJECTIVE: To quantify subdural electrode shift that occurred between the times of implantation (Crani 1), postoperative CT acquisition, and resection surgery (Crani 2). METHODS: Twenty-three patients in this case series undergoing subdural electrode implantation were evaluated. Preoperative magnetic resonance and postoperative CT were acquired and coregistered, and electrode positions were extracted from CT. Intraoperative positions of electrodes and the cortical surface were digitized with a coregistered stereovision system. Movement of the exposed cortical surface was also tracked, and change in electrode positions was calculated relative to both the skull and the cortical surface. RESULTS: In the 23 cases, average shift of electrode positions was 8.0 ± 3.3 mm between Crani 1 and CT, 9.2 ± 3.7 mm between CT and Crani 2, and 6.2 ± 3.0 mm between Crani 1 and Crani 2. The average cortical shift was 4.7 ± 1.4 mm with 2.9 ± 1.0 mm in the lateral direction. The average shift of electrode positions relative to the cortical surface between Crani 1 and Crani 2 was 5.5 ± 3.7 mm. CONCLUSION: The results show that electrodes shifted laterally not only relative to the skull, but also relative to the cortical surface, thereby displacing the electrodes from their initial placement on the cortex. This has significant clinical implications for resection based upon seizure activity and functional mapping derived from intracranial electrodes.

Predictors of disease aggressiveness influence outcome from immunotherapy treatment in renal clear cell carcinoma.

Renal clear cell carcinoma (RCC) is the most common type of kidney cancer and has a high propensity for metastasis. While treatment with immune checkpoint inhibitors, such as anti-PD-1, have shown modest improvements in survival for RCC, it is difficult to identify responders from non-responders. Attempts to elucidate the mechanisms associated with differential response to checkpoint inhibitors have been limited by small sample size making it difficult to detect meaningful associations. We utilized existing large datasets from The Cancer Genome Atlas (TCGA) to first find predictors of disease aggressiveness in the tumor microenvironment (TME) and hypothesized that these same predictors may influence response to immunotherapy. We found primary metastatic (M1-stage IV) tumors exhibit high immune infiltration, and high TP53-inactivation induced senescence activity compared to non-metastatic (M0-Stage I/II) tumors. Moreover, some TME features inferred from deconvolution algorithms, which differ between M0 and M1 tumors, also influence overall survival. A focused analysis identified interactions between tumor TP53-inactivation induced senescence activity and expression of inflammatory molecules in pre-treatment RCC tumors, which predict both change in tumor size and response to checkpoint blockade therapy. We also noted frequency of inactivating mutations in the protein polybromo-1 (PBRM1) gene was found to be negatively associated with TP53-inactivation induced senescence enrichment. Our findings suggest a mechanism by which tumor TP53-inactivation induced senescence can modulate the TME and thereby influence outcome from checkpoint blockade therapy.

Embryonic transcription factor expression in mice predicts medial amygdala neuronal identity and sex-specific responses to innate behavioral cues.

The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.

Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei.

BACKGROUND: Neurons in the hypothalamus function to regulate the state of the animal during both learned and innate behaviors, and alterations in hypothalamic development may contribute to pathological conditions such as anxiety, depression or obesity. Despite many studies of hypothalamic development and function, the link between embryonic development and innate behaviors remains unexplored. Here, focusing on the embryonically expressed homeodomain-containing gene Developing Brain Homeobox 1 (Dbx1), we explored the relationship between embryonic lineage, post-natal neuronal identity and lineage-specific responses to innate cues. We found that Dbx1 is widely expressed across multiple developing hypothalamic subdomains. Using standard and inducible fate-mapping to trace the Dbx1-derived neurons, we identified their contribution to specific neuronal subtypes across hypothalamic nuclei and further mapped their activation patterns in response to a series of well-defined innate behaviors. RESULTS: Dbx1-derived neurons occupy multiple postnatal hypothalamic nuclei including the lateral hypothalamus (LH), arcuate nucleus (Arc) and the ventral medial hypothalamus (VMH). Within these nuclei, Dbx1 (+) progenitors generate a large proportion of the Pmch-, Nesfatin-, Cart-, Hcrt-, Agrp- and ERα-expressing neuronal populations, and to a lesser extent the Pomc-, TH- and Aromatase-expressing populations. Inducible fate-mapping reveals distinct temporal windows for development of the Dbx1-derived LH and Arc populations, with Agrp(+) and Cart(+) populations in the Arc arising early (E7.5-E9.5), while Pmch(+) and Hcrt(+) populations in the LH derived from progenitors expressing Dbx1 later (E9.5-E11.5). Moreover, as revealed by c-Fos labeling, Dbx1-derived cells in male and female LH, Arc and VMH are responsive during mating and aggression. In contrast, Dbx1-lineage cells in the Arc and LH have a broader behavioral tuning, which includes responding to fasting and predator odor cues. CONCLUSION: We define a novel fate map of the hypothalamus with respect to Dbx1 expression in hypothalamic progenitor zones. We demonstrate that in a temporally regulated manner, Dbx1-derived neurons contribute to molecularly distinct neuronal populations in the LH, Arc and VMH that have been implicated in a variety of hypothalamic-driven behaviors. Consistent with this, Dbx1-derived neurons in the LH, Arc and VMH are activated during stress and other innate behavioral responses, implicating their involvement in these diverse behaviors.

Serum low-molecular-weight protein fractionation for biomarker discovery.

Protein biomarkers provide the key diagnostic information for the detection of disease, risk of disease progression, and a patient's likely response to drug therapy. Potential biomarkers exist in biofluids, such as serum, urine, and cerebrospinal fluid. Unfortunately, discovering and validating protein biomarkers are hindered by the presence of high-molecular-weight proteins, such as serum albumin and immunoglobulins, which comprise 90% of the proteins present in these samples. High-abundance, high-molecular-weight proteins mask the low-molecular-weight (LMW) proteins and peptides using conventional protein detection methods. Candidate biomarkers are believed to exist in very low concentrations and comprise less than 1% of serum proteins, and may be highly labile as well. Therefore, it is imperative to isolate and enrich LMW proteins from complex mixtures for biomarker discovery. This chapter describes a continuous -elution electrophoresis method, based on molecular weight sieving, to isolate specific molecular weight fractions for mass spectrometric, western blotting, or protein array analysis.