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The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

Stem cell research endeavors to generate specific subtypes of classically defined cell types. Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

IntroductionBrain tumors are the most common solid malignancy and leading cause of cancer-related deaths in infants. Current epidemiological data is limited by low numbers of reported cases. This study used a population-based approach with analysis of contemporary and historical survival curves to provide up-to-date prognostication.MethodsObservational cohort analysis was performed using the Surveillance, Epidemiology and End Results (SEER) database. Infants with brain tumors diagnosed from 1973 to 2013 were categorized by the most common tumor types (diffuse astrocytic and oligodendroglioma, choroid plexus, embryonal, ependymal, medulloblastoma and pilocytic astrocytoma). The 1, 5 and 10 year survival was stratified by decade, with trends in management and outcomes analyzed.ResultsWe identified 2996 affected infants satisfying inclusion criteria. All tumor types, except embryonal and choroid plexus, demonstrated improving survival with time. Infants with embryonal tumors showed a decline in survival from the 1970s to 1990s (p = 0.009), whereas infants with choroid plexus tumors had no change in survival. Infants with ependymal tumors experienced the greatest improvement in survival from 1980s to 1990s and 1990s to 2000s (p = 0.0001, p = 0.01), with 5-year survival probability improving from 28% (95% CI 15-42%) in the 1980s to 77% (95% CI 69-83%) the 2000s. The use of radiation declined from 1970 to 2000 for all tumors; however, radiation treatment for embryonal and ependymal subtypes increased after 2000.ConclusionsWhile overall survival for infants with brain tumors has improved from the 1970s onwards, not every tumor type has seen a statistically significant change. Given changes in management and survival, prognostication of infants with brain tumor should be updated.

[This corrects the article DOI: 10.1371/journal.pone.0223051.].

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

Life-threatening acute liver failure can be triggered by a variety of factors, including common drugs such as acetaminophen. Positron emission tomography (PET) is rarely used to monitor liver function, in part because of a lack of specific imaging agents for liver function. Here we report a new PET probe, 2-deoxy-2-[(18)F]fluororibose ([(18)F]-2-DFR), for use in imaging liver function. [(18)F]-2-DFR was synthesized and validated as a competitive substrate for the ribose salvage pathway. [(18)F]-2-DFR was prepared through an efficient late stage radiofluorination. The desired selectivity of fluorination was achieved using an unorthodox protecting group on the precursor, which could withstand harsh SN2 reaction conditions with no side reactions. [(18)F]-2-DFR accumulated preferentially in the liver and was metabolized by the same enzymes as ribose. [(18)F]-2-DFR could distinguish between healthy liver and liver damaged by acetaminophen. [(18)F]-2-DFR is expected to be a useful PET probe for imaging and quantifying liver functions in vivo, with likely significant clinical utility.

PET is a powerful technique for quantifying and visualizing biochemical pathways in vivo. Here, we develop and validate a novel PET probe, [(18)F]-2-deoxy-2-fluoroarabinose ([(18)F]DFA), for in vivo imaging of ribose salvage. DFA mimics ribose in vivo and accumulates in cells following phosphorylation by ribokinase and further metabolism by transketolase. We use [(18)F]DFA to show that ribose preferentially accumulates in the liver, suggesting a striking tissue specificity for ribose metabolism. We demonstrate that solute carrier family 2, member 2 (also known as GLUT2), a glucose transporter expressed in the liver, is one ribose transporter, but we do not know if others exist. [(18)F]DFA accumulation is attenuated in several mouse models of metabolic syndrome, suggesting an association between ribose salvage and glucose and lipid metabolism. These results describe a tool for studying ribose salvage and suggest that plasma ribose is preferentially metabolized in the liver.

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8α, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NFκB, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3ζ, a subunit of TCR complex; however, overexpression of CD3ζ rescues the phenotype, suggesting that TMX1 is not required for CD3ζ function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3δ, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3δ leads to impaired TCR assembly and subsequent CD3ζ destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)