Integrating phylogenies into single-cell RNA sequencing analysis allows comparisons across species, genes, and cells.

Comparisons of single-cell RNA sequencing (scRNA-seq) data across species can reveal links between cellular gene expression and the evolution of cell functions, features, and phenotypes. These comparisons evoke evolutionary histories, as depicted by phylogenetic trees, that define relationships between species, genes, and cells. This Essay considers each of these in turn, laying out challenges and solutions derived from a phylogenetic comparative approach and relating these solutions to previously proposed methods for the pairwise alignment of cellular dimensional maps. This Essay contends that species trees, gene trees, cell phylogenies, and cell lineages can all be reconciled as descriptions of the same concept-the tree of cellular life. By integrating phylogenetic approaches into scRNA-seq analyses, challenges for building informed comparisons across species can be overcome, and hypotheses about gene and cell evolution can be robustly tested.

Antibody-based immunotherapy of cancer.

By targeting surface antigens expressed on tumor cells, monoclonal antibodies have demonstrated efficacy as cancer therapeutics. Recent successful antibody-based strategies have focused on enhancing antitumor immune responses by targeting immune cells, irrespective of tumor antigens. We discuss these innovative strategies and propose how they will impact the future of antibody-based cancer therapy.

The evolution of ovary-biased gene expression in Hawaiian Drosophila.

With detailed data on gene expression accessible from an increasingly broad array of species, we can test the extent to which our developmental genetic knowledge from model organisms predicts expression patterns and variation across species. But to know when differences in gene expression across species are significant, we first need to know how much evolutionary variation in gene expression we expect to observe. Here we provide an answer by analyzing RNAseq data across twelve species of Hawaiian Drosophilidae flies, focusing on gene expression differences between the ovary and other tissues. We show that over evolutionary time, there exists a cohort of ovary specific genes that is stable and that largely corresponds to described expression patterns from laboratory model Drosophila species. Our results also provide a demonstration of the prediction that, as phylogenetic distance increases, variation between species overwhelms variation between tissue types. Using ancestral state reconstruction of expression, we describe the distribution of evolutionary changes in tissue-biased expression, and use this to identify gains and losses of ovary-biased expression across these twelve species. We then use this distribution to calculate the evolutionary correlation in expression changes between genes, and demonstrate that genes with known interactions in D. melanogaster are significantly more correlated in their evolution than genes with no or unknown interactions. Finally, we use this correlation matrix to infer new networks of genes that share evolutionary trajectories, and we present these results as a dataset of new testable hypotheses about genetic roles and interactions in the function and evolution of the Drosophila ovary.

AhR promotes phosphorylation of ARNT isoform 1 in human T cell malignancies as a switch for optimal AhR activity.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform­specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform­specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

The evolution of siphonophore tentilla for specialized prey capture in the open ocean.

Predator specialization has often been considered an evolutionary "dead end" due to the constraints associated with the evolution of morphological and functional optimizations throughout the organism. However, in some predators, these changes are localized in separate structures dedicated to prey capture. One of the most extreme cases of this modularity can be observed in siphonophores, a clade of pelagic colonial cnidarians that use tentilla (tentacle side branches armed with nematocysts) exclusively for prey capture. Here we study how siphonophore specialists and generalists evolve, and what morphological changes are associated with these transitions. To answer these questions, we: a) Measured 29 morphological characters of tentacles from 45 siphonophore species, b) mapped these data to a phylogenetic tree, and c) analyzed the evolutionary associations between morphological characters and prey-type data from the literature. Instead of a dead end, we found that siphonophore specialists can evolve into generalists, and that specialists on one prey type have directly evolved into specialists on other prey types. Our results show that siphonophore tentillum morphology has strong evolutionary associations with prey type, and suggest that shifts between prey types are linked to shifts in the morphology, mode of evolution, and evolutionary correlations of tentilla and their nematocysts. The evolutionary history of siphonophore specialization helps build a broader perspective on predatory niche diversification via morphological innovation and evolution. These findings contribute to understanding how specialization and morphological evolution have shaped present-day food webs.

Evolution of Gene Expression across Species and Specialized Zooids in Siphonophora.

Siphonophores are complex colonial animals, consisting of asexually produced bodies (zooids) that are functionally specialized for specific tasks, including feeding, swimming, and sexual reproduction. Though this extreme functional specialization has captivated biologists for generations, its genomic underpinnings remain unknown. We use RNA-seq to investigate gene expression patterns in five zooids and one specialized tissue across seven siphonophore species. Analyses of gene expression across species present several challenges, including identification of comparable expression changes on gene trees with complex histories of speciation, duplication, and loss. We examine gene expression within species, conduct classical analyses examining expression patterns between species, and introduce species branch filtering, which allows us to examine the evolution of expression across species in a phylogenetic framework. Within and across species, we identified hundreds of zooid-specific and species-specific genes, as well as a number of putative transcription factors showing differential expression in particular zooids and developmental stages. We found that gene expression patterns tended to be largely consistent in zooids with the same function across species, but also some large lineage-specific shifts in gene expression. Our findings show that patterns of gene expression have the potential to define zooids in colonial organisms. Traditional analyses of the evolution of gene expression focus on the tips of gene phylogenies, identifying large-scale expression patterns that are zooid or species variable. The new explicit phylogenetic approach we propose here focuses on branches (not tips) offering a deeper evolutionary perspective into specific changes in gene expression within zooids along all branches of the gene (and species) trees.

Electroencephalography in young onset dementia.

BACKGROUND: Young onset dementia (YOD) is a major diagnostic and management problem. METHODS: We set out to explore if electroencephalography (EEG) might be useful in the diagnosis of young onset Alzheimer's disease (YOAD) and young onset frontotemporal dementia (YOFTD). The ARTEMIS project is a 25-year prospective study of YOD based in Perth, Western Australia. 231 participants were included: YOAD: n = 103, YOFTD: n = 28, controls: n = 100. EEGs were performed prospectively, with 30-minute recording time for each subject, without knowledge of diagnosis or other diagnostic data. RESULTS: 80.9% of patients with YOD had abnormal EEGs (P < 0.00001). Slow wave changes were more frequent in YOAD that YOFTD (P < 0.00001), but no difference in the frequency of epileptiform activity (P = 0.32), with 38.8% of YOAD and 28.6% of YOFTD patients having epileptiform activity. Slow wave changes were more generalized in YOAD (P = 0.001). Slow wave changes and epileptiform activity were not sensitive to the diagnosis of YOD, but highly specific (97-99%). The absence of slow wave changes and epileptiform activity had a 100% negative predictive value and likelihood radio 0.14 and 0.62 respectively, meaning that those without slow wave changes or epileptiform activity had low probability of having YOD. No relationship was established between EEG findings and the patient's presenting problem. Eleven patients with YOAD developed seizures during the study, and only one with YOFTD. CONCLUSIONS: The EEG is highly specific for the diagnosis of YOD with the absence of slow wave changes and epileptiform phenomena making the diagnosis unlikely, with 100% negative predictive value and with low probability for the dementia diagnosis.

Structural basis for the activation and inhibition of the UCH37 deubiquitylase.

The UCH37 deubiquitylase functions in two large and very different complexes, the 26S proteasome and the INO80 chromatin remodeler. We have performed biochemical characterization and determined crystal structures of UCH37 in complexes with RPN13 and NFRKB, which mediate its recruitment to the proteasome and INO80, respectively. RPN13 and NFRKB make similar contacts to the UCH37 C-terminal domain but quite different contacts to the catalytic UCH domain. RPN13 can activate UCH37 by disrupting dimerization, although physiologically relevant activation likely results from stabilization of a surface competent for ubiquitin binding and modulation of the active-site crossover loop. In contrast, NFRKB inhibits UCH37 by blocking the ubiquitin-binding site and by disrupting the enzyme active site. These findings reveal remarkable commonality in mechanisms of recruitment, yet very different mechanisms of regulating enzyme activity, and provide a foundation for understanding the roles of UCH37 in the unrelated proteasome and INO80 complexes.

Rooting the Animal Tree of Life.

Identifying our most distant animal relatives has emerged as one of the most challenging problems in phylogenetics. This debate has major implications for our understanding of the origin of multicellular animals and of the earliest events in animal evolution, including the origin of the nervous system. Some analyses identify sponges as our most distant animal relatives (Porifera-sister hypothesis), and others identify comb jellies (Ctenophora-sister hypothesis). These analyses vary in many respects, making it difficult to interpret previous tests of these hypotheses. To gain insight into why different studies yield different results, an important next step in the ongoing debate, we systematically test these hypotheses by synthesizing 15 previous phylogenomic studies and performing new standardized analyses under consistent conditions with additional models. We find that Ctenophora-sister is recovered across the full range of examined conditions, and Porifera-sister is recovered in some analyses under narrow conditions when most outgroups are excluded and site-heterogeneous CAT models are used. We additionally find that the number of categories in site-heterogeneous models is sufficient to explain the Porifera-sister results. Furthermore, our cross-validation analyses show CAT models that recover Porifera-sister have hundreds of additional categories and fail to fit significantly better than site-heterogenuous models with far fewer categories. Systematic and standardized testing of diverse phylogenetic models suggests that we should be skeptical of Porifera-sister results both because they are recovered under such narrow conditions and because the models in these conditions fit the data no better than other models that recover Ctenophora-sister.

Validation of NRG Oncology's prognostic nomograms for oropharyngeal cancer in the Veterans Affairs database.

BACKGROUND: To test whether nomograms developed by NRG Oncology for oropharyngeal squamous cell carcinoma (OPSCC) patients could be validated in an independent population-based sample. METHODS: The authors tested nomograms for estimating progression-free survival (PFS) and overall survival (OS) in patients from the Veterans Health Administration with previously untreated locoregionally advanced OPSCC, diagnosed between 2008 and 2017, managed with definitive radiotherapy with or without adjuvant systemic therapy. Covariates were age, performance status, p16 status, T/N category, smoking history, education history, weight loss, marital status, and anemia. We used multiple imputation to handle missing data and performed sensitivity analyses on complete cases. Validation was assessed via Cox proportional hazards models, log-rank tests, and c-indexes. RESULTS: A total of 4007 patients met inclusion criteria (658 patients had complete data). Median follow-up time was 3.20 years, with 967 progression events and 471 noncancer deaths. Each risk score was associated with poorer outcomes per unit increase (PFS score, hazard ratio [HR], 1.42 [1.37-1.47]; OS score, HR, 1.40 [1.34-1.45]). By risk score quartile, 2-year PFS estimates were 89.2%, 78.5%, 65.8%, and 48.3%; OS estimates were 92.6%, 83.6%, 73.9%, and 51.3%, respectively (P < .01 for all comparisons). C-indices for models of PFS and OS were 0.65 and 0.67, for all patients, respectively (0.69 and 0.73 for complete cases). The nomograms slightly overestimated PFS and OS in the overall cohort but exhibited high agreement in complete cases. CONCLUSIONS: NRG nomograms were effective for predicting PFS and OS for patients with OPSCC, supporting their broader applicability in the OPSCC population undergoing definitive radiotherapy.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Incomplete cisplatin regimens in chemoradiation and its effect on outcomes for locally advanced cervical cancer.

OBJECTIVE: To identify factors associated with receipt of incomplete cisplatin during chemoradiation for locally advanced cervical cancer and its impact on outcomes. METHODS: Patients with locally advanced cervical cancer treated with chemoradiation at our institution between November 2015 and August 2020 were retrospectively identified. Patients who received ≤4 cycles were identified as the 'incomplete' cohort and those who received 5-6 cycles as the 'complete' cohort. The primary endpoint of incomplete chemotherapy was evaluated with multivariable logistic regression. Secondary endpoints of locoregional failure, overall survival, and distant failure were evaluated in multivariable Cox and Fine-Gray models. RESULTS: Of 140 patients with locally advanced cervical cancer that underwent chemoradiation, 22 (15.7%) received an incomplete cisplatin regimen (8 with 0 cycles, 14 with 1-4 cycles). The most common reasons for receiving incomplete treatment were comorbidities/infections (41%), unmet laboratory parameters (27%), and cisplatin intolerance (14%). In multivariable models, only poor (2-4) Eastern Cooperative Oncology Group performance status was a significant predictor as these patients were 41 times more likely to receive incomplete chemotherapy (odds ratio (OR), 95% confidence interval (CI) 4.57 to 375.15, p<0.001). Median follow-up time was 20 months (range 4-64). In multivariable models, receipt of incomplete cisplatin was significantly associated with higher recurrence (locoregional failure hazard ratio (HR) 3.02, 95% CI 1.08 to 8.45, p=0.03; distant failure HR 2.71, 95% CI 1.13 to 6.47, p=0.02) and worse survival (overall survival HR 4.91, 95% CI 1.27 to 18.98, p=0.02). CONCLUSION: Incomplete cisplatin regimen was associated with worse oncologic outcomes. Poor performance status was the only factor associated with receiving an incomplete regimen. This notable proportion of patients may be a target for better tolerated novel targeted anticancer agents in order to improve outcomes.

Disparities in time to start of definitive radiation treatment for patients with locally advanced cervical cancer.

BACKGROUND: Chemoradiation or radiation therapy alone are curative standards for patients with locally advanced cervical cancer. OBJECTIVE: To investigate factors that influence time to initiation of chemoradiation or radiation and the subsequent impact of time to treatment on recurrence and survival outcomes. METHODS: Patients with locally advanced cervical cancer treated with definitive chemoradiation or radiation at our institution between November 2015 and August 2020 were retrospectively identified. Time to treatment initiation was defined as the number of days from date of diagnosis (via biopsy) to the start date of radiation. The cohort was stratified by the median time to treatment into early (<75 days) and delayed (≥75 days) cohorts. Multivariable logistic regression was conducted to examine factors associated with delayed time to treatment. RESULTS: We identified 143 patients with locally advanced cervical cancer who underwent definitive chemoradiation or radiation. Median follow-up time was 18 months (range 2-62). A total of 71 (49.7%) patients had time to treatment <75 days and 72 (50.3%) patients had time to treatment ≥75 days. The delayed cohort had a higher proportion of Hispanic patients (51.4% vs 31.0%, p=0.04). In multivariable modeling, Hispanic women were 2.71 times more likely (p=0.04) to undergo delayed time to treatment than non-Hispanic white women. Additionally, patients with stage >IIB disease were less likely to undergo delayed time to treatment (OR 0.26, p=0.02) than patients with stage

Treeio: An R Package for Phylogenetic Tree Input and Output with Richly Annotated and Associated Data.

Phylogenetic trees and data are often stored in incompatible and inconsistent formats. The outputs of software tools that contain trees with analysis findings are often not compatible with each other, making it hard to integrate the results of different analyses in a comparative study. The treeio package is designed to connect phylogenetic tree input and output. It supports extracting phylogenetic trees as well as the outputs of commonly used analytical software. It can link external data to phylogenies and merge tree data obtained from different sources, enabling analyses of phylogeny-associated data from different disciplines in an evolutionary context. Treeio also supports export of a phylogenetic tree with heterogeneous-associated data to a single tree file, including BEAST compatible NEXUS and jtree formats; these facilitate data sharing as well as file format conversion for downstream analysis. The treeio package is designed to work with the tidytree and ggtree packages. Tree data can be processed using the tidy interface with tidytree and visualized by ggtree. The treeio package is released within the Bioconductor and rOpenSci projects. It is available at https://www.bioconductor.org/packages/treeio/.

Immunotherapy and radiation therapy sequencing: State of the data on timing, efficacy, and safety.

Radiation therapy exerts a tumoricidal local effect as well as both local and systemic immunomodulation. Immune checkpoint blockade has become a widely used treatment modality across cancer types with a rapidly growing list of agents and US Food and Drug Administration-approved indications. Moreover, there may be synergy between radiation therapy and immune checkpoint blockade. Various strategies have been used, but the optimal sequencing of these therapies is unclear. In this review, the authors discuss the major mechanisms of available immune checkpoint inhibitors and explore the available preclinical and clinical evidence regarding treatment sequencing. They also review safety considerations and conclude with possible future directions.

Pairwise comparisons across species are problematic when analyzing functional genomic data.

There is considerable interest in comparing functional genomic data across species. One goal of such work is to provide an integrated understanding of genome and phenotype evolution. Most comparative functional genomic studies have relied on multiple pairwise comparisons between species, an approach that does not incorporate information about the evolutionary relationships among species. The statistical problems that arise from not considering these relationships can lead pairwise approaches to the wrong conclusions and are a missed opportunity to learn about biology that can only be understood in an explicit phylogenetic context. Here, we examine two recently published studies that compare gene expression across species with pairwise methods, and find reason to question the original conclusions of both. One study interpreted pairwise comparisons of gene expression as support for the ortholog conjecture, the hypothesis that orthologs tend to have more similar attributes (expression in this case) than paralogs. The other study interpreted pairwise comparisons of embryonic gene expression across distantly related animals as evidence for a distinct evolutionary process that gave rise to phyla. In each study, distinct patterns of pairwise similarity among species were originally interpreted as evidence of particular evolutionary processes, but instead, we find that they reflect species relationships. These reanalyses concretely show the inadequacy of pairwise comparisons for analyzing functional genomic data across species. It will be critical to adopt phylogenetic comparative methods in future functional genomic work. Fortunately, phylogenetic comparative biology is also a rapidly advancing field with many methods that can be directly applied to functional genomic data.

Challenges and Controversies in Foot and Ankle Trauma.

Management of foot and ankle trauma continues to evolve and change. It is important to be informed about the latest challenges and controversies in management of these injuries, which include ankle fractures, calcaneus fractures, Lisfranc injuries, and Jones fracture. Important concepts related to ankle fracture are the changing indications for surgery, utilization of stress radiographs, the role of arthroscopy, repair of the deltoid ligament, fixation of the posterior malleolus, and diagnosis and treatment of syndesmotic injuries. Regarding calcaneus fractures, discussion revolves around defining indications for fixation, factors that influence outcomes, less invasive approaches versus traditional extensile exposures, and the nature of the constant fragment. With Lisfranc injuries, the orthopaedic surgeon should be aware of fixation methods as well as the issue of fixation versus fusion. Discussion of Jones fracture should include evaluation of indications and different fixation techniques.

Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13.

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2-RPN13 interaction are being developed as potential therapeutic anticancer agents. Our current study builds on the recognition that a residue central to the RPN2-RPN13 interaction, RPN2 Tyr-950, is phosphorylated in Jurkat cells. We found that the Tyr-950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2-RPN13 pTyr-950-ubiquitin complex was determined at 1.76-Å resolution and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr-950, have important implications for efforts to develop specific inhibitors of the RPN2-RPN13 interaction, and suggest the existence of a previously unknown stress-response pathway.