Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis.

Cellular heterogeneity in gene expression is driven by cellular processes, such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). By using these data, we developed a novel approach to characterize cell cycle progression. Although standard methods assign cells to discrete cell cycle stages, our method goes beyond this and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell's position on the cell cycle continuum to within 14% of the entire cycle and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

A comparison of gene expression and DNA methylation patterns across tissues and species.

Previously published comparative functional genomic data sets from primates using frozen tissue samples, including many data sets from our own group, were often collected and analyzed using nonoptimal study designs and analysis approaches. In addition, when samples from multiple tissues were studied in a comparative framework, individuals and tissues were confounded. We designed a multitissue comparative study of gene expression and DNA methylation in primates that minimizes confounding effects by using a balanced design with respect to species, tissues, and individuals. We also developed a comparative analysis pipeline that minimizes biases attributable to sequence divergence. Thus, we present the most comprehensive catalog of similarities and differences in gene expression and DNA methylation levels between livers, kidneys, hearts, and lungs, in humans, chimpanzees, and rhesus macaques. We estimate that overall, interspecies and inter-tissue differences in gene expression levels can only modestly be accounted for by corresponding differences in promoter DNA methylation. However, the expression pattern of genes with conserved inter-tissue expression differences can be explained by corresponding interspecies methylation changes more often. Finally, we show that genes whose tissue-specific regulatory patterns are consistent with the action of natural selection are highly connected in both gene regulatory and protein-protein interaction networks.

Reorganization of 3D genome structure may contribute to gene regulatory evolution in primates.

A growing body of evidence supports the notion that variation in gene regulation plays a crucial role in both speciation and adaptation. However, a comprehensive functional understanding of the mechanisms underlying regulatory evolution remains elusive. In primates, one of the crucial missing pieces of information towards a better understanding of regulatory evolution is a comparative annotation of interactions between distal regulatory elements and promoters. Chromatin conformation capture technologies have enabled genome-wide quantifications of such distal 3D interactions. However, relatively little comparative research in primates has been done using such technologies. To address this gap, we used Hi-C to characterize 3D chromatin interactions in induced pluripotent stem cells (iPSCs) from humans and chimpanzees. We also used RNA-seq to collect gene expression data from the same lines. We generally observed that lower-order, pairwise 3D genomic interactions are conserved in humans and chimpanzees, but higher order genomic structures, such as topologically associating domains (TADs), are not as conserved. Inter-species differences in 3D genomic interactions are often associated with gene expression differences between the species. To provide additional functional context to our observations, we considered previously published chromatin data from human stem cells. We found that inter-species differences in 3D genomic interactions, which are also associated with gene expression differences between the species, are enriched for both active and repressive marks. Overall, our data demonstrate that, as expected, an understanding of 3D genome reorganization is key to explaining regulatory evolution.

Visualizing the structure of RNA-seq expression data using grade of membership models.

Grade of membership models, also known as "admixture models", "topic models" or "Latent Dirichlet Allocation", are a generalization of cluster models that allow each sample to have membership in multiple clusters. These models are widely used in population genetics to model admixed individuals who have ancestry from multiple "populations", and in natural language processing to model documents having words from multiple "topics". Here we illustrate the potential for these models to cluster samples of RNA-seq gene expression data, measured on either bulk samples or single cells. We also provide methods to help interpret the clusters, by identifying genes that are distinctively expressed in each cluster. By applying these methods to several example RNA-seq applications we demonstrate their utility in identifying and summarizing structure and heterogeneity. Applied to data from the GTEx project on 53 human tissues, the approach highlights similarities among biologically-related tissues and identifies distinctively-expressed genes that recapitulate known biology. Applied to single-cell expression data from mouse preimplantation embryos, the approach highlights both discrete and continuous variation through early embryonic development stages, and highlights genes involved in a variety of relevant processes-from germ cell development, through compaction and morula formation, to the formation of inner cell mass and trophoblast at the blastocyst stage. The methods are implemented in the Bioconductor package CountClust.

Correction: Visualizing the structure of RNA-seq expression data using grade of membership models.

[This corrects the article DOI: 10.1371/journal.pgen.1006599.].

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell populations. FlowMap-FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F-measure of 0.88 was obtained, indicating high precision and recall of the FR-based population matching results. FlowMap-FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows.

BlindCall: ultra-fast base-calling of high-throughput sequencing data by blind deconvolution.

MOTIVATION: Base-calling of sequencing data produced by high-throughput sequencing platforms is a fundamental process in current bioinformatics analysis. However, existing third-party probabilistic or machine-learning methods that significantly improve the accuracy of base-calls on these platforms are impractical for production use due to their computational inefficiency. RESULTS: We directly formulate base-calling as a blind deconvolution problem and implemented BlindCall as an efficient solver to this inverse problem. BlindCall produced base-calls at accuracy comparable to state-of-the-art probabilistic methods while processing data at rates 10 times faster in most cases. The computational complexity of BlindCall scales linearly with read length making it better suited for new long-read sequencing technologies.

Attenuating ribosome load improves protein output from mRNA by limiting translation-dependent mRNA decay.

Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure yielding the highest protein outputs due to their slow rates of mRNA decay. Here, we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest-expressing mRNAs in our test set have modest initiation/elongation rates and ribosome loads, leading to minimal translation-dependent mRNA decay. These findings are of potential interest for optimization of protein output from therapeutic mRNAs, which may be achieved by attenuating rather than maximizing ribosome load.

Lipid-encapsulated mRNA encoding an extended serum half-life interleukin-22 ameliorates metabolic disease in mice.

OBJECTIVE: Interleukin (IL)-22 is a potential therapeutic protein for the treatment of metabolic diseases such as obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease due to its involvement in multiple cellular pathways and observed hepatoprotective effects. The short serum half-life of IL-22 has previously limited its use in clinical applications; however, the development of mRNA-lipid nanoparticle (LNP) technology offers a novel therapeutic approach that uses a host-generated IL-22 fusion protein. In the present study, the effects of administration of an mRNA-LNP encoding IL-22 on metabolic disease parameters was investigated in various mouse models. METHODS: C57BL/6NCrl mice were used to confirm mouse serum albumin (MSA)-IL-22 protein expression prior to assessments in C57BL/6NTac and CETP/ApoB transgenic mouse models of metabolic disease. Mice were fed either regular chow or a modified amylin liver nonalcoholic steatohepatitis-inducing diet prior to receiving either LNP-encapsulated MSA-IL-22 or MSA mRNA via intravenous or intramuscular injection. Metabolic markers were monitored for the duration of the experiments, and postmortem histology assessment and analysis of metabolic gene expression pathways were performed. RESULTS: MSA-IL-22 was detectable for ≥8 days following administration. Improvements in body weight, lipid metabolism, glucose metabolism, and lipogenic and fibrotic marker gene expression in the liver were observed in the MSA-IL-22-treated mice, and these effects were shown to be durable. CONCLUSIONS: These results support the application of mRNA-encoded IL-22 as a promising treatment strategy for metabolic syndrome and associated comorbidities in human populations.

An optimized messenger RNA vaccine candidate protects non-human primates from Zika virus infection.

Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In-depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation.

Domain-based mRNA vaccines encoding spike protein N-terminal and receptor binding domains confer protection against SARS-CoV-2.

With the success of messenger RNA (mRNA) vaccines against coronavirus disease 2019, strategies can now focus on improving vaccine potency, breadth, and stability. We designed and evaluated domain-based mRNA vaccines encoding the wild-type spike protein receptor binding domain (RBD) or N-terminal domain (NTD) alone or in combination. An NTD-RBD-linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2° to 8°C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In BALB/c mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses from viral challenge were observed against wild-type, beta, delta, or omicron (BA.1) viruses compared with mRNA-1273-immunized mice, especially at lower vaccine dosages. K18-hACE2 mice immunized with mRNA-1283 or mRNA-1273 as a primary series demonstrated similar degrees of protection from challenge with SARS-CoV-2 Delta and Omicron variants at all vaccine dosages. These results support clinical assessment of mRNA-1283, which has now entered clinical trials (NCT05137236).

Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection.

Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)-lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding [immunoglobulin G (IgG) and IgA] and neutralizing antibodies. Intranasally vaccinated hamsters also had decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. Together, this study demonstrates successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.

An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

Selective activation and expansion of regulatory T cells using lipid encapsulated mRNA encoding a long-acting IL-2 mutein.

Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rß/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.

Altering the mRNA-1273 dosing interval impacts the kinetics, quality, and magnitude of immune responses in mice.

For a vaccine to achieve durable immunity and optimal efficacy, many require a multi-dose primary vaccination schedule that acts to first "prime" naive immune systems and then "boost" initial immune responses by repeated immunizations (ie, prime-boost regimens). In the context of the global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 2-dose primary vaccination regimens were often selected with short intervals between doses to provide rapid protection while still inducing robust immunity. However, emerging post-authorization evidence has suggested that longer intervals between doses 1 and 2 for SARS-CoV-2 vaccines may positively impact robustness and durability of immune responses. Here, the dosing interval for mRNA-1273, a messenger RNA based SARS-CoV-2 vaccine administered on a 2-dose primary schedule with 4 weeks between doses, was evaluated in mice by varying the dose interval between 1 and 8 weeks and examining immune responses through 24 weeks after dose 2. A dosing interval of 6 to 8 weeks generated the highest level of antigen-specific serum immunoglobulin G binding antibody titers. Differences in binding antibody titers between mRNA-1273 1 µg and 10 µg decreased over time for dosing intervals of ≥4 weeks, suggesting a potential dose-sparing effect. Longer intervals (≥4 weeks) also increased antibody-dependent cellular cytotoxicity activity and numbers of antibody-secreting cells (including long-lived plasma cells) after the second dose. An interval of 6 to 8 weeks elicited the strongest CD8+ T-cell responses, while an interval of 3 weeks elicited the strongest CD4+ T-cell response. Overall, these results suggest that in a non-pandemic setting, a longer interval (≥6 weeks) between the doses of the primary series for mRNA-1273 may induce more durable immune responses.

Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains.

With the success of mRNA vaccines against coronavirus disease 2019 (COVID-19), strategies can now focus on improving vaccine potency, breadth, and stability. We present the design and preclinical evaluation of domain-based mRNA vaccines encoding the wild-type spike-protein receptor-binding (RBD) and/or N-terminal domains (NTD). An NTD-RBD linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2-8°C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses and protection from viral challenge were observed against wild-type, beta, delta, or omicron (BA. 1) compared with mRNA-1273 immunized mice, especially at lower vaccine dosages. These results support clinical assessment of mRNA-1283 ( NCT05137236 ). One Sentence Summary: A domain-based mRNA vaccine, mRNA-1283, is immunogenic and protective against SARS-CoV-2 and emerging variants in mice.

Assessing the Stages of Change Among African American Women in a Weight Management Program.

The objective of this study was to assess the relationship between stage of change (SOC) and behavioral outcomes among African American women entering obesity treatment in two settings. Fifty-five overweight/obese (body mass index = 26.50-48.13), but otherwise healthy African American women, 23 to 56 years old, attended a 13-week weight loss-treatment program that took place at churches (n = 36) or a university (n = 19). Participants were weighed, completed SOC measures, and had a physical fitness test at pre- and posttreatment. Pretreatment measures of SOC placed 47% of the participants as actors, 31% as contemplators, and 22% as maintainers. Of the 45 women who reported posttreatment SOC, 7% regressed, 44% did not change, and 31% progressed in SOC. Pretreatment SOC predicted posttreatment weight loss in the church setting but not in the university setting. At churches, contemplators lost more weight than actors and maintainers. The church may be a more conducive setting for weight change behaviors for African American women who are categorized as contemplators in the SOC model.

Nasal Microbiota and Infectious Complications After Elective Surgical Procedures.

Importance: The association of the nasal microbiome with outcomes in surgical patients is poorly understood. Objective: To characterize the composition of nasal microbiota in patients undergoing clean elective surgical procedures and to examine the association between characteristics of preoperative nasal microbiota and occurrence of postoperative infection. Design, Setting, and Participants: Using a nested matched case-control design, 53 individuals who developed postoperative infection were matched (approximately 3:1 by age, sex, and surgical procedure) with 144 individuals who were not infected (ie, the control group). The 2 groups were selected from a prospective cohort of patients undergoing surgical procedures at 2 tertiary care university hospitals in Baltimore, Maryland, who were at high risk for postoperative infectious complications. Included individuals were aged 40 years or older; had no history of autoimmune disease, immunocompromised state, immune-modulating medication, or active infection; and were scheduled to undergo elective cardiac, vascular, spinal, or intracranial surgical procedure. Data were analyzed from October 2015 through September 2020. Exposures: Nasal microbiome cluster class served as the main exposure. An unsupervised clustering method (ie, grades of membership modeling) was used to classify nasal microbial samples into 2 groups based on features derived from 16S ribosomal RNA gene sequencing. The microbiome cluster groups were derived independently and agnostic of baseline clinical characteristics and infection status. Main Outcomes and Measures: Composite of surgical site infection, bacteremia, and pneumonia occurring within 6 months after surgical procedure. Results: Among 197 participants (mean [SD] age, 64.1 [10.6] years; 63 [37.7%] women), 553 bacterial taxa were identified from preoperative nasal swab samples. A 2-cluster model (with 167 patients in cluster 1 and 30 patients in cluster 2) accounted for the largest proportion of variance in microbial profiles using grades of membership modeling and was most parsimonious. After adjusting for potential confounders, the probability of assignment to cluster 2 was associated with 6-fold higher odds of infection after surgical procedure (odds ratio [OR], 6.18; 95% CI, 3.33-11.7; P < .001) independent of baseline clinical characteristics, including nasal carriage of Staphylococcus aureus. Intrasample (ie, α) diversity was inversely associated with infectious outcome in both clusters (OR, 0.57; 95% CI, 0.42-0.75; P < .001); however, probability of assignment to cluster 2 was associated with higher odds of infection independent of α diversity (OR, 4.61; 95% CI, 2.78-7.86; P < .001). Conclusions and Relevance: These findings suggest that the nasal microbiome was an independent risk factor associated with infectious outcomes among individuals who underwent elective surgical procedures and may serve as a biomarker associated with infection susceptibility in this population.

Impact of lipid nanoparticle size on mRNA vaccine immunogenicity.

Lipid nanoparticles (LNP) are effective delivery vehicles for messenger RNA (mRNA) and have shown promise for vaccine applications. Yet there are no published reports detailing how LNP biophysical properties can impact vaccine performance. In our hands, a retrospective analysis of mRNA LNP vaccine in vivo studies revealed a relationship between LNP particle size and immunogenicity in mice using LNPs of various compositions. To further investigate this, we designed a series of studies to systematically change LNP particle size without altering lipid composition and evaluated biophysical properties and immunogenicity of the resulting LNPs. While small diameter LNPs were substantially less immunogenic in mice, all particle sizes tested yielded a robust immune response in non-human primates (NHP).

The impact of nurse case management home visitation on birth outcomes in African-American women.

UNLABELLED: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Uniformed Services University of the Health Sciences, Department of the Navy, Department of Defense or the U. S. government. Dr. Wells is a military service member (employee of the U.S. government). This work was prepared as a part of her official duties. Title 17, USC Section 101 defines a U.S. government work as a work prepared by a military service member or employee of the U.S. government as part of the person's official duties. Despite substantial reductions in U.S. infant mortality rates, racial disparities persist, with black Americans experiencing 2.4 times the rate of their white counterparts. Low birthweight and preterm delivery contribute to this disparity. METHODS: To examine the association between antepartum nurse case management home visitation and the occurrence of low birthweight and preterm deliveries in African-American women in Montgomery County, MD, a retrospective cohort study was conducted using existing data from 109 mothers who were enrolled in the Black Babies Start More Infants Living Equally Healthy (SMILE) program. Logistic regression analysis was used. RESULTS: Women who received antepartum home visits were 0.37 (CI 0.15-0.94) times less likely to experience preterm delivery than women who did not receive antepartum home visits. The effect of antepartum home visits on preterm delivery was independent of level of prenatal care, negative life events and number of prior live births. There was no significant association between antepartum home visits and low birthweight. CONCLUSION: Antepartum home visits appeared to be protective against preterm delivery and could contribute to reducing racial disparities in infant mortality. Further study is needed to understand and replicate specific program components that may contribute to improved birth outcomes in African-American women.