Objective: Longitudinal observational data pose a challenge for causal inference when the exposure of interest varies over time alongside time-dependent confounders, which often occurs in trauma ...research. We describe marginal structural models (MSMs) using inverse probability weighting as a useful solution under several assumptions that are well-suited to estimating causal effects in trauma research. Method: We illustrate the application of MSMs by estimating the joint effects of community violence exposure across time on youths' internalizing and externalizing symptoms. Our sample included 4,327 youth (50% female, 50% male; 1.4% Asian American or Pacific Islander, 34.7% Black, 46.9% Hispanic, .8% Native American, 14.3%, White, 1.5%, Other race/ethnicity; Mage at baseline = 8.62, range = 3-15) from the Project on Human Development in Chicago Neighborhoods. Results: Wave 3 internalizing symptoms increased linearly with increases in Wave 2 and Wave 3 community violence exposure, whereas effects on externalizing symptoms were quadratic for Wave 2 community violence exposure and linear for Wave 3. These results fail to provide support for the desensitization model of community violence exposure. Conclusion: MSMs are a useful tool for researchers who rely on longitudinal observational data to estimate causal effects of time-varying exposures, as is often the case in the study of psychological trauma.
Clinical Impact Statement
Marginal structural models allow researchers to test the causal effect of trauma exposure over time even when exposed individuals are different from those who are not exposed, under certain assumptions. This approach can be especially useful for informing whether an intervention that prevents or imposes exposure would have an impact.
Advanced methods for panel data analysis are commonly used in research on family life and relationships, but the fundamental issue of simultaneous time-dependent confounding and mediation has ...received little attention. In this article the authors introduce inverse-probability-weighted estimation of marginal structural models, an approach to causal analysis that (unlike conventional regression modeling) appropriately adjusts for confounding variables on the causal pathway linking the treatment with the outcome. They discuss the need for marginal structural models in social science research and describe their estimation in detail. Substantively, the authors contribute to the ongoing debate on the effects of incarceration on marriage by applying a marginal structural model approach to panel data from the National Longitudinal Survey of Youth 1997 (N = 4,781). In line with the increasing evidence on the collateral consequences of contact with the criminal justice system, the authors find that incarceration is associated with reduced chances of entering marriage.
Conventional analytic approaches for studying diet patterns assume no dietary synergy, which can lead to bias if incorrectly modeled. Machine learning algorithms can overcome these limitations.
We ...estimated associations between fruit and vegetable intake relative to total energy intake and adverse pregnancy outcomes using targeted maximum likelihood estimation (TMLE) paired with the ensemble machine learning algorithm Super Learner, and compared these with results generated from multivariable logistic regression.
We used data from 7572 women in the Nulliparous Pregnancy Outcomes Study: monitoring mothers-to-be. Usual daily periconceptional intake of total fruits and total vegetables was estimated from an FFQ. We calculated the marginal risk of preterm birth, small-for-gestational-age (SGA) birth, gestational diabetes, and pre-eclampsia according to density of fruits and vegetables (cups/1000 kcal) ≥80th percentile compared with <80th percentile using multivariable logistic regression and Super Learner with TMLE. Models were adjusted for confounders, including other Healthy Eating Index-2010 components.
Using logistic regression, higher fruit and high vegetable densities were associated with 1.1% and 1.4% reductions in pre-eclampsia risk compared with lower densities, respectively. They were not associated with the 3 other outcomes. Using Super Learner with TMLE, high fruit and vegetable densities were associated with fewer cases of preterm birth (–4.0; 95% CI: −4.9, −3.0 and −3.7; 95% CI: −5.0, −2.3), SGA (−1.7; 95% CI: −2.9, −0.51 and −3.8; 95% CI: −5.0, −2.5), and pre-eclampsia (−3.2; 95% CI: −4.2, −2.2 and −4.0; 95% CI: −5.2, −2.7) per 100 births, respectively, and high vegetable densities were associated with a 0.9% increase in risk of gestational diabetes.
The differences in results between Super Learner with TMLE and logistic regression suggest that dietary synergy, which is accounted for in machine learning, may play a role in pregnancy outcomes. This innovative methodology for analyzing dietary data has the potential to advance the study of diet patterns.
While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence ...demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaute 2, fails to enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells. Indeed, a number of mammalian VSRs have been described, of which the most prominent is the influenza A virus (IAV) NS1 protein, which has not only been reported to inhibit RNAi in plants and insects but also to prevent the production of viral siRNAs in IAV-infected human cells. Here, we confirm that an IAV mutant lacking NS1 indeed differs from wild-type IAV in that it induces the production of readily detectable levels of Dicer-dependent viral siRNAs in infected human cells. However, we also demonstrate that these siRNAs have little if any inhibitory effect on IAV gene expression. This is likely due, at least in part, to their inefficient loading into RNA-induced silencing complexes.
While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as ...epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of
-methyladenosine (m
A), 5-methylcytosine (m
C), and 2'O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m
A and m
C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m
A and m
C in viral replication, we also demonstrate that overexpression of the key m
A reader protein YTHDF2 enhances MLV replication, while downregulation of the m
C writer NSUN2 inhibits MLV replication.
The data presented in the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m
A, m
C, and Nm, suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m
A or m
C modifications from MLV transcripts inhibits MLV replication in
, and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in
Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.
Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if ...any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus—but not poliovirus—replication in human cells.
The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been ...controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. Importance: Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.
Abstract
There are important challenges to the estimation and identification of average causal effects in longitudinal data with time-varying exposures. Here, we discuss the difficulty in meeting the ...positivity condition. Our motivating example is the per-protocol analysis of the Effects of Aspirin in Gestation and Reproduction (EAGeR) Trial. We estimated the average causal effect comparing the incidence of pregnancy by 26 weeks that would have occurred if all women had been assigned to aspirin and complied versus the incidence if all women had been assigned to placebo and complied. Using flexible targeted minimum loss-based estimation, we estimated a risk difference of 1.27% (95% CI: −9.83, 12.38). Using a less flexible inverse probability weighting approach, the risk difference was 5.77% (95% CI: −1.13, 13.05). However, the cumulative probability of compliance conditional on covariates approached 0 as follow-up accrued, indicating a practical violation of the positivity assumption, which limited our ability to make causal interpretations. The effects of nonpositivity were more apparent when using a more flexible estimator, as indicated by the greater imprecision. When faced with nonpositivity, one can use a flexible approach and be transparent about the uncertainty, use a parametric approach and smooth over gaps in the data, or target a different estimand that will be less vulnerable to positivity violations.
Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is ...the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
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•DDX39B is a potent activator of IL7R exon 6 splicing and a repressor of sIL7R•DDX39B genetic variants are significantly associated with MS risk•The 5′ UTR DDX39B variant reduces protein levels by decreasing translation efficiency•This variant shows strong genetic and functional epistasis with IL7R rs6897932
Two genes interact epistatically in multiple sclerosis risk in humans.
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