Research

Assessing Risk of Opioid Use Disorder Development

The opioid epidemic in the U.S. and worldwide continues to claim lives on a daily basis. Additionally, the costs associated with problematic opioid use is significant. Because of these reasons, I am interested in understanding and identifying the risks associated with the development of Opioid Use Disorder (OUD).

OUD is defined as a problematic pattern of opioid use that leads to serious impairment and distress. It has been shown that a significant amount of individuals exposed to opioid analgesics in healthcare settings develop OUD and there are many factors that can contribute to an individual developing OUD (shown below)

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Figure 1 from
Freda et al. 2019

Determining an individual’s risk for developing OUD after exposure to opioid-based analgesics in clinical settings could lead to better opioid prescribing and OUD treatment practices, enhancement of  prevention efforts, and alleviation of the adverse impacts of OUD on individuals, families, and society.

There are currently two projects that I am working on to assess OUD development risk. The first attempts to use clustering algorithms to look for structure in a subpopulation of individuals in the Penn Medicine system with problematic opioid use diagnoses. Medications, demographics, diagnoses, and procedures are used to determine if certain groups are at higher risks than others to develop OUD. The second project is in collaboration with Geisinger to pinpoint words and phrases that confer risk of OUD in clinical notes using natural language processing (NLP). There are words and phrases that may have significant associations with the development of OUD and our goal is to pinpoint these features in clinical notes so we can apply them to other clinical systems worldwide.

Testing the Adaptive Decoupling Hypothesis

Things change. This is a universal truth for all living things. Despite this, we humans try to impede the relentless force of change whenever we can. But in nature, organisms have to “roll with the punches.”

One of the most successful adaptive strategies that have evolved over the course of natural history in the response to changing biotic and abiotic factors across development is the complex life cycle. Simply put, complex life cycles are those that have distinct life history phases that differ morphologically, behaviorally, and physiologically. Probably one of the most well known examples is that of the butterfly where the caterpillar becomes a beautiful butterfly, but there are many, many more examples. In fact, the majority of multicellular animals undergo complex life cycles, regardless if we are tallying organisms, species, or phyla (Moran, 1994).

In humans, we do change significantly over our lifetimes. Morphologically, we get larger, become more proportional, and gain physically strength as our muscles grow. Of course, we also change in other ways, both behaviorally and physiologically. But our overall body plan and organs remain the same and function in similar ways. From these facts, it is acceptable to make the assumption that the genes that control certain traits when we are young are, for the most part, also control those same traits when get older. However, we can’t make the same assumption in organisms that change so drastically over their life cycles.

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From: “The Human Life Cycle” Infogram

It is easily apparent to even the unskilled observer that larvae and adults of most insect species look and behave very differently – this is usually due to the process of metamorphosis. For example, in holometabolous insects, that is, insects that go through a pupal phase, the overall body plan is reorganized and reconstructed during metamorphosis. Butterflies are an example of these types of insects. It is theorized that metamorphosis evolved for a number of reasons. The first is that it limits competition between each life stage, allowing them to eat different things and live in different environments so that they do not take resources away from the other life stage. Another is that metamorphosis allows each life stage to specialize in different things (i.e. feeding for larvae and dispersal and mating for adults). These theories beg the question: what occurs at the genomic level during these drastic changes? And do the same genes that control a trait in larvae also control that same trait in adults?

We know that the genome does not edit itself over development. In other words, the A’s, C’s, G’s, and T’s largely remain where they are supposed to in most cells. However, the way the genome is expressed could change. There could be changes in the way genes are expressed via shifts in DNA methylation, and alterations to transcription factors and regulatory regions. Also, natural selection could act on different parts of the genome at certain developmental periods. In order to explore this possibility further, I used isogenic lines of Drosophila melanogaster (the common vinegar fly) from a collection known as the Drosophila Genetic Reference Panel, or the DGRP. Because these lines are isogenic (all individuals of a line have the same genetic makeup), and have their genomes sequenced, I can pinpoint what genes are associated with a trait in either life stage. I do this with a process known as Genome-Wide Association (GWA) mapping, which associates certain SNPs (alleles) that segregate within the DGRP population to some trait value that we are studying. In other words, it allows me to determine what alleles either confer high trait values or low trait values in my flies. Then I determine what genes those SNPs are located in, using genomic data available, to identify candidate loci for further study. I also use next-gen sequencing technology, specifically RNA-seq, to determine if changes in gene expression also occur in response to some stress across development.

The trait that I study is thermal hardiness, or the ability to maintain fitness despite acute exposure to temperature extremes. This is essentially the organisms ability to remain able to conduct normal bodily functions and behaviors despite a stressful environmental temperature. I study this because it is something that most organisms experience in most climates across the globe. Organisms usually have to deal with extreme cold, extreme heat or both at some point during the year and/or their life time. Moreover, it is directly related to fitness when we think of ectotherms like insects. If an organism cannot handle harsh temperatures, it won’t be able to ultimately reproduce. Since larvae and adults of Drosophila melanogaster used in this study are from a temperate climate in North Carolina, it is conceivable that ample amounts of genetic variation exists for thermal hardiness in the DGRP as temperate regions experience both heat and cold extremes. Also, larvae and adults of D. melanogaster are subject to unique thermal habitats, making the system an excellent crucible for exploring the potential of genetic independence in thermal strategy across development.

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Findings

Thermal hardiness, specifically cold hardiness, is decoupled across the metamorphic boundary. On average, their is no relationship between a larvae’s thermal hardiness and an adults, within isogenic lines. In other words, one cannot accurately predict the thermal hardiness of a larvae from the value of an adults and vice-versa. Also, using GWA, we found that the vast majority of SNPs found to have a high inclusion probability (statistical association) with the thermal hardiness of one life stage did not in the other. This provides evidence that the genetic architectures for thermal hardiness in larvae and adults are distinct. Evolutionarily, these findings provide evidence that each life stage can respond to natural selection for cold hardiness independently, achieving their own fitness peaks without affecting the other life stage.

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Figure 2 from Freda et al. 2017

A follow-up study was aimed to determine if the same lack of correlation persists when investigating heat hardiness. Indeed, we found the same pattern for heat hardiness (panel B in figure below). 

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Supplemental Image from 
Freda et al. 2019

Not only does this lack of correlation persist but genotype x environment (GxE) interactions are also stage specific. We can think of this phenomenon as genotype x environment x development interaction (GxExD). We tested this by exposing larvae and adults to different rearing temperatures: cold (18 C) and warm (25 C) and measuring their performance after both cold shock (-6.5 C) and heat shock (38 C). 

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Figure 4 from
Freda et al. 2019

These results provide additional evidence that distinct genetic architectures underlie variation in cold stress hardiness in larvae and adults of D. melanogaster and provide evidence of developmental stage independence for heat stress hardiness and acclimation responses as well. Furthermore, these results indicate that there is thermal niche adaptation in the species with larvae being more heat tolerant overall (although not significantly) so and adults being more cold tolerant. This makes sense as larvae are found in rotting fruits, likely exposed to higher temperatures in summer and fall while adults overwinter.

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Figure 2 from
Freda et al. 2019

These findings have more broad-reaching implications as both significant crop pests and medically-relevant insects (i.e. mosquitoes) undergo complex life cycles. The way each life stage responds to thermal shifts in temperatures could dictate their global distributions. Therefore, accurate population modeling should take into account that each life stage could respond to climate differently.

Other Projects and Previous Research

I have also worked on ovarian development and reproductive arrest in an invasive fruit pest to North America and Europe known as Drosophila suzukii. This is not my first project with D. suzukii as I also worked on it during my masters looking at levels of genetic variation in the northeastern U.S. However, this work, a collaborative work with Elizabeth Everman, Ph.D., investigates how harsh cold temperature affect ovarian development in these flies at different points of development. Our findings show that, at least in the midwest, D. suzukii ovarian development responds to both low temperature and short photoperiod, indicated that some form of reproductive development exists in the species. Additionally, we also provide evidence that D. suzukii males and females have large capacity for short-term acclimation through Rapid Cold Hardening (RCH), which was not observed by researchers in more northern latitudes. Taken together, these results provide evidence that this invasive pest is able to adapt to fluctuating climates despite probable genetic bottlenecks incurred during its invasions of North America and Europe.

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Drosophila ovaries. From: The Journal of Biological Chemistry

I am also passionate about cataloging genetic variation in natural populations over time. In the past, I have sequenced a number of microsatellite loci in wild populations of Drosophila simulans in Philadelphia and Lower Merion, Pennsylvania over two years (2011 – 2012).

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Microsatellite peak calls.

Using pairwise tests of genic differentiation, I discovered significantly different allelic distributions at a number of loci with most significant results resulting in pairwise comparisons with dates at the end of one collection season and the beginning of another. In addition, there were significant deviations from Hardy-Weinberg Equilibrium at a number of loci on several collection dates with most occurring at the end or beginning of a collection season. Finally, observed heterozygosity was lowest, on average, at the end of seasons. Taken together, these results indicate a population bottleneck and reductions in population size and genetic variation at the end of a collection season followed by a rebound of the population, genetically distinct at the analyzed loci, the following year. It is unclear whether this was due to neutral processes, selection, migration, or a combination of all three forces. It would be interesting to perform a more detailed study in the future.