Impact of crowding on the diversity of expanding populations.

Crowding effects critically impact the self-organization of densely packed cellular assemblies, such as biofilms, solid tumors, and developing tissues. When cells grow and divide, they push each other apart, remodeling the structure and extent of the population's range. Recent work has shown that crowding has a strong impact on the strength of natural selection. However, the impact of crowding on neutral processes, which controls the fate of new variants as long as they are rare, remains unclear. Here, we quantify the genetic diversity of expanding microbial colonies and uncover signatures of crowding in the site frequency spectrum. By combining Luria-Delbrück fluctuation tests, lineage tracing in a novel microfluidic incubator, cell-based simulations, and theoretical modeling, we find that the majority of mutations arise behind the expanding frontier, giving rise to clones that are mechanically "pushed out" of the growing region by the proliferating cells in front. These excluded-volume interactions result in a clone-size distribution that solely depends on where the mutation first arose relative to the front and is characterized by a simple power law for low-frequency clones. Our model predicts that the distribution depends on a single parameter-the characteristic growth layer thickness-and hence allows estimation of the mutation rate in a variety of crowded cellular populations. Combined with previous studies on high-frequency mutations, our finding provides a unified picture of the genetic diversity in expanding populations over the whole frequency range and suggests a practical method to assess growth dynamics by sequencing populations across spatial scales.

Intestinal lesions and parasites associated with senescence and prespawn mortality in Chinook Salmon (Oncorhynchus tshawytscha).

Prespawn mortality (PSM) presents a major problem for the recovery of spring Chinook Salmon (Oncorhynchus tshawytscha) populations. In the Willamette River, Oregon, PSM exceeds 90% in some years but factors explaining it are not well understood. We examined intestinal tissue samples using histological slides from over 783 spring Chinook Salmon collected between 2009 and 2021, which included tissues from PSM fish, artificially spawned captive broodstock (BS) and normal river run fish, comprised of trapped (Live) and naturally post-spawned river (RPS) fish collected from the river. We observed degeneration of the intestinal epithelium and loss of villous structure, with concurrent severe enteritis. A natural progression of decline in epithelial integrity (EI) through the summer and fall until spawning and subsequent death was also observed. Live fish exhibited high EI scores (mean = 68%), BS exhibited variable EI scores (35%) and RPS exhibited severe loss of EI (14%). PSM fish exhibited prominent loss of intestinal epithelium with EI scores (13%), very similar to RPS fish, despite having been collected earlier in the year. Hence, we argue that low EI scores are strongly linked with PSM. Ceratonova shasta and Enterocytozoon schreckii were common in all groups, but neither were linked to either PSM or a decline in EI.

Aeromonas salmonicida, causative agent of salmonid furunculosis, isolated from the freshwater parasitic copepod, Salmincola californiensis.

Here, we provide evidence that the freshwater parasitic copepod, Salmincola californiensis, acts as a vector for Aeromonas salmonicida. While investigating the effects of S. californiensis on Chinoook salmon (Oncorhynchus tshawytscha), we tangentially observed that fish infected with the copepod developed furunculosis, caused by A. salmonicida. This occurred despite being reared in pathogen-free well water in a research facility with no prior history of spontaneous infection. We further investigated the possibility of S. californiensis to serve as a vector for the bacterium via detection of fluorescently labelled A. salmonicida inside the egg sacs from copepods in which the fish hosts were experimentally infected with GFP-A449 A. salmonicida. We then evaluated copepod egg sacs that were collected from adult Chinook salmon from a freshwater hatchery with A. salmonicida infections confirmed by either culture or PCR. The bacterium was cultured on tryptic soy agar plates from 75% of the egg sacs, and 61% were positive by PCR. These three separate experiments indicate an alternative tactic of transmission in addition to direct transmission of A. salmonicida in captivity. The copepod may play an important role in transmission of the bacterium when fish are more dispersed, such as in the wild.

Laboratory infection rates and associated mortality of juvenile Chinook Salmon (Oncorhynchus tshawytscha) from parasitic copepod (Salmincola californiensis).

Pacific salmon (Oncorhynchus spp.) rearing in lakes and reservoirs above dams have been known to become heavily infected with an ectoparasitic copepod (Salmincola californiensis). Little is known about the factors that affect the parasite infection prevalence and intensity. However, previous research suggests that the parasite may negatively affect the fitness and survival of the host fish. The effect of water temperature, confinement and the density of the free-swimming infectious stage of S. californiensis, the copepodid, on infection prevalence and intensity was evaluated by experimentally exposing juvenile Chinook Salmon (O. tshawytscha). Infection rates observed in wild populations were achieved under warm water (15-16°C) and high copepodid density (150-300/L) treatment conditions. Infection prevalence and intensity were also significantly higher in larger fish. During the infection experiment, 4.5% of infected fish died within 54 days with mortality significantly related to copepod infection intensity. The potential for autoinfection was compared to cross-infection by cohabitation of infected fish with naïve fish. Previously infected fish had significantly greater infection intensity compared with naïve fish, indicating that infected fish can be reinfected and that they may be more susceptible than naïve fish.

Biomechanical Feedback Strengthens Jammed Cellular Packings.

Growth in confined spaces can drive cellular populations through a jamming transition from a fluidlike state to a solidlike state. Experiments have found that jammed budding yeast populations can build up extreme compressive pressures (over 1 MPa), which in turn feed back onto cellular physiology by slowing or even stalling cell growth. Using numerical simulations, we investigate how this feedback impacts the mechanical properties of model jammed cell populations. We find that feedback directs growth toward poorly coordinated regions, resulting in an excess number of cell-cell contacts that rigidify cell packings. Cell packings possess anomalously large shear and bulk moduli that depend sensitively on the strength of feedback. These results demonstrate that mechanical feedback on the single-cell level is a simple mechanism by which living systems may tune their population-level mechanical properties.

Rearing environment affects spatial learning in juvenile Chinook salmon Oncorhynchus tshawytscha.

We tested the prediction that a complex physical rearing environment would enhance short-term spatial memory as assessed by learning ability in a spatial navigation task in juvenile Chinook salmon Oncorhynchus tshawytscha. We reared fish in two low-density treatments, where fish were either in bare fiberglass tanks (bare) or in tanks with physical structure (complex). We also tested conventionally reared high-density hatchery fish to compare with these other experimental treatments. Our reason for including this third hatchery treatment is that the two low-density treatments, aside from the manipulation of structure, followed a rearing programme that is designed to produce fish with more wild-like characteristics. We tested individually marked fish for seven consecutive days and recorded movement and time to exit a testing maze. Stimulus conspecific fish outside the exit of the maze provided positive reinforcement for test fish. Fish from the bare treatment were less likely to exit the start box compared with fish in the complex and hatchery treatments. However, fish in the hatchery treatment were significantly more likely to exit the maze on their own compared with both the bare and complex treatments. Hatchery fish effectively learned the task as shown by a decrease in the number of mistakes over time, but the number of mistakes was significantly greater on the first day of trials. Increasing habitat complexity with structure may not necessarily promote spatial learning ability, but differences between hatchery and experimental treatments in rearing density and motivation to be near conspecifics likely led to observed behavioural differences.

The effect of rearing structures on behaviour and movement of juvenile steelhead Oncorhynchus mykiss.

The relative movement of juvenile steelhead Oncorhynchus mykiss reared on two treatments was investigated to provide insight on the effect of structure in the rearing environment on the behaviour of the fish before potential release into a natural river system. The progeny of wild broodstock were reared either in the presence or absence of structure in the tank environment for 7 months at the Oregon Hatchery Research Center located in Oregon, U.S.A. Behavioural assessments, including movement response to a simulated predator, showed that fish reared on structure moved a similar amount (based on line crosses) as fish reared in bare hatchery tanks. No significant difference was observed in the proportion of time spent near a small structure within the behavioural test tank between the two treatments, but all fish showed decreased movement over time with each subsequent predation event. Fish from both treatments spent 30% of their time in the section of the tank containing the structure, which was one section out of a total of eight sections. In both treatments, fish foraged 20% of the time in the 2 min following the introduction of live tubifex prey (two separate events). Overall, similar movement and foraging responses occurred following mock predation events for juveniles reared either with or without structure. Developing assessment tools such as these, that measures behaviours related to survival based on rearing environment should allow managers to better predict the survival and effect of rearing conditions on the release of hatchery-origin fish into the wild.

Egg size and growth in steelhead Oncorhynchus mykiss.

The effects of egg size on early development and growth of steelhead Oncorhynchus mykiss were recorded for more than 200 days following hatching. Fish from smaller eggs hatched sooner and at a smaller size than fish from larger eggs, but fish from smaller eggs showed consistently higher growth rates than fish from larger eggs. Since many life-history attributes appear to be determined by size or growth rate at age during the first year, egg size could be a significant predictor of important changes in the life history of individuals.

Innate and adaptive immune responses in migrating spring-run adult chinook salmon, Oncorhynchus tshawytscha.

Adult Chinook salmon (Oncorhynchus tshawytscha) migrate from salt water to freshwater streams to spawn. Immune responses in migrating adult salmon are thought to diminish in the run up to spawning, though the exact mechanisms for diminished immune responses remain unknown. Here we examine both adaptive and innate immune responses as well as pathogen burdens in migrating adult Chinook salmon in the Upper Willamette River basin. Messenger RNA transcripts encoding antibody heavy chain molecules slightly diminish as a function of time, but are still present even after fish have successfully spawned. In contrast, the innate anti-bacterial effector proteins present in fish plasma rapidly decrease as spawning approaches. Fish also were examined for the presence and severity of eight different pathogens in different organs. While pathogen burden tended to increase during the migration, no specific pathogen signature was associated with diminished immune responses. Transcript levels of the immunosuppressive cytokines IL-10 and TGF beta were measured and did not change during the migration. These results suggest that loss of immune functions in adult migrating salmon are not due to pathogen infection or cytokine-mediated immune suppression, but is rather part of the life history of Chinook salmon likely induced by diminished energy reserves or hormonal changes which accompany spawning.

Salinity effects on plasma ion levels, cortisol, and osmolality in Chinook salmon following lethal sampling.

Studies on hydromineral balance in fishes frequently employ measurements of electrolytes following euthanasia. We tested the effects of fresh- or salt-water euthanasia baths of tricaine mesylate (MS-222) on plasma magnesium (Mg(2+)) and sodium (Na(+)) ions, cortisol and osmolality in fish exposed to saltwater challenges, and the ion and steroid hormone fluctuations over time following euthanasia in juvenile spring Chinook salmon (Oncorhynchus tshawytscha). Salinity of the euthanasia bath affected plasma Mg(2+) and Na(+) concentrations as well as osmolality, with higher concentrations in fish euthanized in saltwater. Time spent in the bath positively affected plasma Mg(2+) and osmolality, negatively affected cortisol, and had no effect on Na(+) concentrations. The difference of temporal trends in plasma Mg(2+) and Na(+) suggests that Mg(2+) may be more sensitive to physiological changes and responds more rapidly than Na(+). When electrolytes and cortisol are measured as endpoints after euthanasia, care needs to be taken relative to time after death and the salinity of the euthanasia bath.

Combining and aggregating environmental data for status and trend assessments: challenges and approaches.

Increasingly, natural resource management agencies and nongovernmental organizations are sharing monitoring data across geographic and jurisdictional boundaries. Doing so improves their abilities to assess local-, regional-, and landscape-level environmental conditions, particularly status and trends, and to improve their ability to make short- and long-term management decisions. Status monitoring assesses the current condition of a population or environmental condition across an area. Monitoring for trends aims at monitoring changes in populations or environmental condition through time. We wrote this paper to inform agency and nongovernmental organization managers, analysts, and consultants regarding the kinds of environmental data that can be combined with suitable techniques and statistically aggregated for new assessments. By doing so, they can increase the (1) use of available data and (2) the validity and reliability of the assessments. Increased awareness of the difficulties inherent in combining and aggregating data for local- and regional-level analyses can increase the likelihood that future monitoring efforts will be modified and/or planned to accommodate data from multiple sources.

Mapping structural variants to rare disease genes using long-read whole genome sequencing and trait-relevant polygenic scores.

Recent studies have revealed the pervasive landscape of rare structural variants (rSVs) present in human genomes. rSVs can have extreme effects on the expression of proximal genes and, in a rare disease context, have been implicated in patient cases where no diagnostic single nucleotide variant (SNV) was found. Approaches for integrating rSVs to date have focused on targeted approaches in known Mendelian rare disease genes. This approach is intractable for rare diseases with many causal loci or patients with complex, multi-phenotype syndromes. We hypothesized that integrating trait-relevant polygenic scores (PGS) would provide a substantial reduction in the number of candidate disease genes in which to assess rSV effects. We further implemented a method for ranking PGS genes to define a set of core/key genes where a rSV has the potential to exert relatively larger effects on disease risk. Among a subset of patients enrolled in the Genomic Answers for Kids (GA4K) rare disease program (N=497), we used PacBio HiFi long-read whole genome sequencing (lrWGS) to identify rSVs intersecting genes in trait-relevant PGSs. Illustrating our approach in Autism (N=54 cases), we identified 22, 019 deletions, 2,041 duplications, 87,826 insertions, and 214 inversions overlapping putative core/key PGS genes. Additionally, by integrating genomic constraint annotations from gnomAD, we observed that rare duplications overlapping putative core/key PGS genes were frequently in higher constraint regions compared to controls (P = 1×10-03). This difference was not observed in the lowest-ranked gene set (P = 0.15). Overall, our study provides a framework for the annotation of long-read rSVs from lrWGS data and prioritization of disease-linked genomic regions for downstream functional validation of rSV impacts. To enable reuse by other researchers, we have made SV allele frequencies and gene associations freely available.

Pangenome graphs improve the analysis of structural variants in rare genetic diseases.

Rare DNA alterations that cause heritable diseases are only partially resolvable by clinical next-generation sequencing due to the difficulty of detecting structural variation (SV) in all genomic contexts. Long-read, high fidelity genome sequencing (HiFi-GS) detects SVs with increased sensitivity and enables assembling personal and graph genomes. We leverage standard reference genomes, public assemblies (n = 94) and a large collection of HiFi-GS data from a rare disease program (Genomic Answers for Kids, GA4K, n = 574 assemblies) to build a graph genome representing a unified SV callset in GA4K, identify common variation and prioritize SVs that are more likely to cause genetic disease (MAF < 0.01). Using graphs, we obtain a higher level of reproducibility than the standard reference approach. We observe over 200,000 SV alleles unique to GA4K, including nearly 1000 rare variants that impact coding sequence. With improved specificity for rare SVs, we isolate 30 candidate SVs in phenotypically prioritized genes, including known disease SVs. We isolate a novel diagnostic SV in KMT2E, demonstrating use of personal assemblies coupled with pangenome graphs for rare disease genomics. The community may interrogate our pangenome with additional assemblies to discover new SVs within the allele frequency spectrum relevant to genetic diseases.

Complex trait associations in rare diseases and impacts on Mendelian variant interpretation.

Emerging evidence implicates common genetic variation - aggregated into polygenic scores (PGS) - impacting the onset and phenotypic presentation of rare diseases. In this study, we quantified individual polygenic liability for 1,151 previously published PGS in a cohort of 2,374 probands enrolled in the Genomic Answers for Kids (GA4K) rare disease study, revealing widespread associations between rare disease phenotypes and PGSs for common complex diseases and traits, blood protein levels, and brain and other organ morphological measurements. We observed increased polygenic burden in probands with variants of unknown significance (VUS) compared to unaffected carrier parents. We further observed an enrichment in overlap between diagnostic and candidate rare disease genes and large-effect PGS genes. Overall, our study supports and expands on previous findings of complex trait associations in rare disease phenotypes and provides a framework for identifying novel candidate rare disease genes and in understanding variable penetrance of candidate Mendelian disease variants.

Complex trait associations in rare diseases and impacts on Mendelian variant interpretation.

Emerging evidence implicates common genetic variation - aggregated into polygenic scores (PGS) - in the onset and phenotypic presentation of rare diseases. Here, we comprehensively map individual polygenic liability for 1102 open-source PGS in a cohort of 3059 probands enrolled in the Genomic Answers for Kids (GA4K) rare disease study, revealing widespread associations between rare disease phenotypes and PGSs for common complex diseases and traits, blood protein levels, and brain and other organ morphological measurements. Using this resource, we demonstrate increased polygenic liability in probands with an inherited candidate disease variant (VUS) compared to unaffected carrier parents. Further, we show an enrichment for large-effect rare variants in putative core PGS genes for associated complex traits. Overall, our study supports and expands on previous findings of complex trait associations in rare diseases, implicates polygenic liability as a potential mechanism underlying variable penetrance of candidate causal variants, and provides a framework for identifying novel candidate rare disease genes.

Using DNA-driven assembled phospholipid nanodiscs as a scaffold for gold nanoparticle patterning.

Recently, a new class of materials emerged with the assembly of DNA-coated phospholipid nanodiscs into columnar BioNanoStacks. Within these stacks, lipid discs are periodically incorporated, resulting into quasi-one-dimensional superstructures. With each disc surrounded by two recombinant scaffolding proteins, we decided to examine whether the polyhistidine tags of these proteins could be utilized to bind additional molecules or particles to these BioNanoStacks. Here we demonstrate that patterning of gold nanoparticles onto these BioNanoStacks is indeed possible. Binding occurs via a nickel-mediated interaction between the nanogolds nitrilotriacetic acid and the histidine tags of the scaffold proteins surrounding the nanodiscs. Using Monte Carlo simulations, we determine that the binding of the nanogold particles to the stacks is not a random event. By comparing the simulation and experimental results, we find that there are preferred binding sites, which affects the binding statistics.

Effect of short-term decrease in water temperature on body temperature and involvement of testosterone in steelhead and rainbow trout, Oncorhynchus mykiss.

The Pacific salmonid species Oncorhynchus mykiss is separated into a migratory form (steelhead trout) and a non-migratory form (rainbow trout). A decrease in water temperature is likely a cue triggering downstream behavior in the migratory form, and testosterone inhibits onset of this behavior. To elucidate differences in sensitivity to water temperature decreases between the migratory and non-migratory forms and effect of testosterone on the sensitivity, we examined two experiments. In experiment 1, we compared changes in body temperature during a short-term decrease in water temperature between both live and dead steelhead and rainbow trout. In experiment 2, we investigated effects of testosterone on body temperature decrease in steelhead trout. Water temperature was decreased by 3°C in 30min. The body temperature of the steelhead decreased faster than that of the rainbow trout. In contrast, there was no significant difference in the decrease in body temperature between dead steelhead and rainbow trout specimens. The body temperature of the testosterone-treated steelhead trout decreased more slowly than that of control fish. Our results suggest that the migratory form is more sensitive to decreases in water temperature than the non-migratory form. Moreover, testosterone might play an inhibitory role in sensitivity to such decreases.

IRF7 and UNC93B1 variants in an infant with recurrent herpes simplex virus infection.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.

Gut microbiome composition associates with corticosteroid treatment, morbidity, and senescence in Chinook salmon (Oncorhynchus tshawytscha).

Pacific salmon experience prolonged elevation in corticosteroid hormones during important life history events including migration, reproduction, and senescence. These periods of elevated corticosteroids correspond with changes to immunity and energy metabolism; therefore, fish may be particularly vulnerable to mortality at these times. Recent studies found that stress-induced cortisol release associated with microbial community shifts in salmonids, raising the question of how longer-term corticosteroid dynamics that accompany life history transitions affect salmonid microbiomes. In this work, we experimentally evaluated the relationships between gut microbiome composition, chronically elevated corticosteroids, and mortality in juvenile Chinook salmon (Oncorhynchus tshawytscha). We found that treatment with slow-release implants of the corticosteroids cortisol or dexamethasone resulted in changes to the gut microbiome. Morbidity was also associated with microbiome composition, suggesting that the gut microbiome reflects individual differences in susceptibility to opportunistic pathogens. Additionally, we analyzed a small number of samples from adult fish at various stages of senescence. Results from these samples suggest that microbiome composition associated with gut integrity, and that the microbial communities of corticosteroid treated juveniles shift in composition toward those of senescent adults. Overall, findings from this work indicate that the gut microbiome correlates with mortality risk during periods of chronic corticosteroid elevation.

Extravillous trophoblast cell lineage development is associated with active remodeling of the chromatin landscape.

The extravillous trophoblast cell lineage is a key feature of placentation and successful pregnancy. Knowledge of transcriptional regulation driving extravillous trophoblast cell development is limited. Here, we map the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem cells and their transition into extravillous trophoblast cells. We show that integrating chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enables identification of transcription factors and regulatory mechanisms critical for extravillous trophoblast cell development. We elucidate functional roles for TFAP2C, SNAI1, and EPAS1 in the regulation of extravillous trophoblast cell development. EPAS1 is identified as an upstream regulator of key extravillous trophoblast cell transcription factors, including ASCL2 and SNAI1 and together with its target genes, is linked to pregnancy loss and birth weight. Collectively, we reveal activation of a dynamic regulatory network and provide a framework for understanding extravillous trophoblast cell specification in trophoblast cell lineage development and human placentation.