OptoProfilin: A Single Component Biosensor of Applied Cellular Stress.

The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

Habitat Diversity Mitigates the Impacts of Human Pressure on Stream Biodiversity.

Recent decades have witnessed substantial changes in freshwater biodiversity worldwide. Although research has shown that freshwater biodiversity can be shaped by changes in habitat diversity and human-induced pressure, the potentials for interaction between these drivers and freshwater biodiversity at large spatial extents remain unclear. To address these issues, we employed a spatially extensive multitrophic fish and insect database from 3323 stream sites across the United States, to investigate the ability of habitat diversity to modulate the effect of human pressure on the richness and abundance of fish and insects. We found evidence that high levels of habitat diversity were associated with increased richness and abundance of fish and insects (including whole-assemblage and individual trophic guilds). We also show that the effects of human pressure on the richness and abundance of fish and insects tend to become positive at high levels of habitat diversity. Where habitat diversity is low, human pressure strongly reduces insect richness and abundance, whereas these reductions are attenuated at high levels of habitat diversity. Structural equation modeling revealed that human pressure reduced habitat diversity, indirectly negatively affecting the richness and abundance of fish and insects. These findings illustrate that, in addition to promoting greater fish and insect biodiversity, habitat diversity may mitigate the deleterious effects of human pressures on these two stream assemblages. Overall, our study suggests that maintaining high levels of habitat diversity is a useful way to protect freshwater biodiversity from ongoing increases in human pressure. However, if human pressures continue to increase, this will reduce habitat diversity, further threatening stream assemblages.

Omernik's Ecoregion Framework: a Legacy for Understanding Regional Patterns in Attainable Resource Quality.

An initial and comprehensive map of ecological regions across the conterminous United States was provided by Omernik in 1987. Because that paper was the most-cited published by the Annals of the American Association of Geographers, we sought to assess and quantify its contribution to science. To do so, we conducted a scientometric analysis to address the following main questions: 1) What are the temporal and spatial citation trends? We expected that Omernik's paper would still be employed 36 years after its publication, and mostly in the United States of America. 2) For what types of environments and organisms has it been applied? Based on its generality, we expected that it had been applied to both terrestrial and aquatic ecosystems. 3) What are the main applications of Omernik's article? We predicted that it would mostly be used for describing and delineating study sites and management areas, as well as for selecting regional reference sites. The number of citations presented a positive temporal increase, indicating its continued applicability. Most papers dealt with aquatic environments, mainly in streams carried out predominantly in the United States of America, as was one of its earliest applications. The usefulness of ecoregions for assessing and managing biotic and abiotic patterns and distributions were the main topics addressed by scientists. Ecoregions have offered a general framework for developing regional expectations and rational regional management policies across large areas, as was their original intent. In addition, ecoregion maps were used for communicating patterns-or the lack of them-to interested scientists, citizens, and decision-makers. That comprehensiveness of Omernik's ecoregion approach has led to its widespread applicability and continued usefulness to a diverse set of scientific and management disciplines.

CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures.

BAR (Bin, Amphiphysin, and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse BAR) domain containing tools derived from the fusion of the Arabidopsis thaliana cryptochrome 2 photoreceptor and I-BAR protein domains ("CRY-BARs") with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane-binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and phosphatidylinositol 4,5-bisphosphate-binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

Fish and macroinvertebrate assemblages reveal extensive degradation of the world's rivers.

Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.

Human land-uses homogenize stream assemblages and reduce animal biomass production.

Human land-use change is a major threat to natural ecosystems worldwide. Nonetheless, the effects of human land-uses on the structure of plant and animal assemblages and their functional characteristics need to be better understood. Furthermore, the pathways by which human land uses affect ecosystem functions, such as biomass production, still need to be clarified. We compiled a unique dataset of fish, arthropod and macrophyte assemblages from 61 stream ecosystems in two Neotropical biomes: Amazonian rainforest and Uruguayan grasslands. We then tested how the cover of agriculture, pasture, urbanization and afforestation affected the taxonomic richness and functional diversity of those three species assemblages, and the consequences of these effects for animal biomass production. Single trait categories and functional diversity were evaluated, combining recruitment and life-history, resource and habitat-use, and body size. The effects of intensive human land-uses on taxonomic and functional diversities were as strong as other drivers known to affect biodiversity, such as local climate and environmental factors. In both biomes, the taxonomic richness and functional diversity of animal and macrophyte assemblages decreased with increasing cover of agriculture, pasture, and urbanization. Human land-uses were associated with functional homogenization of both animal and macrophyte assemblages. Human land-uses reduced animal biomass through direct and indirect pathways mediated by declines in taxonomic and functional diversities. Our findings indicate that converting natural ecosystems to supply human demands results in species loss and trait homogenization across multiple biotic assemblages, ultimately reducing animal biomass production in streams.

Hydrology and water quality shape macroinvertebrate patterns and facilitate non-native species dispersals in an inter-basin water transfer system.

Understanding biotic assemblage variations resulting from water diversions and other pressures is critical for aquatic ecosystem conservation, but hampered by limited research. Mechanisms driving macroinvertebrate assemblages were determined across five lakes along China's South-to-North Water Diversion Project, an over 900-km water transfer system connecting four river basins. We assessed macroinvertebrate patterns from 59 sites in relation to water quality, climatic, spatial, and hydrologic factors. Macroinvertebrate density, biomass, and species richness increased from upriver to downriver lakes, and were higher during the water transfer period than in the non-water transfer period. Non-native species including Nephtys sp., Paranthura japonica, Potamillacf acuminata, Capitekkidae spp. and Novaculina chinensis, were distributed along the entire study system, some become dominant in upriver lakes. High species turnover occurred in two upriver lakes. Hydrology and water quality are critical factors in shaping these macroinvertebrate patterns. Hydrological disturbance by water transfer boosted macroinvertebrate abundance during the water transfer period while facilitated non-native species dispersals and increased biotic homogenization. This study indicates the need for: 1) an effective ecosystem monitoring system; 2) unified system management standards; 3) external pollution controls; and 4) limiting the dispersal of non-native species.

A Cost-Utility Analysis of Remote Pulse-Oximetry Monitoring of Patients With COVID-19.

OBJECTIVES: Since 2020, COVID-19 has infected tens of millions and caused hundreds of thousands of fatalities in the United States. Infection waves lead to increased emergency department utilization and critical care admission for patients with respiratory distress. Although many individuals develop symptoms necessitating a ventilator, some patients with COVID-19 can remain at home to mitigate hospital overcrowding. Remote pulse-oximetry (pulse-ox) monitoring of moderately ill patients with COVID-19 can be used to monitor symptom escalation and trigger hospital visits, as needed. METHODS: We analyzed the cost-utility of remote pulse-ox monitoring using a Markov model with a 3-week time horizon and daily cycles from a US health sector perspective. Costs (US dollar 2020) and outcomes were derived from the University Hospitals' real-world evidence and published literature. Costs and quality-adjusted life-years (QALYs) were used to determine the incremental cost-effectiveness ratio at a cost-effectiveness threshold of $100 000 per QALY. We assessed model uncertainty using univariate and probabilistic sensitivity analyses. RESULTS: Model results demonstrated that remote monitoring dominates current standard care, by reducing costs ($11 472 saved) and improving outcomes (0.013 QALYs gained). There were 87% fewer hospitalizations and 77% fewer deaths among patients with access to remote pulse-ox monitoring. The incremental cost-effectiveness ratio was not sensitive to uncertainty ranges in the model. CONCLUSIONS: Patient with COVID-19 remote pulse-ox monitoring increases the specificity of those requiring follow-up care for escalating symptoms. We recommend remote monitoring adoption across health systems to economically manage COVID-19 volume surges, maintain patients' comfort, reduce community infection spread, and carefully monitor needs of multiple individuals from one location by trained experts.

Physical habitat in conterminous US streams and rivers, Part 1: Geoclimatic controls and anthropogenic alteration.

Anthropogenic alteration of physical habitat structure in streams and rivers is increasingly recognized as a major cause of impairment worldwide. As part of their assessment of the status and trends in the condition of rivers and streams in the U.S., the U.S. Environmental Protection Agency's (USEPA) National Aquatic Resource Surveys (NARS) quantify and monitor channel size and slope, substrate size and stability, instream habitat complexity and cover, riparian vegetation cover and structure, anthropogenic disturbance activities, and channel-riparian interaction. Like biological assemblages and water chemistry, physical habitat is strongly controlled by natural geoclimatic factors that can obscure or amplify the influence of human activities. We developed a systematic approach to estimate the deviation of observed river and stream physical habitat from that expected in least-disturbed reference conditions. We applied this approach to calculate indices of anthropogenic alteration of three aspects of physical habitat condition in the conterminous U.S. (CONUS): streambed sediment size and stability, riparian vegetation cover, and instream habitat complexity. The precision and responsiveness of these indices led the USEPA to use them to evaluate physical habitat condition in CONUS rivers and streams. The scores of these indices systematically decreased with greater anthropogenic disturbance at river and stream sites in the CONUS and within ecoregions, which we interpret as a response of these physical habitat indices to anthropogenic influences. Although anthropogenic activities negatively influenced all three physical habitat indices in the least-disturbed sites within most ecoregions, natural geoclimatic and geomorphic factors were the dominant influences. For sites over the full range of anthropogenic disturbance, analyses of observed/expected sediment characteristics showed augmented flood flows and basin and riparian agriculture to be the leading predictors of streambed instability and excess fine sediments. Similarly, basin and riparian agriculture and non-agricultural riparian land uses were the leading predictors of reduced riparian vegetation cover complexity in the CONUS and within ecoregions. In turn, these reductions in riparian vegetation cover and complexity, combined with reduced summer low flows, were the leading predictors of instream habitat simplification. We conclude that quantitative measures of physical habitat structure are useful and important indicators of the impacts of human activities on stream and river condition.

Physical habitat in conterminous US streams and Rivers, part 2: A quantitative assessment of habitat condition.

Rigorous assessments of the ecological condition of water resources and the effect of human activities on those waters require quantitative physical, chemical, and biological data. The U.S. Environmental Protection Agency's river and stream surveys quantify river and stream bed particle size and stability, instream habitat complexity and cover, riparian vegetation cover and structure, and anthropogenic disturbance activities. Physical habitat is strongly controlled by natural geoclimatic factors that co-vary with human activities. We expressed the anthropogenic alteration of physical habitat as O/E ratios of observed habitat metric values divided by values expected under least-disturbed reference conditions, where site-specific expected values vary given their geoclimatic and geomorphic context. We set criteria for good, fair, and poor condition based on the distribution of O/E values in regional least-disturbed reference sites. Poor conditions existed in 22-24% of the 1.2 million km of streams and rivers in the conterminous U.S. for riparian human disturbance, streambed sediment and riparian vegetation cover, versus 14% for instream habitat complexity. Based on the same four indicators, the percentage of stream length in poor condition within 9 separate U.S. ecoregions ranged from 4% to 42%. Associations of our physical habitat indices with anthropogenic pressures demonstrate the scope of anthropogenic habitat alteration; habitat condition was negatively related to the level of anthropogenic disturbance nationally and in nearly all ecoregions. Relative risk estimates showed that streams and rivers with poor sediment, riparian cover complexity, or instream habitat cover conditions were 1.4 to 2.6 times as likely to also have fish or macroinvertebrate assemblages in poor condition. Our physical habitat condition indicators help explain deviations in biological conditions from those observed among least-disturbed sites and inform management actions for rehabilitating impaired waters and mitigating further ecological degradation.

Habitat and river riparian assessment in the Hyrcanian Forest Ecoregion in Iran: providing basic information for the river management and rehabilitation.

The Hyrcanian Forest holds broad leaf forest remnants dating back to the early Cenozoic Era, which once covered a vast area of the North Temperate Zone. Today, many rivers within this region have been altered by human activities and urgently need rehabilitation. In this regard, 35 wadeable rivers including 14 reference and impacted sites were investigated to determine how different human pressures altered riverine landscapes and habitats. Hence, five common human pressures (agriculture, urbanization, aquaculture, dams, aggregate mining) were identified, then the riverine landscape and habitat condition of each site were assessed. At each site, 17 aquatic, riparian, and terrestrial features, including abiotic and biotic substrate types, were investigated. The number and ratio of pressure-influenced channel features and substrate types differed from those in reference sites. Reference sites were dominated by microlithal, mesolithal, and macrolithal abiotic substrates and large wood, algae, and coarse particulate organic matter biotic substrates. Urbanized sites were most altered and dominated by single channels, steep unvegetated riprap banks, and algae substrate. The results provide valuable information for managers and decision-makers to restore riverine ecosystems considering the impaired parameters resulting from human pressures.

CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells.

The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as ß-amyloid (Aß) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.

Ghost nets: A poorly known threat to Brazilian freshwater biodiversity.

Ghost nets constitute a serious threat to aquatic biodiversity, because they entangle animals as long as they persist in the environment. However, scientific literature in Brazil is virtually silent about this issue in inland ecosystems. Concerned with this gap, we conducted searches on YouTube BR to gather information about ghost nets in Brazilian freshwaters. Through our search, we compiled 33 independent videos showing ghost nets in different aquatic environments. In several cases, we identified entangled animals (i.e., fishes, reptiles, and birds). In this work we also provide recommendations to better understand and mitigate this problem in Brazilian freshwater ecosystems.

Correspondence between a recreational fishery index and ecological condition for U.S.A. streams and rivers.

Sport fishing is an important recreational and economic activity, especially in Australia, Europe and North America, and the condition of sport fish populations is a key ecological indicator of water body condition for millions of anglers and the public. Despite its importance as an ecological indicator representing the status of sport fish populations, an index for measuring this ecosystem service has not been quantified by analyzing actual fish taxa, size and abundance data across the U.S.A. Therefore, we used game fish data collected from 1,561 stream and river sites located throughout the conterminous U.S.A. combined with specific fish species and size dollar weights to calculate site-specific recreational fishery index (RFI) scores. We then regressed those scores against 38 potential site-specific environmental predictor variables, as well as site-specific fish assemblage condition (multimetric index; MMI) scores based on entire fish assemblages, to determine the factors most associated with the RFI scores. We found weak correlations between RFI and MMI scores and weak to moderate correlations with environmental variables, which varied in importance with each of 9 ecoregions. We conclude that the RFI is a useful indicator of a stream ecosystem service, which should be of greater interest to the USA public and traditional fishery management agencies than are MMIs, which tend to be more useful for ecologists, environmentalists and environmental quality agencies.

Sampling Efforts for Estimating Fish Species Richness in Western USA River Sites.

Fish species richness is an indicator of river ecological condition but it is particularly difficult to estimate in large unwadeable rapidly flowing rivers. Intensive multi-gear sampling is time consuming, logistically complex and expensive. However, insufficient sampling effort underestimates species richness and yields inaccurate data about the ecological condition of river sites. We raft-electrofished 10 river sites in 10 different ecoregions and six western USA states for distances equal to 300 times their mean wetted channel widths (MCWs) to estimate the effort needed to approach asymptotes in fish species richness. To collect 90% of the observed fish species at the sites, we found that an average of 150 MCWs (ranging 80-210 MCWs) were needed, with the number of MCWs increasing in rivers with a higher proportion of spatially rare species. Frequently, the second or third additional 100 MCWs produced only one or two additional singletons or doubletons (species occurring only once or twice at a site). Before initiating sampling programs for adequately estimating species richness, we recommend assessing sampling effort, particularly if rare or uncommon species are expected or desired.

A compendium of chemical and genetic approaches to light-regulated gene transcription.

On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.

Imaging of morphological and biochemical hallmarks of apoptosis with optimized optogenetic tools.

Creation of optogenetic switches for specific activation of cell death pathways can provide insights into apoptosis and could also form a basis for noninvasive, next-generation therapeutic strategies. Previous work has demonstrated that cryptochrome 2 (Cry2)/cryptochrome-interacting ß helix-loop-helix (CIB), a blue light-activated protein-protein dimerization module from the plant Arabidopsis thaliana, together with BCL2-associated X apoptosis regulator (BAX), an outer mitochondrial membrane-targeting pro-apoptotic protein, can be used for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we further developed the original light-activated Cry2-BAX system (hereafter referred to as OptoBAX) by improving the photophysical properties and light-independent interactions of this optogenetic switch. The resulting optogenetic constructs significantly reduced the frequency of light exposure required for membrane permeabilization activation and also decreased dark-state cytotoxicity. We used OptoBAX in a series of experiments in Neuro-2a and HEK293T cells to measure the timing of the dramatic morphological and biochemical changes occurring in cells after light-induced MOMP. In these experiments, we used OptoBAX in tandem with fluorescent reporters to image key events in early apoptosis, including membrane inversion, caspase cleavage, and actin redistribution. We then used these data to construct a timeline of biochemical and morphological events in early apoptosis, demonstrating a direct link between MOMP-induced redistribution of actin and apoptosis progression. In summary, we created a next-generation Cry2/CIB-BAX system requiring less frequent light stimulation and established a timeline of critical apoptotic events, providing detailed insights into key steps in early apoptosis.

TrkA overexpression in non-tumorigenic human breast cell lines confers oncogenic and metastatic properties.

BACKGROUND/PURPOSE: TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS: To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS: TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS: Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.