The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability.

Salinity is an increasing problem in coastal areas affected by saltwater intrusion, with deleterious effects on tree health and forest growth. Ectomycorrhizal (ECM) fungi may improve the salinity tolerance of host trees, but the impact of external potassium (K+ ) availability on these effects is still unclear. Here, we performed several experiments with the ECM fungus Paxillus ammoniavirescens and loblolly pine (Pinus taeda L.) in axenic and symbiotic conditions at limited or sufficient K+ and increasing sodium (Na+ ) concentrations. Growth rate, biomass, nutrient content, and K+ transporter expression levels were recorded for the fungus, and the colonization rate, root development parameters, biomass, and shoot nutrient accumulation were determined for mycorrhizal and non-mycorrhizal plants. P. ammoniavirescens was tolerant to high salinity, although growth and nutrient concentrations varied with K+ availability and increasing Na+ exposure. While loblolly pine root growth and development decreased with increasing salinity, ECM colonization was unaffected by pine response to salinity. The mycorrhizal influence on loblolly pine salinity response was strongly dependent on external K+ availability. This study reveals that P. ammoniavirescens can reduce Na+ accumulation of salt-exposed loblolly pine, but this effect depends on external K+ availability.

Split down the middle: studying arbuscular mycorrhizal and ectomycorrhizal symbioses using split-root assays.

Most land plants symbiotically interact with soil-borne fungi to ensure nutrient acquisition and tolerance to various environmental stressors. Among these symbioses, arbuscular mycorrhizal and ectomycorrhizal associations can be found in a large proportion of plants, including many crops. Split-root assays are widely used in plant research to study local and systemic signaling responses triggered by local treatments, including nutrient availability, interaction with soil microbes, or abiotic stresses. However, split-root approaches have only been occasionally used to tackle these questions with regard to mycorrhizal symbioses. This review compiles and discusses split-root assays developed to study arbuscular mycorrhizal and ectomycorrhizal symbioses, with a particular emphasis on colonization by multiple beneficial symbionts, systemic resistance induced by mycorrhizal fungi, water and nutrient transport from fungi to colonized plants, and host photosynthate allocation from the host to fungal symbionts. In addition, we highlight how the use of split-root assays could result in a better understanding of mycorrhizal symbioses, particularly for a broader range of essential nutrients, and for multipartite interactions.

The Ectomycorrhizal Fungus Laccaria bicolor Produces Lipochitooligosaccharides and Uses the Common Symbiosis Pathway to Colonize Populus Roots.

Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

Physiological and transcriptomic response of Medicago truncatula to colonization by high- or low-benefit arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi form a root endosymbiosis with many agronomically important crop species. They enhance the ability of their host to obtain nutrients from the soil and increase the tolerance to biotic and abiotic stressors. However, AM fungal species can differ in the benefits they provide to their host plants. Here, we examined the putative molecular mechanisms involved in the regulation of the physiological response of Medicago truncatula to colonization by Rhizophagus irregularis or Glomus aggregatum, which have previously been characterized as high- and low-benefit AM fungal species, respectively. Colonization with R. irregularis led to greater growth and nutrient uptake than colonization with G. aggregatum. These benefits were linked to an elevated expression in the roots of strigolactone biosynthesis genes (NSP1, NSP2, CCD7, and MAX1a), mycorrhiza-induced phosphate (PT8), ammonium (AMT2;3), and nitrate (NPF4.12) transporters and the putative ammonium transporter NIP1;5. R. irregularis also stimulated the expression of photosynthesis-related genes in the shoot and the upregulation of the sugar transporters SWEET1.2, SWEET3.3, and SWEET 12 and the lipid biosynthesis gene RAM2 in the roots. In contrast, G. aggregatum induced the expression of biotic stress defense response genes in the shoots, and several genes associated with abiotic stress in the roots. This suggests that either the host perceives colonization by G. aggregatum as pathogen attack or that G. aggregatum can prime host defense responses. Our findings highlight molecular mechanisms that host plants may use to regulate their association with high- and low-benefit arbuscular mycorrhizal symbionts.

Correlation between the analysis of the dentoalveolar square and the photometric clinical diagnosis of smile.

AIM: Increased vertical growth of the maxilla is a condition that affects a large part of the population. The condition reveals a skeletal alteration of the cranio-masticatory system. One of the effects generated by the excessive vertical growth of the maxilla is a gingival smile pattern that can affect esthetic patterns as well as alter the masticatory biomechanics, which is a primary etiologic factor in temporomandibular dysfunction (TMD). Contemporary imaging aids help to optimize diagnostic analysis; perform treatment; and make an evaluation before, during, and after treatment. The present study aims to compare the clinical diagnosis of gingival smile with the dimensions of the dentoalveolar square, digitally calculated in the panoramic projection of the CBCT. MATERIALS AND METHODS: In a sample of 37 patients, an analysis was performed of the correlation between the dimensions of the dentoalveolar square of the Tatis panoramic cephalometry and the clinical photometry, applying the Tjan gingival smile analysis. RESULTS: The results show that there is high correlation and agreement between the cephalometric measurement method of the dentoalveolar square and Tjan's photometric measurement method. Both methods can be used to classify the smile type as high, medium or low. CONCLUSIONS: Analysis of the dentoalveolar square of the panoramic cephalometry provides an accurate diagnosis of the anterior vertical dimension of the maxilla as it relates to the clinical diagnosis of smile.

Detection of nucleotides in hydrated ssDNA via 2D h-BN nanopore with ionic-liquid/salt-water interface.

Accomplishing slow translocation speed with high sensitivity has been the most critical mission for solid-state nanopore (SSN) device to electrically detect nucleobases in ssDNA. In this study, a method to detect nucleobases of ssDNA using a 2D SSN is introduced by considerably reducing the translocation speed and effectively increasing its sensitivity. The ultra-thin titanium dioxide coated hexagonal boron nitride nanopore was fabricated, along with an ionic-liquid 1-butyl-3-methylimidazolium hexafluorophosphate/2.0 M KCl aqueous (cis/trans) interface, for increasing both the spatial and the temporal resolutions. As the ssDNA molecules entered the nanopore, a brief surge of electrical conductivity occurred, which was followed by multiple resistive pulses from nucleobases during the translocation of ssDNA and another brief current surge flagging the exit of the molecule. The continuous detection of nucleobases using a 2D SSN device is a novel achievement: the water molecules bound to ssDNA increased the molecular conductivity and amplified electrical signals during the translocation. Along with the experiment, computational simulations using COMSOL Multiphysics are presented to explain the pivotal role of water molecules bound to ssDNA to detect nucleobases using a 2D SSN.

Performance characteristics of Fungitell STAT™, a rapid (1→3)-ß-D-glucan single patient sample in vitro diagnostic assay.

Serum (1→3)-ß-D-glucan (BDG), is an adjunct test in the diagnosis of invasive fungal disease (IFD). Fungitell STAT™, a facile, rapid, single patient option, executable for one or more patient specimens in approximately an hour, has been developed to address a need for rapid in-house testing. This method presents qualitative information concerning serum BDG levels, using an index value that allows the rapid categorization of patients as positive, negative, or indeterminate relative to serum BDG titer. The categorical and analytical performance of Fungitell STAT was evaluated. The categorical agreement between methods was established by testing patient samples which had been previously categorized with Fungitell. Receiver Operating Characteristic curves were used to identify cut-offs using 93 de-identified patient specimens. Subsequently, using these cutoffs, an independent group of 488 patient specimens was analyzed. Positive percent agreement (PPA) with, and without, indeterminate results was 74% and 99%, respectively. Negative percent agreement (NPA) was 91% and 98% with, and without, indeterminate results, respectively. Additionally, commercially available normal off-the-clot sera were spiked with Saccharomyces cerevisiae-derived (1→3)-ß-D-glucan to produce analytical samples. Analytical reproducibility using spiked samples was excellent with 94% of the CV (coefficient of variation) values ≤10% among three independent laboratories. Good correlation with the predicate method was demonstrated with correlation coefficients of 0.90 or better with patient samples and 0.99 with spiked samples. The Fungitell STAT index assay provides a rapid and suitable method for serum BDG testing.

Benefits provided by four ectomycorrhizal fungi to Pinus taeda under different external potassium availabilities.

Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.

Nickel-Catalyzed Addition of Aryl Bromides to Aldehydes To Form Hindered Secondary Alcohols.

Transition-metal-catalyzed addition of aryl halides across carbonyls remains poorly developed, especially for aliphatic aldehydes and hindered substrate combinations. We report here that simple nickel complexes of bipyridine and PyBox can catalyze the addition of aryl halides to both aromatic and aliphatic aldehydes using zinc metal as the reducing agent. This convenient approach tolerates acidic functional groups that are not compatible with Grignard reactions, yet sterically hindered substrates still couple in high yield (33 examples, 70% average yield). Mechanistic studies show that an arylnickel, and not an arylzinc, adds efficiently to cyclohexanecarboxaldehyde, but only in the presence of a Lewis acid co-catalyst (ZnBr2).

Nutrient demand and fungal access to resources control the carbon allocation to the symbiotic partners in tripartite interactions of Medicago truncatula.

Legumes form tripartite interactions with arbuscular mycorrhizal fungi and rhizobia, and both root symbionts exchange nutrients against carbon from their host. The carbon costs of these interactions are substantial, but our current understanding of how the host controls its carbon allocation to individual root symbionts is limited. We examined nutrient uptake and carbon allocation in tripartite interactions of Medicago truncatula under different nutrient supply conditions, and when the fungal partner had access to nitrogen, and followed the gene expression of several plant transporters of the Sucrose Uptake Transporter (SUT) and Sugars Will Eventually be Exported Transporter (SWEET) family. Tripartite interactions led to synergistic growth responses and stimulated the phosphate and nitrogen uptake of the plant. Plant nutrient demand but also fungal access to nutrients played an important role for the carbon transport to different root symbionts, and the plant allocated more carbon to rhizobia under nitrogen demand, but more carbon to the fungal partner when nitrogen was available. These changes in carbon allocation were consistent with changes in the SUT and SWEET expression. Our study provides important insights into how the host plant controls its carbon allocation under different nutrient supply conditions and changes its carbon allocation to different root symbionts to maximize its symbiotic benefits.

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum.

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.

The Hebeloma cylindrosporum HcPT2 Pi transporter plays a key role in ectomycorrhizal symbiosis.

Through a mutualistic relationship with woody plant roots, ectomycorrhizal fungi provide growth-limiting nutrients, including inorganic phosphate (Pi), to their host. Reciprocal trades occur at the Hartig net, which is the symbiotic interface of ectomycorrhizas where the two partners are symplasmically isolated. Fungal Pi must be exported to the symbiotic interface, but the proteins facilitating this transfer are unknown. In the present study, we combined transcriptomic, microscopy, whole plant physiology, X-ray fluorescence mapping, 32 P labeling and fungal genetic approaches to unravel the role of HcPT2, a fungal Pi transporter, during the Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizal association. We localized HcPT2 in the extra-radical hyphae and the Hartig net and demonstrated its determinant role for both the establishment of ectomycorrhizas and Pi allocation towards P. pinaster. We showed that the host plant induces HcPT2 expression and that the artificial overexpression of HcPT2 is sufficient to significantly enhance Pi export towards the central cylinder. Together, our results reveal that HcPT2 plays an important role in ectomycorrhizal symbiosis, affecting both Pi influx in the mycelium and efflux towards roots under the control of P. pinaster.

Physiological Responses and Gene Co-Expression Network of Mycorrhizal Roots under K+ Deprivation.

Arbuscular mycorrhizal (AM) associations enhance the phosphorous and nitrogen nutrition of host plants, but little is known about their role in potassium (K+) nutrition. Medicago truncatula plants were cocultured with the AM fungus Rhizophagus irregularis under high and low K+ regimes for 6 weeks. We determined how K+ deprivation affects plant development and mineral acquisition and how these negative effects are tempered by the AM colonization. The transcriptional response of AM roots under K+ deficiency was analyzed by whole-genome RNA sequencing. K+ deprivation decreased root biomass and external K+ uptake and modulated oxidative stress gene expression in M. truncatula roots. AM colonization induced specific transcriptional responses to K+ deprivation that seem to temper these negative effects. A gene network analysis revealed putative key regulators of these responses. This study confirmed that AM associations provide some tolerance to K+ deprivation to host plants, revealed that AM symbiosis modulates the expression of specific root genes to cope with this nutrient stress, and identified putative regulators participating in these tolerance mechanisms.

Synthesis of Spirocyclic Diphosphite-Supported Gold Metallomacrocycles via a Protodeauration/Cyclization Strategy: Mechanistic and Binding Studies.

A spirocylic diphosphite was used to generate P-metalated bimetallic complexes through protodeauration reactions involving LAuC6H4tBu (L = JohnPhos, tBuXPhos) and metallomacrocycles through protodeauration/cyclization using tBuC6H4AuP^PAuC6H4tBu precursors (P^P = flexible diphosphine). While the synthesis of the bimetallic complexes followed a stepwise process, generation of the metallomacrocycles was highly complex because of a series of reversible ligand redistribution reactions. The self-assembly was monitored, and key intermediates were identified by NMR spectroscopy and high-resolution mass spectrometry. The mechanistic investigation showed that using flexible diphosphine linkers was critical to the selective synthesis of metallomacrocycles because rigid diphosphines generated intractable mixtures of linear and cyclic compounds. The X-ray structure of a 32-membered metallomacrocycle revealed that the compound crystallized in an unsymmetrical collapsed form that was held together by two supported aurophilic interactions while the flexible diphosphines were folded along opposite sides of the metallomacrocycle. The solution structure was consistent with a symmetric species, which suggested interconversion between an open and collapsed form and/or rapid twisting of a collapsed form. The 32-membered metallomacrocycle was used to bind estrogen primarily through the formation of AuP-O-···H-OR hydrogen bonds.

Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes.

Access to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2 to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.

Molecular signals required for the establishment and maintenance of ectomycorrhizal symbioses.

Ectomycorrhizal (ECM) symbioses are among the most widespread associations between roots of woody plants and soil fungi in forest ecosystems. These associations contribute significantly to the sustainability and sustainagility of these ecosystems through nutrient cycling and carbon sequestration. Unfortunately, the molecular mechanisms controlling the mutual recognition between both partners are still poorly understood. Elegant work has demonstrated that effector proteins from ECM and arbuscular mycorrhizal (AM) fungi regulate host defenses by manipulating plant hormonal pathways. In parallel, genetic and evolutionary studies in legumes showed that a 'common symbiosis pathway' is required for the establishment of the ancient AM symbiosis and has been recruited for the rhizobia-legume association. Given that genes of this pathway are present in many angiosperm trees that develop ectomycorrhizas, we propose their potential involvement in some but not all ECM associations. The maintenance of a successful long-term relationship seems strongly regulated by resource allocation between symbiotic partners, suggesting that nutrients themselves may serve as signals. This review summarizes our current knowledge on the early and late signal exchanges between woody plants and ECM fungi, and we suggest future directions for decoding the molecular basis of the underground dance between trees and their favorite fungal partners.

Potassium nutrition of ectomycorrhizal Pinus pinaster: overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K(+) and phosphorus in the host plant.

Mycorrhizal associations are known to improve the hydro-mineral nutrition of their host plants. However, the importance of mycorrhizal symbiosis for plant potassium nutrition has so far been poorly studied. We therefore investigated the impact of the ectomycorrhizal fungus Hebeloma cylindrosporum on the potassium nutrition of Pinus pinaster and examined the involvement of the fungal potassium transporter HcTrk1. HcTrk1 transcripts and proteins were localized in ectomycorrhizas using in situ hybridization and EGFP translational fusion constructs. Importantly, an overexpression strategy was performed on a H. cylindrosporum endogenous gene in order to dissect the role of this transporter. The potassium nutrition of mycorrhizal pine plants was significantly improved under potassium-limiting conditions. Fungal strains overexpressing HcTrk1 reduced the translocation of potassium and phosphorus from the roots to the shoots of inoculated plants in mycorrhizal experiments. Furthermore, expression of HcTrk1 and the phosphate transporter HcPT1.1 were reciprocally linked to the external inorganic phosphate and potassium availability. The development of these approaches provides a deeper insight into the role of ectomycorrhizal symbiosis on host plant K(+) nutrition and in particular, the K(+) transporter HcTrk1. The work augments our knowledge of the link between potassium and phosphorus nutrition via the mycorrhizal pathway.

Resolved P-metalated nucleoside phosphoramidites.

The synthesis of resolved P-metalated nucleoside phosphoramidites is described. These rare compounds were initially prepared with gold as the metal center; however, the gold can be removed using basic phosphines or solid-supported triphenylphosphine. Treatment of the free nucleoside phosphoramidite with a platinum source generated a unique platinated dinucleoside species with a diastereomeric ratio of >99:1.

Fungal biomass and ectomycorrhizal community assessment of phosphorus responsive Pinus taeda plantations.

Ectomycorrhizal fungi and non-ectomycorrhizal fungi are responsive to changes in environmental and nutrient availabilities. Although many species of ectomycorrhizas are known to enhance the uptake of phosphorus and other nutrients for Pinus taeda, it is not understood how to optimize these communities to have tangible effects on plantation silviculture and P use efficiency. The first step of this process is the identification of native fungi present in the system that are associated with P. taeda and influence P uptake efficiency. We used sand-filled mesh bags baited with finely ground apatite to sample ectomycorrhizal and non-ectomycorrhizal fungi associated with the rhizosphere of P-responsive P. taeda under several field conditions. Mesh bags were assessed for biomass accumulation over three years using a single three-month burial period pre-harvest and three six-month burial periods post-planting. Amplicon sequencing assessed ectomycorrhizal and non-ectomycorrhizal communities between phosphorus treatments, sites, mesh bags, and the rhizosphere of actively growing P. taeda in the field. We found biomass accumulation within the mesh bags was inversely related to increasing phosphorus fertilization (carryover) rates from pre-harvest to post-planting. Up to 25% increases in total biomass within the bags were observed for bags baited with P. Taxonomic richness was highest in Alfisol soils treated with phosphorus from the previous rotation and lowest in the Spodosol regardless of phosphorus treatment.

Substance use typologies among young people experiencing homelessness in seven cities across the United States: A latent class analysis.

BACKGROUND: Substance use and other health-risk risk factors, including mental health, trauma, and sexual-risk behaviors, often co-occur among youth experiencing homelessness (YEH). The present study aimed to identify subgroups of YEH based on polysubstance use and the linkages to sociodemographic and health-risk characteristics. METHODS: From June 2016 to July 2017, 1,426 YEH (aged 18-26 years) were recruited from seven cities (Houston, Los Angeles, Denver, Phoenix, New York City, St. Louis, San Jose). Participants provided information via a self-administered electronic survey on substance use, mental health, trauma, sexual risk behaviors, and sociodemographic characteristics. The majority of YEH identified as Black (37.3%), cisgender (92.8%), and heterosexual (69.2%). On average, YEH were 20.9 years (SD = 2.1). This study employed latent class analysis (LCA) to identify subgroups of YEH according to their substance use. Multinomial logistic regression analyses were conducted to identify sociodemographic and health-risk characteristics associated with class-membership. RESULTS: Four latent classes of YEH substance use were identified: (1) high polysubstance use; (2) moderate stimulant and high marijuana, alcohol, and prescription drug use; (3) high marijuana, alcohol, and prescription drug use; (4) low/moderate marijuana and alcohol use. Multinomial logistic regression models indicated that geographic location, gender, race/ethnicity, mental health, trauma history, and sexual risk behaviors were significant correlates of substance use class membership among YEH. CONCLUSIONS: These findings offer important implications for the prevention and treatment of substance use among YEH. Screening protocols should consider co-occurring risk factors such as traumatic experiences, sexual risk behaviors, and mental health history as indicators of polysubstance use.