Essentiality of dietary cholesterol and its interactions with phospholipid in juvenile slipper lobster (Thenus australiensis).

This study was conducted to verify the essentiality of dietary cholesterol for early juvenile slipper lobster, Thenus australiensis (initial weight 4.50 ± 0.72 g, mean ± SD, CV = 0.16), and to explore the potential for interactions between dietary cholesterol and phospholipid. An 8-week experiment was conducted using six experimental feeds containing three supplemental cholesterol concentrations (0, 0.2 and 0.4% dry matter) at two supplemental phospholipid concentrations (0% and 1.0% dry matter). Dietary cholesterol concentrations of ≥ 0.2% resulted in up to threefold greater weight gain compared to 0% dietary cholesterol, but without any significant main or interactive dietary phospholipid effect. An interaction was observed for lobster survival with lowest survival (46%) recorded for combined 0% cholesterol and 0% phospholipid compared to every other treatment (71-100%). However, all surviving lobsters at 0% dietary cholesterol, regardless of dietary phospholipid level, were in poor nutritional condition. Apparent feed intake (AFI) was significantly higher at dietary cholesterol ≥ 0.2% but was lower for each corresponding dietary cholesterol level at 1% dietary phospholipid. This implied that the feed conversion ratio was improved with supplemental phospholipid. In conclusion, this study confirms the essential nature of dietary cholesterol and that dietary phospholipid can provide additional benefits.

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Plant Development in the Garden Pea as Revealed by Mutations in the Crd/PsYUC1 Gene.

In common with other plant species, the garden pea (Pisum sativum) produces the auxin indole-3-acetic acid (IAA) from tryptophan via a single intermediate, indole-3-pyruvic acid (IPyA). IPyA is converted to IAA by PsYUC1, also known as Crispoid (Crd). Here, we extend our understanding of the developmental processes affected by the Crd gene by examining the phenotypic effects of crd gene mutations on leaves, flowers, and roots. We show that in pea, Crd/PsYUC1 is important for the initiation and identity of leaflets and tendrils, stamens, and lateral roots. We also report on aspects of auxin deactivation in pea.

Sex-dimorphism in human serum endocannabinoid and n-acyl ethanolamine concentrations across the lifespan.

The endocannabinoid (ECB) system has recently been considered a potential treatment target for various clinical disorders. However, research around age- and sex-related changes within the ECB system is relatively limited. To improve our understanding of these changes, the current study measured arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG), oleoylethanolamine (OEA), palmitoylethanolamine (PEA), arachidonic acid (AA), cortisol, and progesterone in pooled serum samples stratified by sex (male and female) and age groups (5-15; 15-30; 30-45; 45-60; 60-75; 85+), using liquid-chromatography tandem mass spectrometry. Serum progesterone levels significantly increased in females of the 15-30 and 30-45 age groups, before declining. Significantly higher cortisol, AEA, 2-AG, OEA, and PEA were found in males and in older age, while significantly higher AA was found in females. Our results indicate that ECBs and related hormones exhibit sexual dimorphism in the age ranges that correspond with female pregnancy, menopause, and post menopause. Male testosterone levels most likely influences male ECB changes throughout the lifespan. Future research could capitalise on these findings by performing repeated measurements in individuals in a longitudinal style, to further refine the temporal profile of age-specific changes to the ECB system identified here.

Epithelial Yap/Taz are required for functional alveolar regeneration following acute lung injury.

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

Hardware Salvage in the Lower Extremity after Flap Coverage: 10-Year Single Center Outcomes Analysis.

An unanswered question with open tibial fractures is whether the type of flap used affects hardware retention. Flap survival may not equate hardware retention or limb salvage. In this study, we performed a 10-year single institution review and analysis of all patients who had placement of hardware for open tibial fractures followed by flap coverage. Methods: Inclusion criteria consisted of patients who underwent pedicled or free flap coverage of Gustilo IIIB or IIIC tibial fractures requiring open reduction and internal fixation. Outcomes and complications were statistically analyzed based on flap type. Flap type was stratified into free versus pedicled flaps and muscle versus fasciocutaneous flaps. Primary outcome measures included hardware failure and infection requiring hardware removal. Secondary outcome measures included limb salvage, flap success, and fracture union. Results: Overall primary outcome measures were better for pedicled flaps (n = 31), with lower rates of hardware failure and infection (25.8%; 9.7%) compared with free flaps (n = 27) (51.9%; 37.0%). Limb salvage and flap success was not different comparing pedicled and free flaps. There was no significant difference in outcomes between muscle and fasciocutaneous flaps. Multivariable analysis showed that patients who had free versus pedicled flaps or muscle versus fasciocutaneous flaps had a higher chance of hardware failure. A formal orthoplastic team was established in the period from 2017 to 2022, after which flap numbers were higher and hardware failure less for pedicled and fasciocutaneous flaps. Conclusions: Pedicled flaps were associated with lower rates of hardware failure and infection requiring hardware removal. A formal orthoplastic team improves hardware-related outcomes.

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO 2 ), we developed a model of repetitive exposure to SO 2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO 2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO 2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

Hair endocannabinoids predict physiological fear conditioning and salivary endocannabinoids predict subjective stress reactivity in humans.

On the basis of substantial preclinical evidence, the endogenous cannabinoid system has been proposed to be closely involved in stress reactivity and extinction of fear. Existing human research supports this proposal to some extent, but existing studies have used only a narrow range of tools and biomatrices to measure endocannabinoids during stress and fear experiments. In the present study we collected hair and saliva samples from 99 healthy participants who completed a fear conditioning and intrusive memory task. Subjective, physiological and biological stress reactivity to a trauma film, which later served as unconditional stimulus during fear conditioning, was also measured. We found that salivary endocannabinoid concentrations predicted subjective responses to stress, but not cortisol stress reactivity, and replicated previous findings demonstrating a sex dimorphism in hair and salivary endocannabinoid levels. Hair 2-arachidonoyl glycerol levels were significantly associated with better retention of safety learning during extinction and renewal phases of fear conditioning, while hair concentrations of oleoylethanolamide and palmitoylethanolamide were associated with overall physiological arousal, but not conditional learning, during fear conditioning. This study is the first to test the relationship between hair and salivary endocannabinoids and these important psychological processes. Our results suggest that these measures may serve as biomarkers of dysregulation in human fear memory and stress.

Fear conditioning depends on the nature of the unconditional stimulus and may be related to hair levels of endocannabinoids.

The replicability of fear conditioning research has come under recent scrutiny, with increasing acknowledgment that the use of differing materials and methods may lead to incongruent results. Direct comparisons between the main two unconditional stimuli used in fear conditioning - an electric shock or a loud scream-are scarce, and yet these stimuli are usually used interchangeably. In the present study, we tested whether a scream, a shock, or an unpredictable combination of the two affected fear acquisition, extinction, and return of fear amongst healthy participants (N = 109, 81 female). We also collected hair samples and tested the relationship between fear conditioning and hair endocannabinoid levels. Our findings suggest that, although subjective ratings of pleasantness, arousal, and anxiety were similar regardless of the unconditional stimuli used, skin conductance responses were significantly lower for stimuli paired with the scream compared to a shock alone. Further, reducing the predictability of the unconditional stimulus reduced habituation of skin conductance responses during acquisition and reacquisition, but did not produce stronger conditioning compared to shock alone. Exploratory analyses suggested that hair endocannabinoids were associated with overall physiological arousal during fear conditioning, as well as higher return of fear to the threat cue, but not to the safety cue. These findings have multiple implications for the design and replicability of fear conditioning research and provide the first evidence for an association between hair levels of endocannabinoids and human fear conditioning.

Image-based spatial transcriptomics identifies molecular niche dysregulation associated with distal lung remodeling in pulmonary fibrosis.

The human lung is structurally complex, with a diversity of specialized epithelial, stromal and immune cells playing specific functional roles in anatomically distinct locations, and large-scale changes in the structure and cellular makeup of this distal lung is a hallmark of pulmonary fibrosis (PF) and other progressive chronic lung diseases. Single-cell transcriptomic studies have revealed numerous disease-emergent/enriched cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain. Using sub-cellular resolution image-based spatial transcriptomics, we analyzed the gene expression of more than 1 million cells from 19 unique lungs. Through complementary cell-based and innovative cell-agnostic analyses, we characterized the localization of PF-emergent cell-types, established the cellular and molecular basis of classical PF histopathologic disease features, and identified a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Using machine-learning and trajectory analysis methods to segment and rank airspaces on a gradient from normal to most severely remodeled, we identified a sequence of compositional and molecular changes that associate with progressive distal lung pathology, beginning with alveolar epithelial dysregulation and culminating with changes in macrophage polarization. Together, these results provide a unique, spatially-resolved characterization of the cellular and molecular programs of PF and control lungs, provide new insights into the heterogeneous pathobiology of PF, and establish analytical approaches which should be broadly applicable to other imaging-based spatial transcriptomic studies.

Analysis of Citrus Bioflavonoid Content and Dipeptidyl Peptidase-4 Inhibitory Potential of Commercially Available Supplements.

Citrus bioflavonoids are polyphenolic plant-derived pigments found in high levels in oranges, lemons, grapefruits and other citrus fruits. The three most abundant types of citrus bioflavonoids are hesperidin, naringenin and eriocitrin. Citrus bioflavonoids have long been known to possess powerful free radical-scavenging properties and cardioprotective effects. The study involved the analysis of 10 commercially available citrus bioflavonoid supplements from three different countries: Australia, the United States and Canada. The supplements were tested for their citrus bioflavonoid content which varied from 0.8 to 33.3% w/w. The daily bioflavonoid dose varied from 19 mg to 560 mg. Hesperidin was the major citrus bioflavonoid in nine out of ten supplements. One supplement was found to contain less than 10% of the quantity of rutin claimed to have been added. The DPP-4 inhibitory potential, compared through an estimation of rutin equivalence, ranged from 1.9 mg to 400 mg per day. This data highlights the variability between the supplements in their potential to inhibit DPP-4 for subsequent health benefits.

Multi-omics reveals mechanisms of resistance to potato root infection by Spongospora subterranea.

The pathogen Spongospora subterranea infects potato roots and developing tubers resulting in tuber yield and quality losses. Currently, there are no fully effective treatments for disease control. Host resistance is an important tool in disease management and understanding the molecular mechanisms of defence responses in roots of potato plants is required for the breeding of novel resistant cultivars. Here, we integrated transcriptomic and proteomic datasets to uncover these mechanisms underlying S. subterranea resistance in potato roots. This multi-omics approach identified upregulation of glutathione metabolism at the levels of RNA and protein in the resistant cultivar but not in the susceptible cultivar. Upregulation of the lignin metabolic process, which is an important component of plant defence, was also specific to the resistant cultivar at the transcriptome level. In addition, the inositol phosphate pathway was upregulated in the susceptible cultivar but downregulated in the resistant cultivar in response to S. subterranea infection. We provide large-scale multi-omics data of Spongospora-potato interaction and suggest an important role of glutathione metabolism in disease resistance.

Large-Scale Protein and Phosphoprotein Profiling to Explore Potato Resistance Mechanisms to Spongospora subterranea Infection.

Potato is one of the most important food crops for human consumption. The soilborne pathogen Spongospora subterranea infects potato roots and tubers, resulting in considerable economic losses from diminished tuber yields and quality. A comprehensive understanding of how potato plants respond to S. subterranea infection is essential for the development of pathogen-resistant crops. Here, we employed label-free proteomics and phosphoproteomics to quantify systemically expressed protein-level responses to S. subterranea root infection in potato foliage of the susceptible and resistant potato cultivars. A total of 2,669 proteins and 1,498 phosphoproteins were quantified in the leaf samples of the different treatment groups. Following statistical analysis of the proteomic data, we identified oxidoreductase activity, electron transfer, and photosynthesis as significant processes that differentially changed upon root infection specifically in the resistant cultivar and not in the susceptible cultivar. The phosphoproteomics results indicated increased activity of signal transduction and defense response functions in the resistant cultivar. In contrast, the majority of increased phosphoproteins in the susceptible cultivar were related to transporter activity and sub-cellular localization. This study provides new insight into the molecular mechanisms and systemic signals involved in potato resistance to S. subterranea infection and has identified new roles for protein phosphorylation in the regulation of potato immune response.

Shotgun Proteomics as a Powerful Tool for the Study of the Proteomes of Plants, Their Pathogens, and Plant-Pathogen Interactions.

The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant-pathogen interaction. Mass spectrometry (MS)-based proteomics has become the preferred method for characterizing proteins at the proteome and sub-proteome (e.g., the phosphoproteome) levels. MS-based proteomics can reveal changes in the quantitative state of a proteome and provide a foundation for understanding the mechanisms involved in plant-pathogen interactions. This review is intended as a primer for biologists that may be unfamiliar with the diverse range of methodology for MS-based shotgun proteomics, with a focus on techniques that have been used to investigate plant-pathogen interactions. We provide a summary of the essential steps required for shotgun proteomic studies of plants, pathogens and plant-pathogen interactions, including methods for protein digestion, identification, separation, and quantification. Finally, we discuss how protein PTMs may directly participate in the interaction between a pathogen and its host plant.

Modelling growth and histamine formation of Klebsiella aerogenes TI24 isolated from Indonesian pindang.

Indonesian salted-boiled fish (pindang) is a popular traditional food in Indonesia, which is made from Scombroid fish such as tuna and mackerel. As with other traditionally prepared fish products, pindang has important economic and social values, especially for those living in the coastal areas of Indonesia. However, pindang is a major cause of histamine fish poisoning (HFP) for consumers. Klebsiella aerogenes T124, a relatively high histamine-producing isolate from pindang, was used to describe lag time (λ), growth rate (µmax), maximum population density (Nmax), and histamine production in histidine broth and artificially contaminated Grey mackerel. Broth was adjusted to 1.5, 6, 10 and 20% w/v NaCl; mackerel was treated with 6% w/w NaCl, a level common to Indonesian industry practice, or not treated with additional NaCl. Samples were incubated at 10, 15, 20 and 30 °C. In broth, µmax and Nmax were significantly affected by temperature and NaCl, respectively, with λ influenced by both parameters. In control fish, µmax was significantly affected by temperature and NaCl, except at 10 and 15 °C; for 6% NaCl treatment, growth was only observed at 20 and 30 °C. Under similar incubation conditions for broth and fish, histamine formation was markedly affected by NaCl concentration. In broth, -5.1 to -6.6 log µg of histamine was produced per CFU, versus -4.6 to -6.6 log µg per CFU in fish. This study demonstrated that mackerel treated with 6% NaCl and stored at 10-15 °C prevents growth of K. aerogenes strain TI24 and formation of toxic levels of histamine.

Protein levels, air pollution and vitamin D deficiency: links with allergy.

This study provides novel insights into mechanisms of traffic-related air pollution-induced allergy by down-regulation via complement regulators (CFI, PROS1 and PLG) and its interaction with vitamin D deficiency via the complement inhibitor PLG https://bit.ly/3x0jYOw.

In Planta Transcriptome and Proteome Profiles of Spongospora subterranea in Resistant and Susceptible Host Environments Illuminates Regulatory Principles Underlying Host-Pathogen Interaction.

Spongospora subterranea is an obligate biotrophic pathogen, causing substantial economic loss to potato industries globally. Currently, there are no fully effective management strategies for the control of potato diseases caused by S. subterranea. To further our understanding of S. subterranea biology during infection, we characterized the transcriptome and proteome of the pathogen during the invasion of roots of a susceptible and a resistant potato cultivar. A total of 7650 transcripts from S. subterranea were identified in the transcriptome analysis in which 1377 transcripts were differentially expressed between two cultivars. In proteome analysis, we identified 117 proteins with 42 proteins significantly changed in comparisons between resistant and susceptible cultivars. The functional annotation of transcriptome data indicated that the gene ontology terms related to the transportation and actin processes were induced in the resistant cultivar. The downregulation of enzyme activity and nucleic acid metabolism in the resistant cultivar suggests a probable influence of these processes in the virulence of S. subterranea. The protein analysis results indicated that the majority of differentially expressed proteins were related to the metabolic processes and transporter activity. The present study provides a comprehensive molecular insight into the multiple layers of gene regulation that contribute to S. subterranea infection and development in planta and illuminates the role of host immunity in affecting pathogen responses.

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species.

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0-4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27-59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

Spore Germination of the Obligate Biotroph Spongospora subterranea: Transcriptome Analysis Reveals Germination Associated Genes.

For soilborne pathogens, germination of the resting or dormant propagule that enables persistence within the soil environment is a key point in pathogenesis. Spongospora subterranea is an obligate soilborne protozoan that infects the roots and tubers of potato causing root and powdery scab disease for which there are currently no effective controls. A better understanding of the molecular basis of resting spore germination of S. subterranea could be important for development of novel disease interventions. However, as an obligate biotroph and soil dwelling organism, the application of new omics techniques for the study of the pre-infection process in S. subterranea has been problematic. Here, RNA sequencing was used to analyse the reprogramming of S. subterranea resting spores during the transition to zoospores in an in-vitro model. More than 63 million mean high-quality reads per sample were generated from the resting and germinating spores. By using a combination of reference-based and de novo transcriptome assembly, 6,664 unigenes were identified. The identified unigenes were subsequently annotated based on known proteins using BLAST search. Of 5,448 annotated genes, 570 genes were identified to be differentially expressed during the germination of S. subterranea resting spores, with most of the significant genes belonging to transcription and translation, amino acids biosynthesis, transport, energy metabolic processes, fatty acid metabolism, stress response and DNA repair. The datasets generated in this study provide a basic knowledge of the physiological processes associated with spore germination and will facilitate functional predictions of novel genes in S. subterranea and other plasmodiophorids. We introduce several candidate genes related to the germination of an obligate biotrophic soilborne pathogen which could be applied to the development of antimicrobial agents for soil inoculum management.

Chloroform-based liquid-liquid extraction and LC-MS/MS quantification of endocannabinoids, cortisol and progesterone in human hair.

Understanding the role of endogenous cannabinoids (endocannabinoids) in disease is of increasing importance. However, tools to investigate endocannabinoid levels in humans are limited. In the current study, we report a simplified sample preparation method for quantifying endocannabinoids and steroid hormones in hair using liquid-liquid extraction combined with ultra performance liquid chromatography coupled to tandem mass spectrometry. The fully validated method is at least R2 = 0.99 linear between 5 and 1,000 pg/mg for each analyte and the detection limits are at or below 0.50 pg/mg for cortisol, progesterone, oleoylethanolamide, and arachidonoyl ethanolamide, and 2.65 pg/mg for 2-arachidonoyl glycerol. Sequential extraction of hair samples revealed that multiple extractions may be required for quantitative recovery of steroids. However endogenous cannabinoids were efficiently recovered using a single sample extraction. The method was applied to a psychosocial stress study where participants provided samples of both hair and saliva. Endogenous hair arachidonoyl ethanolamide levels were negatively associated with resting, but not stressed, salivary cortisol levels in healthy participants. This simplified method enables the detailed study of hormonal and endocannabinoids in human hair with high sensitivity.