Search Results (218)

Plant growth-promoting bacteria (PGPB)-based inoculants represent a promising alternative to mineral fertilizers. However, their application may be limited by constraints associated with the use of living microorganisms, particularly under field conditions. The objective of this study was to select a bacterial strain and a suitable carrier for the inoculation of rice cv. Selección 1. The effect of inoculation with 3 Rhizobium spp. strains on rice growth was evaluated under greenhouse conditions, enabling selection of the most promising strain. This strain was further characterized based on its motility, production of indolic compounds in the presence of tryptophan, and antagonistic activity against 3 rice phytopathogenic fungi. In addition, the effects of culture media based on aqueous extracts of soybean and rice seeds on bacterial growth and chemotactic response were evaluated, along with the shelf-life stability of the resulting inoculant formulations. Rhizobium sp. strain 5P1 significantly increased plant height (33%), root length (21%), shoot dry weight (30%), and root dry weight (17%) of rice cultivar Selección 1 under greenhouse conditions. The strain exhibited motility predominantly via swarming and twitching, produced indolic compounds (23.9 ± 0.8 µg mL⁻¹), and showed antagonistic activity against Magnaporthe oryzae (32.5% radial growth inhibition at 16 days), Curvularia oryzae (20.0%), and Bipolaris oryzae (6.6%) under in vitro conditions. Culture media based on molasses and soybean or rice seed extracts did not enhance bacterial growth relative to the conventional medium; however, they elicited a stronger chemotactic response. Formulations supplemented with sodium alginate and carboxymethylcellulose maintained cell viability above 10⁸ CFU mL⁻¹ after 105 days of storage at 4 °C. These findings propose Rhizobium sp. strain 5P1 and a molasses-based carrier formulation as strong candidates for the development of an effective bioinoculant for rice in Ferric Gleysol soils. Full article

Depression is a common mental disorder that substantially impairs patients’ daily life and work. To identify natural and safe preventive options, we investigated the preventive effect and underlying mechanism of the Ganoderma lucidum and Rosa roxburghii Tratt formula (GLRRTF) on depression. A total of 72 chemical components in GLRRTF were identified by UHPLC-ESI-Q-Exactive Plus Orbitrap-MS Analysis. GLRRTF (containing 400 mg/kg of G. lucidum extract and 800 mg/kg of R. roxburghii extract per day), administered as a 1-week preventive intervention followed by 4 weeks of co-administration with chronic unpredictable mild stress, prevented the development of depression-like behaviors in male C57BL/6J mice and reduced neuronal damage in the hippocampus. Airflow-assisted desorption electrospray ionization mass spectrometry imaging and enzyme-linked immunosorbent assays showed that GLRRTF corrected abnormalities in neurotransmitter levels. The 16S rRNA sequencing indicated that GLRRTF restored dysbiosis of the gut microbiota. Metabolomic profiling revealed that GLRRTF increased the level of tryptophan and promoted tryptophan metabolism towards the 5-HT and indole pathways in feces and the brain. Western blot demonstrated that GLRRTF increased 5-HT production from tryptophan in the brain by regulating tryptophan hydroxylase 2 and DOPA decarboxylase. GLRRTF activated the PI3K/AKT pathway by regulating brain-derived neurotrophic factor and its receptor tropomyosin receptor kinase B. This research provides a comprehensive mechanistic understanding of GLRRTF’s preventive effect against depression, highlighting its potential as a novel, safe, and preventive functional food formulation. Full article

Tuberculosis (TB) remains a major global cause of morbidity and mortality. Current tools for monitoring treatment response rely on sputum-based microscopy and culture, which are often insensitive, time-consuming, and impractical in extrapulmonary or pediatric TB and in individuals unable to produce sputum. Metabolomics has emerged as a promising approach for identifying host-derived biomarkers that reflect treatment-associated immunometabolic changes; however, the available evidence remains heterogeneous and has not been comprehensively synthesized. We conducted a comprehensive literature review of human studies evaluating metabolomic biomarkers in relation to TB treatment response or outcomes. PubMed, Scopus, and EMBASE were searched for human studies evaluating targeted or untargeted metabolomics (NMR, LC-MS, GC-MS, CE-MS) in relation to treatment response or outcomes. Two reviewers independently screened studies, extracted data, and assessed risk of bias using QUIPS and PROBAST. Findings were synthesized using a structured framework organized across treatment stages and outcomes. Of 218 records identified, 139 titles and abstracts were screened and 42 full texts assessed; 15 studies met the inclusion criteria. Recurrent treatment-associated signals involved amino acid metabolism, particularly the tryptophan–kynurenine pathway, as well as vitamin and cofactor metabolites (pyridoxate, nicotinamide, trigonelline). Plasma studies frequently reported lipid remodeling and bile acid perturbations, whereas urine studies highlighted polyamine metabolism (e.g., N¹,N¹²-diacetylspermine) and fatty acid β-oxidation markers. Common limitations included inadequate adjustment for confounders and, in prediction models, small sample sizes and limited external validation. Metabolomics reveals reproducible but heterogeneous immunometabolic changes during TB therapy. Key pathways include tryptophan–kynurenine metabolism, vitamin and cofactor metabolism, lipid remodeling, and urine polyamine pathways. Standardization and prospective multicenter validation are needed for clinical translation. Full article

Pancreatic lipase (PL) inhibition is a promising dietary strategy for obesity management. In this study, the inhibitory mechanisms and structural basis of polyphenols extracted from different sugarcane fractions were investigated using in vitro enzyme assays, spectroscopy, and molecular docking analyses. PL inhibitory activity was evaluated using p-nitrophenyl laurate (pNPL) as the substrate, with all assays performed in triplicate and results statistically analyzed. Among the extracts, sugarcane peel polyphenols (SP) exhibited the strongest inhibition, with a half-maximal inhibitory concentration (IC₅₀) of 31.56 mg/mL, significantly lower than that of sugarcane juice polyphenols (SJ, 55.86 mg/mL) and sugarcane bagasse polyphenols (SB, 65.31 mg/mL). Enzyme kinetic analyses revealed a reversible mixed-type inhibition mechanism. In contrast to crude extracts, individual phenolic monomers showed substantially lower IC₅₀ values (0.13–1.33 mg/mL), highlighting the intrinsic dilution. Compositional analysis identified ferulic acid, gallic acid, chlorogenic acid, and schaftoside as key contributors to PL inhibition. Fourier transform infrared (FTIR) and fluorescence spectroscopy demonstrated that polyphenols altered PL secondary structure by modulating α-helix and β-sheet contents and perturbed the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. Molecular docking further indicated that these compounds bind within or near the substrate-binding channel via hydrogen bonding and hydrophobic interactions, engaging critical residues including Ser152, His263, and Phe77, and potentially influencing conformational elements involved in active-site accessibility. Collectively, these results suggest that sugarcane, particularly its peel, represents a valuable natural source of PL inhibitors. Despite the relatively high IC₅₀ values of crude extracts, their inhibitory activity arises from multicomponent contributions and supports their potential application as dietary modulators of fat digestion rather than as pharmaceutical lipase inhibitors. Full article

Kynurenic acid (KYNA), a metabolite of the L-kynurenine pathway of L-tryptophan degradation, is an endogenous blocker of glutamate ionotropic excitatory amino acid (EAA) receptors and nicotinic acetylcholine receptors (nAChRs). KYNA plays a significant role in various neuropsychiatric disorders and the aging process. Some researchers have suggested that KYNA may contribute to memory impairment. In this study, we examined the impact of L-kynurenine (a KYNA substrate) and the anti-dementia drugs D-cycloserine and Cerebrolysin on kynurenine aminotransferase (KAT) activity, an enzyme forming KYNA, in liver homogenates of Helix pomatia snails. Furthermore, a memory model was established using these snails, wherein tentacle shortening served as an indicator of learning activity. In vitro experiments on Helix pomatia demonstrated the significant impact of L-kynurenine and anti-dementia drugs on KYNA synthesis. KYNA levels increased significantly in the presence of L-kynurenine in liver homogenate. However, KYNA formation decreased when anti-dementia drugs, including Cerebrolysin or D-cycloserine, were administered to the snails’ liver homogenate. L-kynurenine has been shown to impair the learning process in vivo in snails, but an anti-dementia drug has been demonstrated to reverse this effect. Significant inhibition of tentacle lowering was observed in response to L-kynurenine treatment, which corresponded with elevated KYNA levels in the central nervous system. Administering D-cycloserine or Cerebrolysin alongside L-kynurenine reversed its effects. The Helix pomatia memory model is a valuable tool for studying learning and memory formation in various conditions and in the presence of different pharmacological agents. A drug or natural extract that blocks KYNA synthesis has the ability to increase tentacle lowering and could be considered an anti-dementia agent. Furthermore, this metabolite may also protect against aging and delay damage to the central nervous system related to memory. Full article

Conventional extraction of indigo, a vital natural dye, provides low yields and has a negative environmental impact. However, microbial synthesis has emerged as a sustainable alternative. In this study, we describe the optimization of indigo biosynthesis in an engineered Escherichia coli strain called E216. This strain carries, on a replicative plasmid, the styAB genes originating from Pseudomonas putida that constitute the monooxygenase biosynthetic pathway of indigo, as well as mdh, encoding malate dehydrogenase, which plays a role in reducing power generation. In this strain, the overexpression of mtr (a gene encoding a transporter of tryptophan (Trp), the precursor of indigo biosynthesis) and acrA (a gene encoding a protein involved in indigo efflux) was found to substantially enhance indigo yields. Consistently, knocking out these two genes using CRISPR-Cas9 significantly reduced indigo production, whereas it was restored through the complementation of these mutants. This study thus revealed that stimulating tryptophan uptake and indigo efflux, the latter of which limits indigo’s toxic intracellular accumulation, has a positive impact on indigo yields. Furthermore, a comparative mass spectrometry-based proteomic analysis of E216 grown in fermentation medium with or without tryptophan supplementation, integrated with data-independent acquisition (DIA), revealed the global impact of tryptophan supplementation on cellular metabolism. This analysis identified upregulation of key proteins and enriched metabolic pathways under conditions of tryptophan supplementation. Integrating the results of the genetic engineering and proteomic analysis establishes a strong scientific and practical basis for developing a highly efficient method for the green industrial production of indigo using engineered E. coli strains. Full article

Tyrosinase promotes excessive deposition of melanin, which may lead to severe skin diseases. Passiflora edulis f. edulis seeds have been reported to be rich in diverse bioactive constituents exhibiting potential tyrosinase inhibitory activity. However, the principal bioactive constituents responsible for tyrosinase inhibitory activity and its underlying mechanisms remain largely unclear. Therefore, this study aimed to: (1) optimize SC-CO₂ extraction of Passiflora edulis f. edulis seed oil (PFSO) for maximum yield and bioactive preservation; (2) comprehensively characterize its physicochemical and phytochemical profile; (3) elucidate the tyrosinase inhibition mechanism through kinetic, spectroscopic, and computational approaches; and (4) validate its safety, antioxidant, and anti-pigmentation efficacy in a zebrafish model. PFSO exhibited a yield of 24.96%, with a high content of unsaturated fatty acids (88.03%, mainly linoleic acid at 74.40%). The oil inhibited tyrosinase via a reversible mixed-type mechanism (IC₅₀ = 1.12 mg/mL). Fluorescence spectroscopy and molecular docking revealed that linoleic acid binds to LYS180 and β-sitosterol binds to TYR78, mainly driven by hydrogen bonding and hydrophobic interaction, which changed the microenvironment of tryptophan residues and indicated static quenching. Further validation experiments revealed that the major constituent, linoleic acid, exhibited only weak inhibitory activity against tyrosinase (IC₅₀ = 29.44 mg/mL), whereas the key component β-sitosterol markedly suppressed tyrosinase activity (IC₅₀ = 46.43 μg/mL). In vitro assays demonstrated PFSO’s significant efficacy in reducing the melanin content and tyrosinase activity in α-MSH-stimulated B16F10 murine melanoma cells. In vivo experiments in zebrafish that received dietary supplementation with PFSO confirmed that PFSO (≤5 mg/mL) reduced ROS production, suppressed melanin deposition, inhibited tyrosinase activity, and downregulated the expression of melanogenesis-related genes (TYR, TYRP1, TYRP2, MITF). This study provides, for the first time, a comprehensive elucidation of PFSO’s potential as a natural tyrosinase inhibitor, integrating extraction optimization, multicomponent characterization, multimodal inhibition analysis, and in vivo validation. Full article

Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder with a high incidence of anxiety and depression. However, the underlying mechanisms of these symptoms remain to be fully elucidated. This study investigated the effects and mechanisms of a 20% ethanolic extract of Petasites japonicus leaves (EPJ) on dextran sulfate sodium (DSS)-induced colitis and depression-like behaviors. The physiological compounds identified in the EPJ were citric acid, chlorogenic acid, caffeic acid, fukinolic acid, 3,5-dicaffeoylquinic acid, quercetin 3-O-β-D-glucose-6″-acetate, 4,5-dicaffeoylquinic acid, kaempferol-3-O-(6″-acetyl)-β-glucopyranoside, and pedunculoside. EPJ significantly alleviated DSS-induced colitis, as evidenced by improvements in body weight loss (87.41% vs. 76.02% in the DSS group), colon length (5.75 vs. 4.34 cm), intestinal permeability (52.80 vs. 163.01 μg/mL), and myeloperoxidase (MPO) activity (0.24 vs. 0.67 U/mg) (p < 0.05). Histological analysis further confirmed recovery of goblet cells and attenuation of muscle layer thickening. EPJ also reversed DSS-induced gut microbiota dysbiosis and contributed to the restoration of microbial homeostasis. Behavioral assessments showed that EPJ effectively ameliorated depression-like behaviors. EPJ improved antioxidant systems in colon and brain tissues by modulating malondialdehyde (MDA) levels and reduced glutathione (GSH) and superoxide dismutase (SOD) activity. EPJ further upregulated tight junction protein expression and suppressed TLR4/NF-κB inflammatory pathway activation in both colon and brain tissues. Moreover, EPJ modulated serum stress-related hormones, normalized hypothalamic–pituitary–adrenal (HPA) axis dysregulation, regulated the BDNF/TrkB signaling pathway, and modulated tryptophan–kynurenine metabolism. Collectively, these findings suggest that EPJ exerts protective effects against DSS-induced colitis and depression-like behaviors. Full article

Moringa oleifera leaves are recognized as a nutrient-dense plant material of compositional and nutritional interest. This study aimed to characterize the nutritional and physicochemical properties of M. oleifera dried leaves through nutritional assessment and spectroscopic fingerprinting. Amino acid profiling, antioxidant activity assessment using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and oxygen radical absorbance capacity (ORAC) assays, chromatographic analysis of organic acids and sugars, color measurement, techno-functional characterization, and vibrational spectroscopy including Fourier Transform infrared with attenuated total reflectance (FT-IR/ATR) and Raman were employed. The crude protein content was 16.13 ± 0.43%. Moringa leaves contained all essential amino acids, with notably high tryptophan content (amino acid score, AAS = 200.00%). The amino acids limiting the nutritional value of the protein were primarily sulfur-containing amino acids (AAS = 49.57%) and lysine (AAS = 49.79%). Histidine, leucine, and valine also showed levels below the reference protein. Antioxidant activity exhibited solvent-dependent patterns: the 80% ethanolic extract demonstrated significantly higher FRAP activity (27.05 ± 1.05 mg Trolox Equivalent (TxE)/g dry matter (DM)) and ORAC values (107.24 ± 6.80 mg TxE/g DM), while no statistically significant differences between extracts were observed for DPPH, ABTS, or total phenolic content. Chromatographic profiling identified fructose and glucose as the predominant sugars, alongside citric, succinic, lactic, and acetic acids. The leaves exhibited favorable techno-functional properties, including high water holding capacity and water solubility index. Spectroscopic analysis revealed bands consistent with proteins, lipids, carbohydrates, and glycoside-related structures, while the preserved green-yellow coloration (hue angle 101.68°) indicated retention of pigment-related features during processing. These findings provide compositional and physicochemical characteristics of Moringa leaves relevant to their evaluation as a plant-derived food material. Full article

Aerobic granular sludge (AGS) represents a promising alternative to the conventional activated sludge process for wastewater treatment, owing to its advantages in reducing land area requirements, operational costs, and carbon footprint. With the increasing global implementation of full-scale AGS systems, recent studies have increasingly concentrated on the recovery of valuable resources from waste AGS. AGS is an effective carrier for a variety of valuable substances, including alginate-like exopolymers, polyhydroxyalkanoates, phosphorus, tryptophan, xanthan, curdlan, and cellulose. This study seeks to offer a thorough review of the recovery potential, extraction methodologies and current state of knowledge regarding each of these materials. To improve economic viability, future studies should focus on developing strategies for the sequential recovery of multiple resources from AGS. Furthermore, integrating AGS with other emerging technologies, such as microalgal treatment and a partial nitritation/anammox process, may enhance the reclamation of organic carbon from wastewater. Full article

This study selected the typical eutrophication associated algae species Chlorella pyrenoidosa and Microcystis aeruginosa, from which alginate-like expolymers (ALEs) were extracted. Their composition, structural characteristics, and potential as biofertilizers were systematically analyzed. Results indicate that both C. pyrenoidosa-ALE (Cp-ALE) and M. aeruginosa-ALE (Ma-ALE) primarily comprise proteins and polysaccharides as functional components. Cp-ALE exhibited higher extraction yields (35.34 ± 4.32 mg·g⁻¹ VSS, volatile suspended solids) and richer growth-promoting constituents such as tryptophan, while Ma-ALE demonstrated higher aromaticity in its structure. Pot experiments further demonstrated that both ALEs exhibited a “low-concentration promotion, high-concentration inhibition” effect on ryegrass growth: at the optimal concentration (1:10,000), Cp-ALE and Ma-ALE increased ryegrass dry weight by 61.2% and 59.8%, respectively, with no significant difference compared to the algal whole-cell fertilizer (CF). This study has established a simple, environmentally friendly pathway for resource utilization of microalgal waste. Extracting ALEs effectively preserves plant-promoting components within microalgae, providing not only a sustainable solution for high-value utilization of eutrophication associated algae, but also a viable pathway for green agriculture and circular economic development. Full article

In recent years, plant proteins recycled from agricultural waste have gained increasing attention in food manufacturing due to the relatively low environmental and economic cost. Safflower (Carthamus tinctorius L.) is an important edible oil crop which generates a large amount of seed meal as by-products. Thus, this study aimed to investigate the properties of proteins extracted from the safflower seed meal as food materials. The physicochemical properties and functional characteristics of safflower seed meal protein (SMP) were analyzed at different NaCl concentrations and pH. Results showed that the extraction rate of SMP is 55% and SMP contains 86% protein with an isoelectric point of 4.5. The molecular weight of proteins in SMP predominantly ranged from 10 to 43 kilodaltons (kDa), with a maximum weight loss temperature of 317 °C. Glutamic acid exhibited the highest, while lysine served as the primary limiting amino acid. All seven essential amino acids were present except for tryptophan, which was not included in the testing scope. Additionally, SMP exhibited its highest solubility (59.55%) and emulsifying capacity (62.63 m²/g) at pH 11, and its highest foaming capacity (70.67%) at pH 9. The highest solubility (41.56%) was observed at 1 mol/L NaCl; the highest emulsifying capacity (16.88 m²/g) was observed at 0.6 mol/L NaCl; and the highest foaming capacity (90.67%) was observed at 0.7 mol/L NaCl. This study demonstrates that SMP has excellent nutritional value and a variety of functional properties, making it a promising plant-based protein source for the food processing industry. Subsequent processing involving adjustment to a high pH and increased NaCl concentration can help SMP to exhibit its processing characteristics. Full article

Hypertension is a major global health challenge, with excessive dietary salt intake recognized as a key environmental factor contributing to its pathogenesis. However, safe and effective dietary interventions for salt-sensitive hypertension remain limited. Vine tea (Ampelopsis grossedentata), a traditional herbal tea widely consumed for centuries in southern China, has been reported to exhibit antioxidant, anti-inflammatory, and hepatoprotective activities, yet its antihypertensive efficacy and underlying mechanisms remain unclear. In this study, the chemical profile of vine tea aqueous extract (VTE) was characterized by UPLC–Q–TOF–MS, identifying dihydromyricetin, isoquercitrin, and myricetin as the predominant flavonoids. The protective effects of VTE were evaluated in C57BL/6J mice with high-salt-diet (HSD)-induced hypertension. VTE treatment significantly lowered systolic blood pressure and ameliorated cardiac and renal injury, accompanied by reduced inflammation, fibrosis, and cardiac stress-related gene expression. Gut microbiota analysis using 16S rRNA gene sequencing revealed that VTE restored microbial richness and diversity, enriching short-chain fatty acid-producing taxa while suppressing pathogenic Desulfovibrio and Ruminococcus torques. Untargeted plasma metabolomic profiling based on UPLC–Q–TOF–MS further showed that VTE normalized tryptophan, bile acid, and glycerophospholipid metabolism, decreasing the uremic toxin indoxyl sulfate while increasing tauroursodeoxycholic acid. Notably, these protective effects were abolished under antibiotic-induced microbiota depletion, confirming that VTE acts through a gut microbiota-dependent mechanism. Collectively, VTE mitigates salt-induced hypertension and cardiorenal injury by remodeling the gut microbiota–metabolite axis, supporting its potential as a natural dietary intervention for managing hypertension. Full article

Major depressive disorder remains a leading cause of disability worldwide, with conventional antidepressants offering incomplete and often transient relief. Mounting evidence highlights disturbances in tryptophan (Trp) metabolism as a key biological axis linking inflammation, neuroplasticity, and mood regulation. Plant-derived compounds that modulate this pathway, including 5-hydroxytryptophan, isoflavones, berberine, and polyphenols, have emerged as promising candidates for integrative treatment strategies. Yet, despite encouraging preclinical and clinical findings, knowledge gaps persist regarding long-term efficacy, mechanistic specificity, and standardized therapeutic protocols. This narrative review explores how Trp modulators influence central and peripheral mechanisms relevant to depression, from serotonergic synthesis and kynurenine shunting to gut–brain–immune interactions. Evidence from animal models and randomized clinical trials is critically synthesized, with particular attention to outcomes on mood stabilization, anxiety reduction, cognitive function, and sleep regulation. Special emphasis is placed on translational potential, methodological limitations, and the need for harmonized research frameworks. Here we highlight that phytochemical interventions represent a mechanistically informed and biocompatible strategy for advancing depression management. By bridging neurobiology and clinical psychiatry, these insights may pave the way for next-generation therapeutics that integrate dietary, microbiota-targeted, and anti-inflammatory approaches. Broader application of this research could ultimately refine personalized psychiatry, expand therapeutic horizons, and contribute to global mental health resilience. Full article

Background/Objectives: Nutritional patterns influence the gut–brain axis and immune signaling with potential consequences for depression and anxiety. We conducted a review focused on clinically meaningful psychiatric outcomes (symptom severity/diagnosis) to synthesize recent evidence (2020–2025) on Mediterranean-style dietary interventions; ultra-processed food (UPF) exposure; and psychobiotic/prebiotic strategies, integrating mechanistic insights relevant to practice. Methods: Searches in PubMed/MEDLINE, Scopus, and Web of Science (January 2020–October 2025) combined terms for diet, Mediterranean diet (MD), UPF, microbiota, probiotics, psychobiotics, depression, and anxiety. Eligible designs were randomized/controlled trials (RCTs), prospective cohorts, and systematic reviews/meta-analyses reporting clinical psychiatric outcomes in adults. We prioritized high-quality quantitative syntheses and recent RCTs; data were extracted into a prespecified matrix and synthesized narratively. Results: Recent systematic reviews/meta-analyses support that MD interventions reduce depressive symptoms in adults with major or subthreshold depression, although large, long-term, multicenter RCTs remain a gap. Exposure to UPF is consistently associated with higher risk of common mental disorders and depressive outcomes in large prospective cohorts. Psychobiotics (specific probiotic strains and prebiotics) show small-to-moderate benefits on depressive symptoms across clinical and nonclinical samples, with heterogeneity in strains, dosing, and duration. Mechanistic reviews implicate microbiota-derived metabolites (short-chain fatty acids) and immune–inflammatory signaling (including tryptophan–kynurenine pathways) as plausible mediators. Conclusions: Clinically, emphasizing Mediterranean-style dietary patterns, reducing UPF intake, and considering targeted psychobiotics may complement standard psychiatric care for depression. Future work should prioritize adequately powered, longer RCTs with standardized dietary protocols and microbiome-informed stratification to clarify responders and mechanisms. Full article