Selective Modulation of the Human Glucocorticoid Receptor Compromises GR Chromatin Occupancy and Recruitment of p300/CBP and the Mediator Complex.

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.

Dynamic turnover of paused Pol II complexes at human promoters.

Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5' paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5' ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

First-in-Human Phase I Study of Minnelide in Patients With Advanced Gastrointestinal Cancers: Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity.

BACKGROUND: Minnelide is a water-soluble prodrug of triptolide. Triptolide is an anticancer agent that targets cancer resistance through several mechanisms. Minnelide was evaluated in a phase I study in patients with advanced GI carcinomas to establish the safety, pharmacodynamic, antitumor activity, and recommended phase II dose (RP2D). PATIENTS AND METHODS: Patients with refractory GI carcinoma and with measurable disease on CT scan were eligible. The study used a 3 + 3 dose-escalation scheme. Due to neutropenia toxicity, 2 dosing schedules were evaluated to determine the RP2D for future studies. Response was assessed using RECIST 1.1 and Choi criteria. Minnelide and triptolide PK were evaluated. Patients who completed the first 28-day treatment cycle without DLTs continued treatment until disease progression or unacceptable toxicity. RESULTS: Forty-five patients were enrolled (23 pancreatic cancer, 10 colorectal, and the remaining 9 had other GI tumors); 42 patients received at least one dose of Minnelide. Grade ≥ 3 toxicities occurred in 69% of patients, most common neutropenia (38%). 2 patients with severe cerebellar toxicity who had a 2-fold higher triptolide concentration than other participants. ORR was 4%; the disease control rate (DCR) was 54% (15/28). Choi criteria demonstrated a decrease in average tumor density in 57% (16/28) patients. CONCLUSIONS: This first-in-human, phase I clinical study identified a dose and schedule of Minnelide in patients with refractory GI cancers. The primary toxicity experienced was hematologic. Evidence of efficacy of Minnelide treatment in this group of patients was observed. The DCR ranged from ~2 to 6 months in 14/28 (50%) of evaluable patients. Studies in monotherapy and combination treatments are underway.

Metagenomic and proteomic insights into the self-adaptive cell surface hydrophobicity of Sphingomonas sp. strain PAH02 reducing the migration of cadmium-phenanthrene co-pollutant in rice.

Cell surface hydrophobicity (CSH) dominates the interactions between rhizobacteria and pollutants at the soil-water interface, which is critical for understanding the dissipation of pollutants in the rhizosphere microzone of rice. Herein, we explored the effects of self-adaptive CSH of Sphingomonas sp. strain PAH02 on the translocation and biotransformation behaviour of cadmium-phenanthrene (Cd-Phe) co-pollutant in rice and rhizosphere microbiome. We evidenced that strain PAH02 reduced the adsorption of Cd-Phe co-pollutant on the rice root surface while enhancing the degradation of Phe and adsorption of Cd via its self-adaptive CSH in the hydroponic experiment. The significant upregulation of key protein expression levels such as MerR, ARHDs and enoyl-CoA hydratase/isomerase, ensures self-adaptive CSH to cope with the stress of Cd-Phe co-pollutant. Consistently, the bioaugmentation of strain PAH02 promoted the formation of core microbiota in the rhizosphere soil of rice (Oryza sativa L.), such as Bradyrhizobium and Streptomyces and induced gene enrichment of CusA and PobA that are strongly associated with pollutant transformation. Consequently, the contents of Cd and Phe in rice grains at maturity decreased by 17.2% ± 0.2% and 65.7% ± 0.3%, respectively, after the bioaugmentation of strain PAH02. These findings present new opportunities for the implementation of rhizosphere bioremediation strategies of co-contaminants in paddy fields.

Suppression of retinal neovascularization by intravitreal injection of cryptotanshinone.

Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.

Triptolide promotes differentiation of human monocytes into immunosuppressive MDSCs.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) negatively modulate immune activity. Prior investigations have shown much promise in using MDSCs-assisted immunotherapy for organ transplantation patients. Additionally, owing to its immunosuppressive activity, MDSCs can also be used to manage immune-associated disorders. METHODS: Granulocyte-macrophage colony-stimulating factor (GM-CSF) was employed to stimulate myeloid progenitor cell differentiation. Triptolide (PG490) was introduced toward the later phases of in vitro MDSCs induction. Lastly, real-time PCR (RT-PCR) and flow cytometry were used to assess transcript expression and cell phenotype, and a mouse skin transplantation model was established to evaluate the MDSCs-mediated immune suppression in vivo. RESULTS: Co-stimulation with PG490 and GM-CSF potently induced myeloid-derived monocytes to form MDSCs, with remarkable immune-suppressive activity. The underlying mechanism involved downregulation of T cell proliferation, activation, enhancement of inflammatory cytokine release, as well as T cell conversion to Treg cells. PG490 strongly enhanced iNOS expression in MDSCs, and iNOS inhibition successfully reversed the immune-suppression. The PG490- and GM-CSF-induced MDSCs substantially extended survival duration of murine skin grafts, thereby validating their strong immune-suppressive activity in vivo. CONCLUSIONS: Herein, we presented a new approach involving MDSCs-based immunosuppression in vitro. PG490 and GM-CSF co-treatment strongly induced immuno-suppressive activity in MDSCs both in vitro and in vivo. Our findings highlight the promise of applying MDSCs-based therapy in clinical organ transplantation treatment.

Determination of soil phenanthrene degradation through a fungal-bacterial consortium.

Fungal-bacterial consortia enhance organic pollutant removal, but the underlying mechanisms are unclear. We used stable isotope probing (SIP) to explore the mechanism of bioaugmentation involved in polycyclic aromatic hydrocarbon (PAH) biodegradation in petroleum-contaminated soil by introducing the indigenous fungal strain Aspergillus sp. LJD-29 and the bacterial strain Pseudomonas XH-1. While each strain alone increased phenanthrene (PHE) degradation, the simultaneous addition of both strains showed no significant enhancement compared to treatment with XH-1 alone. Nonetheless, the assimilation effect of microorganisms on PHE was significantly enhanced. SIP revealed a role of XH-1 in PHE degradation, while the absence of LJD-29 in 13C-DNA indicated a supporting role. The correlations between fungal abundance, degradation efficiency, and soil extracellular enzyme activity indicated that LJD-29, while not directly involved in PHE assimilation, played a crucial role in the breakdown of PHE through extracellular enzymes, facilitating the assimilation of metabolites by bacteria. This observation was substantiated by the results of metabolite analysis. Furthermore, the combination of fungus and bacterium significantly influenced the diversity of PHE degraders. Taken together, this study highlighted the synergistic effects of fungi and bacteria in PAH degradation, revealed a new fungal-bacterial bioaugmentation mechanism and diversity of PAH-degrading microorganisms, and provided insights for in situ bioremediation of PAH-contaminated soil.IMPORTANCEThis study was performed to explore the mechanism of bioaugmentation by a fungal-bacterial consortium for phenanthrene (PHE) degradation in petroleum-contaminated soil. Using the indigenous fungal strain Aspergillus sp. LJD-29 and bacterial strain Pseudomonas XH-1, we performed stable isotope probing (SIP) to trace active PHE-degrading microorganisms. While inoculation of either organism alone significantly enhanced PHE degradation, the simultaneous addition of both strains revealed complex interactions. The efficiency plateaued, highlighting the nuanced microbial interactions. SIP identified XH-1 as the primary contributor to in situ PHE degradation, in contrast to the limited role of LJD-29. Correlations between fungal abundance, degradation efficiency, and extracellular enzyme activity underscored the pivotal role of LJD-29 in enzymatically facilitating PHE breakdown and enriching bacterial assimilation. Metabolite analysis validated this synergy, unveiling distinct biodegradation mechanisms. Furthermore, this fungal-bacterial alliance significantly impacted PHE-degrading microorganism diversity. These findings advance our understanding of fungal-bacterial bioaugmentation and microorganism diversity in polycyclic aromatic hydrocarbon (PAH) degradation as well as providing insights for theoretical guidance in the in situ bioremediation of PAH-contaminated soil.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

Activity-Based Protein Profiling to Probe Relationships between Cytochrome P450 Enzymes and Early-Age Metabolism of Two Polycyclic Aromatic Hydrocarbons (PAHs): Phenanthrene and Retene.

A growing body of literature has linked early-life exposures to polycyclic aromatic hydrocarbons (PAH) with adverse neurodevelopmental effects. Once in the body, metabolism serves as a powerful mediator of PAH toxicity by bioactivating and detoxifying PAH metabolites. Since enzyme expression and activity vary considerably throughout human development, we evaluated infant metabolism of PAHs as a potential contributing factor to PAH susceptibility. We measured and compared rates of phenanthrene and retene (two primary PAH constituents of woodsmoke) metabolism in human hepatic microsomes from individuals ≤21 months of age to a pooled sample (n = 200) consisting primarily of adults. We used activity-based protein profiling (ABPP) to characterize cytochrome P450 enzymes (CYPs) in the same hepatic microsome samples. Once incubated in microsomes, phenanthrene demonstrated rapid depletion. Best-fit models for phenanthrene metabolism demonstrated either 1 or 2 phases, depending on the sample, indicating that multiple enzymes could metabolize phenanthrene. We observed no statistically significant differences in phenanthrene metabolism as a function of age, although samples from the youngest individuals had the slowest phenanthrene metabolism rates. We observed slower rates of retene metabolism compared with phenanthrene also in multiple phases. Rates of retene metabolism increased in an age-dependent manner until adult (pooled) metabolism rates were achieved at ∼12 months. ABPP identified 28 unique CYPs among all samples, and we observed lower amounts of active CYPs in individuals ≤21 months of age compared to the pooled sample. Phenanthrene metabolism correlated to CYPs 1A1, 1A2, 2C8, 4A22, 3A4, and 3A43 and retene metabolism correlated to CYPs 1A1, 1A2, and 2C8 measured by ABPP and vendor-supplied substrate marker activities. These results will aid efforts to determine human health risk and susceptibility to PAHs exposure during early life.

A Phenanthrenequinone-Based Ratiometric Fluorescent Probe for Rapid Detection of Peroxynitrite with Imaging in Osteoblast Precursor Cells.

In the current situation, peroxynitrite (ONOO-) is drawing the increasing attention of researchers for its pivotal role in diverse pathological and physiological processes on grounds of robust oxidation and nitrification. Herein, we have successfully designed and synthesized a phenanthrenequinone benzyl borate-based chemosensor for fast and selective detection of ONOO-. The probe PTDP itself had an orange fluorescence, which was changed to strong blue fluorescence upon the addition of ONOO-, indicating the ratiometric response of the probe. This is so because of the cleavage of the benzyl boronate-protecting group of PTDP upon the addition of ONOO- with simultaneous releasing of pyridinyl-based chemosensor PPI. The PTDP showed outstanding performance in the various photophysical studies such as good selectivity, excellent sensitivity with a very low detection limit of 2.74 nM, and a very fast response time (<15 s). Furthermore, for practical applicability, it was successfully applied in the ratiometric detection of ONOO- in osteoblast precursor cells.

CHIR-98014, a GSK 3ß Inhibitor, Protects Against Triptolide/Lipopolysaccharide-Induced Hepatotoxicity by Mitochondria-Dependent Apoptosis Inhibition.

Triptolide (TP) is a remarkable anti-inflammatory and immunosuppressive component separated from Tripterygium wilfordii Hook. F. However, its hepatotoxicity limits its application in the clinical. Our group has proposed a new perspective on TP-induced hepatotoxicity, in which TP enhances liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. Because the cause of the disease is unknown, there is currently no uniform treatment available. In this study, we attempted to determine whether the GSK-3ß-JNK pathway affects liver damage and its regulatory mechanism in response to TP/LPS costimulation. In addition, we investigated the effect of CsA or the GSK 3ß inhibitor CHIR-98014 on TP/LPS-induced hepatotoxicity. The results showed that the TP/LPS cotreatment mice exhibited obvious hepatotoxicity, as indicated by a remarkable increase in the serum ALT and AST levels, glycogen depletion, GSK 3ß-JNK upregulation, and increased apoptosis. Instead of the specific knockdown of JNK1, the specific knockdown of JNK2 had a protective effect. Additionally, 40 mg/kg of CsA and 30 mg/kg of CHIR-98014 might provide protection. In summary, CHIR-98014 could protect against TP/LPS- or TP/TNF-α-induced activation of the GSK 3ß-JNK pathway and mitochondria-dependent apoptosis, improving the indirect hepatotoxicity induced by TP.

Detrimental Role of CXCR3 in α-Naphthylisothiocyanate- and Triptolide-Induced Cholestatic Liver Injury.

The chemokine receptor CXCR3 is functionally pleiotropic, not only recruiting immune cells to the inflamed liver but also mediating the pathological process of cholestatic liver injury (CLI). However, the mechanism of its involvement in the CLI remains unclear. Both alpha-naphthylisothiocyanate (ANIT) and triptolide are hepatotoxicants that induce CLI by bile acid (BA) dysregulation, inflammation, and endoplasmic reticulum (ER)/oxidative stress. Through molecular docking, CXCR3 is a potential target of ANIT and triptolide. Therefore, this study aimed to investigate the role of CXCR3 in ANIT- and triptolide-induced CLI and to explore the underlying mechanisms. Wild-type mice and CXCR3-deficient mice were administered with ANIT or triptolide to compare CLI, BA profile, hepatic recruitment of IFN-γ/IL-4/IL-17+CD4+T cells, IFN-γ/IL-4/IL-17+iNKT cells and IFN-γ/IL-4+NK cells, and the expression of ER/oxidative stress pathway. The results showed that CXCR3 deficiency ameliorated ANIT- and triptolide-induced CLI. CXCR3 deficiency alleviated ANIT-induced dysregulated BA metabolism, which decreased the recruitment of IFN-γ+NK cells and IL-4+NK cells to the liver and inhibited ER stress. After triptolide administration, CXCR3 deficiency ameliorated dysregulation of BA metabolism, which reduced the migration of IL-4+iNKT cells and IL-17+iNKT cells and reduced oxidative stress through inhibition of Egr1 expression and AKT phosphorylation. Our findings suggest a detrimental role of CXCR3 in ANIT- and triptolide-induced CLI, providing a promising therapeutic target and introducing novel mechanisms for understanding cholestatic liver diseases.

Quantifying the Lymphatic Transport of Model Therapeutics from the Brain in Rats.

In recent years, the drainage of fluids, immune cells, antigens, fluorescent tracers, and other solutes from the brain has been demonstrated to occur along lymphatic outflow pathways to the deep cervical lymph nodes in the neck. To the best of our knowledge, no studies have evaluated the lymphatic transport of therapeutics from the brain. The objective of this study was to determine the lymphatic transport of model therapeutics of different molecular weights and lipophilicity from the brain using cervical lymph cannulation and ligation models in rats. To do this, anesthetized Sprague-Dawley rats were cannulated at the carotid artery and cannulated, ligated, or left intact at the cervical lymph duct. Rats were administered 14C-ibuprofen (206.29 g/mol, logP 3.84), 3H-halofantrine HCl (536.89 g/mol, logP 8.06), or 3H-albumin (∼65,000 g/mol) via direct injection into the brain striatum at a rate of 0.5 µL/min over 16 min. Plasma or cervical lymph samples were collected for up to 6-8 h following dosing, and brain and lymph nodes were collected at 6 or 8 h. Samples were subsequently analyzed for radioactivity levels via scintillation counting. For 14C-ibuprofen, plasma concentrations over time (plasma AUC0-6h) were >2 fold higher in lymph-ligated rats than in lymph-intact rats, suggesting that ibuprofen is cleared from the brain primarily via nonlymphatic routes (e.g., across the blood-brain barrier) but that this clearance is influenced by changes in lymphatic flow. For 3H-halofantrine, >73% of the dose was retained at the brain dosing site in lymph-intact and lymph-ligated groups, and plasma AUC0-8h values were low in both groups (<0.3% dose.h/mL), consistent with the high retention in the brain. It was therefore not possible to determine whether halofantrine undergoes lymphatic transport from the brain within the duration of the study. For 3H-albumin, plasma AUC0-8h values were not significantly different between lymph-intact, lymph-ligated, and lymph-cannulated rats. However, >4% of the dose was recovered in cervical lymph over 8 h. Lymph/plasma concentration ratios of 3H-albumin were also very high (up to 53:1). Together, these results indicate that 3H-albumin is transported from the brain not only via lymphatic routes but also via the blood. Similar to other tissues, the lymphatics may thus play a significant role in the transport of macromolecules, including therapeutic proteins, from the brain but are unlikely to be a major transport pathway from the brain for small molecule drugs that are not lipophilic. Our rat cervical lymph cannulation model can be used to quantify the lymphatic drainage of different molecules and factors from the brain.

Bioremediation of petroleum refinery wastewater using Bacillus subtilis IH-1 and assessment of its toxicity.

Environmental contamination from petroleum refinery operations has increased due to the rapid population growth and modernization of society, necessitating urgent repair. Microbial remediation of petroleum wastewater by prominent bacterial cultures holds promise in circumventing the issue of petroleum-related pollution. Herein, the bacterial culture was isolated from petroleum-contaminated sludge samples for the valorization of polyaromatic hydrocarbons and biodegradation of petroleum wastewater samples. The bacterial strain was screened and identified as Bacillus subtilis IH-1. After six days of incubation, the bacteria had degraded 25.9% of phenanthrene and 20.3% of naphthalene. The treatment of wastewater samples was assessed using physico-chemical and Fourier-transform infrared spectroscopy analysis, which revealed that the level of pollutants was elevated and above the allowed limits. Following bacterial degradation, the reduction in pollution parameters viz. EC (82.7%), BOD (87.0%), COD (80.0%), total phenols (96.3%), oil and grease (79.7%), TKN (68.8%), TOC (96.3%) and TPH (52.4%) were observed. The reduction in pH and heavy metals were also observed after bacterial treatment. V. mungo was used in the phytotoxicity test, which revealed at 50% wastewater concentration the reduction in biomass (30.3%), root length (87.7%), shoot length (93.9%), and seed germination (30.0%) was observed in comparison to control. When A. cepa root tips immersed in varying concentrations of wastewater samples, the mitotic index significantly decreased, suggesting the induction of cytotoxicity. However, following the bacterial treatment, there was a noticeable decrease in phytotoxicity and cytotoxicity. The bacterial culture produces lignin peroxidase enzyme and has the potential to degrade the toxic pollutants of petroleum wastewater. Therefore the bacterium may be immobilised or directly used at reactor scale or pilot scale study to benefit the industry and environmental safety.

Physiology and comparative genomics of the haloalkalitolerant and hydrocarbonoclastic marine strain Rhodococcus ruber MSA14.

Marine hydrocarbonoclastic bacteria can use polycyclic aromatic hydrocarbons as carbon and energy sources, that makes these bacteria highly attractive for bioremediation in oil-polluted waters. However, genomic and metabolic differences between species are still the subject of study to understand the evolution and strategies to degrade PAHs. This study presents Rhodococcus ruber MSA14, an isolated bacterium from marine sediments in Baja California, Mexico, which exhibits adaptability to saline environments, a high level of intrinsic pyrene tolerance (> 5 g L- 1), and efficient degradation of pyrene (0.2 g L- 1) by 30% in 27 days. Additionally, this strain demonstrates versatility by using naphthalene and phenanthrene as individual carbon sources. The genome sequencing of R. ruber MSA14 revealed a genome spanning 5.45 Mbp, a plasmid of 72 kbp, and three putative megaplasmids, lengths between 110 and 470 Kbp. The bioinformatics analysis of the R. ruber MSA14 genome revealed 56 genes that encode enzymes involved in the peripheral and central pathways of aromatic hydrocarbon catabolism, alkane, alkene, and polymer degradation. Within its genome, R. ruber MSA14 possesses genes responsible for salt tolerance and siderophore production. In addition, the genomic analysis of R. ruber MSA14 against 13 reference genomes revealed that all compared strains have at least one gene involved in the alkanes and catechol degradation pathway. Overall, physiological assays and genomic analysis suggest that R. ruber MSA14 is a new haloalkalitolerant and hydrocarbonoclastic strain toward a wide range of hydrocarbons, making it a promising candidate for in-depth characterization studies and bioremediation processes as part of a synthetic microbial consortium, as well as having a better understanding of the catabolic potential and functional diversity among the Rhodococci group.

From Antagonism to Enhancement: Triton X-100 Surfactant Affects Phenanthrene Interfacial Biodegradation by Mycobacteria through a Shift in Uptake Mechanisms.

Polycyclic aromatic hydrocarbons (PAHs), as persistent environmental pollutants, often reside in nonaqueous-phase liquids (NAPLs). Mycobacterium sp. WY10, boasting highly hydrophobic surfaces, can adsorb to the oil-water interface, stabilizing the Pickering emulsion and directly accessing PAHs for biodegradation. We investigated the impact of Triton X-100 (TX100) on this interfacial uptake of phenanthrene (PHE) by Mycobacteria, using n-tetradecane (TET) and bis-(2-ethylhexyl) phthalate (DEHP) as NAPLs. Interfacial tension, phase behavior, and emulsion stability studies, alongside confocal laser scanning microscopy and electron microscope observations, unveiled the intricate interplay. In surfactant-free systems, Mycobacteria formed stable W/O Pickering emulsions, directly degrading PHE within the NAPLs because of their intimate contact. Introducing low-dose TX100 disrupted this relationship. Preferentially binding to the cells, the surfactant drastically increased the cell hydrophobicity, triggering desorption from the interface and phase separation. Consequently, PAH degradation plummeted due to hindered NAPL access. Higher TX100 concentrations flipped the script, creating surfactant-stabilized O/W emulsions devoid of interfacial cells. Surprisingly, PAH degradation remained efficient. This paradox can be attributed to NAPL emulsification, driven by the surfactant, which enhanced mass transfer and brought the substrate closer to the cells, despite their absence at the interface. This study sheds light on the complex effect of surfactants on Mycobacteria and PAH uptake, revealing an antagonistic effect at low concentrations that ultimately leads to enhanced degradation through emulsification at higher doses. These findings offer valuable insights into optimizing bioremediation strategies in PAH-contaminated environments.

Golexanolone improves fatigue, motor incoordination and gait and memory in rats with bile duct ligation.

BACKGROUND AND AIMS: Many patients with the chronic cholestatic liver disease primary biliary cholangitis (PBC) show fatigue and cognitive impairment that reduces their quality of life. Likewise, rats with bile duct ligation (BDL) are a model of cholestatic liver disease. Current PBC treatments do not improve symptomatic alterations such as fatigue or cognitive impairment and new, more effective treatments are therefore required. Golexanolone reduces the potentiation of GABAA receptors activation by neurosteroids. Golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in rats with chronic hyperammonemia. The aims of the present study were to assess if golexanolone treatment improves fatigue and cognitive and motor function in cholestatic BDL rats and if this is associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. METHODS: Rats were subjected to bile duct ligation. One week after surgery, oral golexanolone was administered daily to BDL and sham-operated controls. Fatigue was analysed in the treadmill, motor coordination in the motorater, locomotor gait in the Catwalk, and short-term memory in the Y-maze. We also analysed peripheral inflammation, neuroinflammation, and GABAergic neurotransmission markers by immunohistochemistry and Western blot. RESULTS: BDL induces fatigue, impairs memory and motor coordination, and alters locomotor gait in cholestatic rats. Golexanolone improves these alterations, and this was associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. CONCLUSION: Golexanolone may have beneficial effects to treat fatigue, and motor and cognitive impairment in patients with the chronic cholestatic liver disease PBC.

Lactobacillus rhamnosus GG ameliorates triptolide-induced liver injury through modulation of the bile acid-FXR axis.

Triptolide (TP) is the principal bioactive compound of Tripterygium wilfordii with significant anti-tumor, anti-inflammatory and immunosuppressive activities. However, its severe hepatotoxicity greatly limits its clinical use. The underlying mechanism of TP-induced liver damage is still poorly understood. Here, we estimate the role of the gut microbiota in TP hepatotoxicity and investigate the bile acid metabolism mechanisms involved. The results of the antibiotic cocktail (ABX) and fecal microbiota transplantation (FMT) experiment demonstrate the involvement of intestinal flora in TP hepatotoxicity. Moreover, TP treatment significantly perturbed gut microbial composition and reduced the relative abundances of Lactobacillus rhamnosus GG (LGG). Supplementation with LGG reversed TP-induced hepatotoxicity by increasing bile salt hydrolase (BSH) activity and reducing the increased conjugated bile acids (BA). LGG supplementation upregulates hepatic FXR expression and inhibits NLRP3 inflammasome activation in TP-treated mice. In summary, this study found that gut microbiota is involved in TP hepatotoxicity. LGG supplementation protects mice against TP-induced liver damage. The underlying mechanism was associated with the gut microbiota-BA-FXR axis. Therefore, LGG holds the potential to prevent and treat TP hepatotoxicity in the clinic.

Inflammation-Responsive Mesoporous Silica Nanoparticles with Synergistic Anti-inflammatory and Joint Protection Effects for Rheumatoid Arthritis Treatment.

PURPOSE: Joint destruction is a major burden and an unsolved problem in rheumatoid arthritis (RA) patients. We designed an intra-articular mesoporous silica nanosystem (MSN-TP@PDA-GlcN) with anti-inflammatory and joint protection effects. The nanosystem was synthesized by encapsulating triptolide (TP) in mesoporous silica nanoparticles and coating it with pH-sensitive polydopamine (PDA) and glucosamine (GlcN) grafting on the PDA. The nano-drug delivery system with anti-inflammatory and joint protection effects should have good potency against RA. METHODS: A template method was used to synthesize mesoporous silica (MSN). MSN-TP@PDA-GlcN was synthesized via MSN loading with TP, coating with PDA and grafting of GlcN on PDA. The drug release behavior was tested. A cellular inflammatory model and a rat RA model were used to evaluate the effects on RA. In vivo imaging and microdialysis (MD) system were used to analyze the sustained release and pharmacokinetics in RA rats. RESULTS: TMSN-TP@PDA-GlcN was stable, had good biocompatibility, and exhibited sustained release of drugs in acidic environments. It had excellent anti-inflammatory effects in vitro and in vivo. It also effectively repaired joint destruction in vivo without causing any tissue toxicity. In vivo imaging and pharmacokinetics experiments showed that the nanosystem prolonged the residence time, lowered the Cmax value and enhanced the relative bioavailability of TP. CONCLUSIONS: These results demonstrated that MSN-TP@PDA-GlcN sustained the release of drugs in inflammatory joints and produced effective anti-inflammatory and joint protection effects on RA. This study provides a new strategy for the treatment of RA.