Relative humidity-dependent evolution of molecular composition of α-pinene secondary organic aerosol upon heterogeneous oxidation by hydroxyl radicals.

Heterogeneous oxidation by gas-phase oxidants is an important chemical transformation pathway of secondary organic aerosol (SOA) and plays an important role in controlling the abundance, properties, as well as climate and health impacts of aerosols. However, our knowledge on this heterogeneous chemistry remains inadequate. In this study, the heterogeneous oxidation of α-pinene ozonolysis SOA by hydroxyl (OH) radicals was investigated under both low and high relative humidity (RH) conditions, with an emphasis on the evolution of molecular composition of SOA and its RH dependence. It is found that the heterogeneous oxidation of SOA at an OH exposure level equivalent to 12 hr of atmospheric aging leads to particle mass loss of 60% at 25% RH and 95% at 90% RH. The heterogeneous oxidation strongly changes the molecular composition of SOA. The dimer-to-monomer signal ratios increase dramatically with rising OH exposure, in particular under high RH conditions, suggesting that aerosol water stimulates the reaction of monomers with OH radicals more than that of dimers. In addition, the typical SOA tracer compounds such as pinic acid, pinonic acid, hydroxy pinonic acid and dimer esters (e.g., C17H26O8 and C19H28O7) have lifetimes of several hours against heterogeneous OH oxidation under typical atmospheric conditions, which highlights the need for the consideration of their heterogeneous loss in the estimation of monoterpene SOA concentrations using tracer-based methods. Our study sheds lights on the heterogeneous oxidation chemistry of monoterpene SOA and would help to understand their evolution and impacts in the atmosphere.

Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis.

Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Unraveling the effective inhibition of α-terpinol and terpene-4-ol against Aspergillus carbonarius: Antifungal mechanism, ochratoxin A biosynthesis inhibition and degradation perspectives.

Aspergillus carbonarius, a common food-contaminating fungus, produces ochratoxin A (OTA) and poses a risk to human health. This study aimed to assess the inhibitory activity of tea tree essential oil and its main components, Terpene-4-ol (T4), α-terpineol (αS), and 3-carene (3C) against A. carbonarius. The study showed αS and T4 were the main antifungal components of tea tree essential oil, which primarily inhibit A. carbonarius growth through cell membrane disruption, reducing antioxidant enzyme activities (catalase, peroxidase, superoxide dismutase) and interrupting the tricarboxylic acid cycle. Furthermore, αS and T4 interacted with enzymes related to OTA biosynthesis. Molecular docking and molecular dynamics show that they bound mainly to P450 with a minimum binding energy of -7.232 kcal/mol, we infered that blocking the synthesis of OTA precursor OTß. Our hypothesis was preliminarily verified by the detection of key substances in the OTA synthesis pathway. The results of UHPLC-QTOF-MS2 analysis demonstrated that T4 achieved a degradation rate of 43 % for OTA, while αS reached 29.6 %, resulting in final breakdown products such as OTα and phenylalanine. These results indicated that α-terpinol and Terpene-4-ol have the potential to be used as naturally safe and efficient preservatives or active packaging to prevent OTA contamination.

Paeoniflorin protects hepatocytes from APAP-induced damage through launching autophagy via the MAPK/mTOR signaling pathway.

BACKGROUND: Drug-induced liver injury (DILI) is gradually becoming a common global problem that causes acute liver failure, especially in acute hepatic damage caused by acetaminophen (APAP). Paeoniflorin (PF) has a wide range of therapeutic effects to alleviate a variety of hepatic diseases. However, the relationship between them is still poorly investigated in current studies. PURPOSE: This work aimed to explore the protective effects of PF on APAP-induced hepatic damage and researched the potential molecular mechanisms. METHODS: C57BL/6J male mice were injected with APAP to establish DILI model and were given PF for five consecutive days for treatment. Aiming to clarify the pharmacological effects, the molecular mechanisms of PF in APAP-induced DILI was elucidated by high-throughput and other techniques. RESULTS: The results demonstrated that serum levels of ALP, γ-GT, AST, TBIL, and ALT were decreased in APAP mice by the preventive effects of PF. Moreover, PF notably alleviated hepatic tissue inflammation and edema. Meanwhile, the results of TUNEL staining and related apoptotic factors coincided with the results of transcriptomics, suggesting that PF inhibited hepatocyte apoptosis by regulated MAPK signaling. Besides, PF also acted on reactive oxygen species (ROS) to regulate the oxidative stress for recovery the damaged mitochondria. More importantly, transmission electron microscopy showed the generation of autophagosomes after PF treatment, and PF was also downregulated mTOR and upregulated the expression of autophagy markers such as ATG5, ATG7, and BECN1 at the mRNA level and LC3, p62, ATG5, and ATG7 at the protein level, implying that the process by which PF exerted its effects was accompanied by the occurrence of autophagy. In addition, combinined with molecular dynamics simulations and western blotting of MAPK, the results suggested p38 as a direct target for PF on APAP. Specifically, PF-activated autophagy through the downregulation of MAPK/mTOR signaling, which in turn reduced APAP injury. CONCLUSIONS: Paeoniflorin mitigated liver injury by activating autophagy to suppress oxidative stress and apoptosis via the MAPK/mTOR signaling pathway. Taken together, our findings elucidate the role and mechanism of paeoniflorin in DILI, which is expected to provide a new therapeutic strategy for the development of paeoniflorin.

Lemongrass essential oil and its major component citronellol: evaluation of larvicidal activity and acetylcholinesterase inhibition against Anopheles sinensis.

Mosquito-borne diseases, such as malaria, dengue fever, and the Zika virus, pose significant global health challenges, affecting millions annually. Due to increasing insecticide resistance, there is a growing interest in natural alternatives for mosquito control. Lemongrass essential oil, derived from Cymbopogon citratus, has shown promising repellent and larvicidal properties against various mosquito species. In this study, we investigated the larvicidal effect of lemongrass oil and its major compounds on Anopheles sinensis, the primary malaria vector in China. GC-MS analysis identified the major compounds of lemongrass oil as ( +)-citronellal (35.60%), geraniol (21.84%), and citronellol (13.88%). Lemongrass oil showed larvicidal activity against An. sinensis larvae, with an LC50 value of 119.20 ± 3.81 mg/L. Among the major components, citronellol had the lowest LC50 value of 42.76 ± 3.18 mg/L. Moreover, citronellol demonstrated inhibitory effects on acetylcholinesterase (AChE) activity in An. sinensis larvae, assessed by homogenizing larvae at different time points following treatment. Molecular docking studies further elucidated the interaction between citronellol and AChE, revealing the formation of hydrogen bonds and Pi-Sigma bonds. Aromatic amino acid residues such as Tyr71, Trp83, Tyr370, and Tyr374 played a pivotal role in these interactions. These findings may contribute to understanding lemongrass oil's larvicidal activity against An. sinensis and the mechanisms underlying these effects.

Activation of the Nrf2/Keap1 signaling pathway mediates the neuroprotective effect of Perillyl alcohol against cerebral hypoxic-ischemic damage in neonatal rats.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe disease with a poor prognosis, whose clinical treatment is still limited to therapeutic hypothermia with limited efficacy. Perillyl alcohol (POH), a natural monoterpene found in various plant essential oils, has shown neuroprotective properties, though its effects on HIE are not well understood. This study investigates the neuroprotective effects of POH on HIE both in vitro and in vivo. We established an in vitro model using glucose deprivation and hypoxia/reperfusion (OGD/R) in PC12 cells, alongside an in vivo model via the modified Rice-Vannucci method. Results indicated that POH acted as an indirect antioxidant, reducing inducible nitric oxide synthase and malondialdehyde production, maintaining content of antioxidant molecules and enzymes in OGD/R-induced PC12 cells. In vivo, POH remarkably lessened infarct volume, reduced cerebral edema, accelerated tissue regeneration, and blocked reactive astrogliosis after hypoxic-ischemic brain injury. POH exerted antiapoptotic activities through both the intrinsic and extrinsic apoptotic pathways. Mechanistically, POH activated Nrf2 and inactivated its negative regulator Keap1. The use of ML385, a Nrf2 inhibitor, reversed these effects. Overall, POH mitigates neuronal damage in HIE by combating oxidative stress, reducing inflammation, and inhibiting apoptosis via the Nrf2/Keap1 pathway, suggesting its potential for HIE treatment.

Transcriptome analysis of Reticulitermes flaviceps exposed to Mentha spicata essential oil and carvone indicates a potential neurotoxic mechanism of action characterized by intracellular calcium influx mediated by dopamine receptors.

Reticulitermes flaviceps is an economically important pest in agriculture, forestry, and construction. Recent studies have shown an increase in research focusing on the anti-termite properties of plant essential oils, however, there remains a lack of information regarding the specific molecular mechanism involved. In this study, RNA-seq analysis was conducted on termites exposed to Mentha spicata essential oil (EO) and carvone, leading to the discovery of various genes that were expressed differentially under different treatment conditions. Numerous genes that exhibited a response to M. spicata EO and carvone found to be associated with stress-related pathways, such as drug metabolism cytochrome P450, glutathione metabolism, fatty acid metabolism, citric acid cycle, neuroactive ligand-receptor interaction, cell apoptosis, the AMPK signalling pathway, the mTOR signalling pathway, the longevity regulation pathway, ubiquitin-mediated protein hydrolysis, and the calcium signalling pathway. The up-regulation of genes (SPHK) associated with calcium channels, such as SPHK, indicates a potential mechanism of neurotoxicity, while the up-regulation of apoptosis-associated genes, including ACTB_G1, PYG, SQSTM1, RNF31, suggests a potential mechanism of cytotoxicity. The metabolism of M. spicata EO induces oxidative stress, elevates free Ca2+ levels in mitochondria, and initiates the generation of reactive oxygen species (ROS), ultimately resulting in programmed cell necrosis and apoptosis, as well as facilitating cellular autophagy. The monoterpenes exhibited neurotoxic and cytotoxic effects on R. flaviceps and could be exploited to advance termiticide development and eco-friendly termite control.

Inhibitory activity and antioomycete mechanism of citral against Phytophthora capsici.

The natural terpenoid citral has antifungal activity against multiple fungi, but its bioactivity against oomycetes is unclear. Therefore, this study investigated the antioomycete activity and mechanism of citral against Phytophthora capsici, a highly destructive invasive oomycete. Results showed that citral not only had a great inhibition on the mycelial growth of P. capsici (EC50 = 94.15 mg/L), but also had a significant inhibition on multiple spores, such as sporangia formation, zoospore discharge and zoospore germination. Citral at 4000 mg/L exhibited favorable protective (73.33%) and curative efficacy (55.11%) against pepper Phytophthora blight. Citral significantly damaged the hyphal morphology, disrupted the cell membrane integrity, increased the permeability of cell membrane, and increased the glycerol content in P. capsici. A total of 250 upregulated and 288 downregulated proteins were identified in iTRAQ-based quantitative proteomic analysis. Downregulated proteins were mostly enriched in pathways of ABC transporters, cyanoamino acid metabolism and starch and sucrose metabolism, suggesting an inhibition of citral on transmembrane transporter (e.g., ABC transporters) and pathogenicity (e.g., ß-glucosidases) proteins. Upregulated proteins were enriched in biosynthesis of unsaturated fatty acids, pyruvate metabolism and glycolysis/gluconeogenesis, suggesting an activation of citral on energy generation proteins, including acyl-CoA oxidase, D-lactate dehydrogenase, pyruvate kinase, acetyl-CoA synthetase and phosphoenolpyruvate carboxykinase. Biochemical and iTRAQ analysis suggested that cell membrane may be the target of citral in P. capsici.

Plant-derived citronellol can significantly disrupt cell wall integrity maintenance of Colletotrichum camelliae.

Anthracnose, a fungal disease, commonly infects tea plants and severely impacts the yield and quality of tea. One method for controlling anthracnose is the application of citronellol, a plant extract that exhibits broad-spectrum antimicrobial activity. Herein, the physiological and biochemical mechanism by which citronellol controls anthracnose caused by Colletotrichum camelliae was investigated. Citronellol exhibited excellent antifungal activity based on direct and indirect mycelial growth inhibition assays, with EC50 values of 76.88 mg/L and 29.79 µL/L air, respectively. Citronellol also exhibited good control effects on C. camelliae in semi-isolated leaf experiments. Optical and scanning electron microscopy revealed that citronellol caused C. camelliae mycelia to thin, fracture, fold and deform. Transmission electron microscopy revealed that the mycelial cell walls collapsed inward and separated, and the organelles became blurred after treatment with citronellol. The sensitivity of C. camelliae to calcofluor white staining was significantly enhanced by citronellol, while PI staining showed minimal fluorescence, and the relative conductivity of mycelia were not significantly different. Under citronellol treatment, the expression levels of ß-1,3-glucanase, chitin synthase, and chitin deacetylase-related genes were significantly decreased, while the expression levels of chitinase genes were increased, leading to lower chitinase activity and increased ß-1,3-glucanase activity. Therefore, citronellol disrupted the cell wall integrity of C. camelliae and inhibited normal mycelial growth.

Toxicity and enzymatic mechanism of Citrus spp. essential oils and major constituents on Haemaphysalis longicornis (Acari: Ixodidae) and non-target Harmonia axyridis (Coleoptera: Coccinellidae).

Plant essential oils (EOs)-based acaricides have been recognized as environmentally-friendly alternatives to synthetic acaricides because of their low toxicity against non-target species. Despite this, there are knowledge gaps regarding the toxicity mechanisms of plant EOs against non-target species. Here, the toxicology and enzymatic mechanism of Citrus reticulata and Citrus lemon EOs were evaluated against the vector pest, Haemaphysalis longicornis, and non-target ladybird beetle, Harmonia axyridis. Both EOs were mainly composed of d-Limonene, followed by ß-Myrcene and γ-Terpinene in C. reticulata, and (-)-ß-Pinene and γ-Terpinene in C. lemon. Citrus reticulata and C. lemon EOs were toxic to Hae. longicornis, with 50 % lethal concentration (LC50) values estimated at 0.43 and 0.98 µL/mL via nymphal immersion test, and 42.52 and 46.38 µL/mL via spray application, respectively. Among the constituents tested, ß-Myrcene was the most effective, with LC50 values of 0.17 and 47.87 µL/mL via immersion and spray treatment, respectively. A significant mortality of non-target Har. axyridis was found when treated by the EOs at concentrations two times greater than LC50 estimated against H. longicornis. The biochemical assay revealed that the EOs induced changes in the antioxidant enzyme activity of superoxide dismutases, catalase, and glutathione peroxidase in Hae. longicornis and Har. axyridis. The results demonstrated the acaricidal potential of citrus EOs and their major constituents for tick control, revealed the risk of the EOs to non-target species, and provided relevant insights into the mechanisms underlying their toxicity.

Evaluation of antifungal and apoptotic effects of linalool, citral, and carvacrol separately and in combination with nystatin against clinical isolates of Pichia kudriavzevii.

Pichia kudriavzevii (formerly Candida krusei) poses a significant threat to immunocompromised patients due to its inherent resistance to various antifungal drugs. This study explored the anticandidal potential of citral, linalool, and carvacrol in combination with nystatin against P. kudriavzevii strains.Using the microdilution method following CLSI guidelines, Minimum Inhibitory Concentrations (MICs) and fungicidal concentrations (MFCs) were determined. Citral exhibited MIC values ranging from 50 to 100 µg/ml, averaging 70.24 ± 16.99 µg/ml, while carvacrol had MIC values of 50 to 100 µg/ml, averaging 86.90 ± 16.99 µg/ml. Linalool demonstrated weaker antifungal activity, with MIC values between 100 and 200 µg/ml, averaging 150 ± 38.73 µg/ml. The study assessed the synergistic effectsof these phenols with nystatin through fractional inhibitory concentration indices (FICIS). In addition, flow cytometry was employed to assess apoptosis induction in P. kudriavzevii cells.Carvacrol displayed a remarkable synergistic effect in combination with nystatin against all 21 isolates tested. Conversely, linalool showed synergy in 17 isolates, while citral exhibited synergy in only 2 isolates. These findings highlight distinct patterns of synergy between the different compounds and nystatin against P. kudriavzevii. Also, Carvacrol emerged as the most potent inducer of apoptosis across all P. kudriavzevii strains, followed by citral and linalool. This suggests that carvacrol not only possesses a stronger antifungal effect but also has a more pronounced ability to trigger programmed cell death in P. kudriavzevii. In conclusion, the study supports the potential of carvacrol, citral and linalool, as anticandidal agents, suggesting their supplementation with nystatin for treating P. kudriavzevii infections.

Can Alpha-Pinene Prevent Methotrexate-Induced Cardiac and Hepatic Damage?

The effects of alpha-pinene (AP), a monoterpenoid, known for its antioxidant, anti-inflammatory, and anti-apoptotic properties, on methotrexate (MTX)-induced cardiac and hepatic damage were investigated in this study. Male Sprague-Dawley rats were divided into Control, Vehicle, AP, MTX, and AP+MTX groups (n=7). AP (50 mg/kg/day, 14 days) was applied subcutaneously in the AP and AP+MTX groups. MTX (20 mg/kg) was injected three days before sacrification. Serum CK-MB, troponin T, ALT, and AST levels, as well as cardiac and hepatic MDA, GSH, caspase-3, and p53 levels, were measured by ELISA. Histological changes in tissues were evaluated by scoring in terms of tissue damage and cellular degeneration parameters after hematoxylin-eosin staining. MTX caused significant increase in serum CK-MB, troponin T, ALT, and AST levels, hepatic and cardiac lipid peroxidation, GSH depletion, and caspase-3 level. However, tissue levels of p53 did not change significantly. MTX-induced histological deterioration was observed in both tissues. These MTX-induced changes were significantly reduced in the AP+MTX group. Present results show that MTX-induced cardiac and hepatic damage is prevented by AP pretreatment. This protection can be attributed to the antioxidant and anti-apoptotic properties of AP. Considering the importance of MTX in cancer treatment, AP appears to have highly promising potential as a cardioprotective and hepatoprotective agent in anti-tumoral therapy. Key words: MDA, GSH, Caspase-3, p53, Oxidative stress, Apoptosis.

In Situ Antimicrobial Properties of Sabinene Hydrate, a Secondary Plant Metabolite.

The objective of this research was to investigate natural products for their potential against pathogenic microorganisms. Sabinene hydrate (SH), a monoterpenoid, is synthesised by numerous different plants as a secondary metabolite. At present, there is a lack of definite investigations regarding the antimicrobial activity of SH itself and its different isomers. The antimicrobial effects of commercially available SH (composed mainly of trans-isomer) were evaluated within a range of concentrations in three types of contact tests: solid and vapor diffusion and the macro-broth dilution method. Moreover, the effects of SH on the rate of linear growth and spore germination were also examined. Ethanolic SH solutions were tested against an array of microorganisms, including blue-stain fungi (Ceratocystis polonica, Ophiostoma bicolor, O. penicillatum), frequently originating from bark beetle galleries; three fungal strains (Musicillium theobromae, Plectosphaerella cucumerina, and Trichoderma sp.) isolated from a sapwood underneath bark beetle galleries (Ips typographus) on spruce (Picea abies) stems; Verticillium fungicola, isolated from diseased I. typographus larvae; two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa); five yeasts (Candida albicans, C. krusei, C. parapsilosis, Saccharomyces cerevisiae, and Rhodotorula muscilaginosa), and two saprophytic fungi (Aspergillus niger and Penicillium notatum). In solid agar disc diffusion tests, Gram-positive bacteria exhibited greater susceptibility to SH than Gram-negative bacteria, followed by yeasts and fungi. The most resistant to SH in both the disc diffusion and broth macro-dilution methods were P. aeruginosa, A. niger, and Trichoderma sp. strains. Blue-stain fungi and fungi isolated from the Picea sapwood were the most resistant among the fungal strains tested. The minimum inhibition concentrations (MICs) generated by SH and determined using a disc volatilization method were dependent on the fungal species and played an important role in the development of microorganism inhibition. The two Gram-positive bacteria, B. subtilis and S. aureus (whose MICs were 0.0312 and 0.0625 mg/mL, respectively), were the organisms most susceptible to SH, followed by the Gram-negative bacterium, E. coli (MIC = 0.125 mg/mL) and two yeasts, C. albicans and C. kruei (MIC was 0.125 mg/mL and 0.25 mg/mL, respectively). C. parapsilosis (MIC = 0.75 mg/mL) was the yeast most resistant to SH. The investigation of antimicrobial properties of plant secondary metabolites is important for the development of a new generation of fungicides.

Antimicrobial cyclodextrin-assisted electrospun fibers loaded with carvacrol, citronellol and cinnamic acid for wound healing.

This work aimed to explore an alternative to the use of antibiotics for prevention and treatment of wounds infection caused by two common bacterial pathogens Staphylococcus aureus and Pseudomonas aeruginosa. For this purpose, three different essential oil components (EOCs), namely carvacrol, citronellol and cinnamic acid, were loaded into electrospun fibers of poly-ε-caprolactone (PCL) aided by alpha-cyclodextrin (αCD) and hydroxypropyl-ß-cyclodextrin (HPßCD). Electrospun-fibers prepared with each EOC and their mixtures were screened for antimicrobial capability and characterized regarding morphological, mechanical, thermal, surface polarity, antibiofilm and antioxidant properties. αCD formed poly(pseudo)rotaxanes with PCL and weakly interacted with EOCs, while HPßCD facilitated EOC encapsulation and formation of homogeneous fibers (500-1000 nm diameter) without beads. PCL/HPßCD fibers with high concentration of EOCs (mainly carvacrol and cinnamic acid) showed strong antibiofilm (>3 log CFU reduction) and antioxidant activity (10-50% DPPH scavenging effects). Different performances were recorded for the EOCs and their mixtures; cinnamic acid migrated to fiber surface and was released faster. Fibers biocompatibility was verified using hemolysis tests and in ovo tissue integration and angiogenesis assays. Overall, HPßCD facilitates complete release of EOCs from the fibers to the aqueous medium, being an environment-friendly and cost-effective strategy for the treatment of infected wounds.

The combination of paeoniflorin and metformin synergistically inhibits the progression of liver fibrosis in mice.

Liver fibrosis is a pathological process that endangers human health, for which effective treatments remain elusive to date. Paeoniflorin (PAE), a pineane-type monoter penoid compound from the traditional Chinese medicine PaeoniaeRubra Radix, and metformin (MET), an oral biguanide hypoglycemic agent, both demonstrate anti-inflammatory and hepatoprotective effects. In current work, we first discovered that the combined treatment of PAE and MET synergistically inhibited the progression of liver fibrosis in two different animal models: therapeutic and preventive. This therapeutic effect is evidenced by a reduction in the expression levels of liver fibrosis markers and an improvement in histopathological characteristics. Mechanistic exploration further revealed that this combination therapy downregulated the expression of TGF-ß1 and p-Smad2, while upregulating Smad7 expression in both models. Importantly, we also found that this combinatorial approach significantly reduced hepatotoxicity and nephrotoxicity in both models. Our findings suggest an effective combination therapy for liver fibrosis and provide the possibility of therapeutic improvement for patients with liver fibrosis.

Systematic Engineering to Enhance ß-Myrcene Production in Yeast.

ß-Myrcene is an important monoterpene compound widely used in the fragrance, agricultural, and food industries. The microbial production of ß-myrcene conforms to the trend of green biological manufacturing, which has great potential for development. The poor catalytic activity of ß-myrcene synthase (MS) and the insufficient supply of precursors are considered to be the bottlenecks of ß-myrcene production. Here, source screening, subcellular localization, enzyme fusion, and precursor-enhancing strategies were integrated for ß-myrcene biosynthesis with Saccharomyces cerevisiae. The ß-myrcene titer gradually increased by 218-fold (up to 63.59 mg/L) compared to that of the initial titer of the shake flask. Moreover, the titer reached 66.82 mg/L after the addition of antioxidants (1 mM glutathione, GSH, and 1% butylated hydroxytoluene, BHT). Ultimately, 142.64 mg/L ß-myrcene in S. cerevisiae was achieved in 5.0 L of fed-batch fermentation under a carbon restriction strategy, which was the highest reported titer in yeast thus far. This study not only established a platform for ß-myrcene production but also provided a reference for the efficient biosynthesis of other monoterpene compounds.

Bioinformatics Analysis of the Genome of Rhodococcus rhodochrous IEGM 1362, an (-)-Isopulegol Biotransformer.

A genome of Rhodococcus rhodochrous IEGM 1362 was sequenced and annotated. This strain can transform monoterpene alcohol (-)-isopulegol with the formation of two novel pharmacologically promising metabolites. Nine genes encoding cytochrome P450, presumably involved in (-)-isopulegol transformation, were found in the genome of R. rhodochrous IEGM 1362. Primers and PCR conditions for their detection were selected. The obtained data can be used for the further investigation of genes encoding enzymes involved in monoterpene biotransformation.

Paeoniflorin suppresses ferroptosis after traumatic brain injury by antagonizing P53 acetylation.

BACKGROUND: Traumatic brain injury (TBI) could induce multiple forms of cell death, ferroptosis, a novel form of cell death distinct from apoptosis and autophagy, plays an important role in disease progression in TBI. Therapies targeting ferroptosis are beneficial for recovery from TBI. Paeoniflorin (Pae) is a water-soluble monoterpene glycoside and the active ingredient of Paeonia lactiflora pall. It has been shown to exert anti-inflammatory and antioxidant effects. However The effects and mechanisms of paeoniflorin on secondary injury after TBI are unknown. PURPOSE: To investigate the mechanism by which Pae regulates ferroptosis after TBI. METHODS: The TBI mouse model and cortical primary neurons were utilized to study the protective effect of paeoniflorin on the brain tissue after TBI. The neuronal cell ferroptosis model was established by treating cortical primary neurons with erastin. Liproxstatin-1(Lip-1) was used as a positive control drug. Immunofluorescence staining, Nissl staining, biochemical analyses, pharmacological analyses, and western blot were used to evaluate the effects of paeoniflorin on TBI. RESULTS: Pae significantly ameliorated neuronal damage after TBI, inhibited mitochondrial damage, increased glutathione peroxidase 4 (GPX4) activity, decreased malondialdehyde (MDA) production, restored neurological function and inhibited cerebral edema. Pae promotes the degradation of P53 in the form of proteasome, promotes its ubiquitination, and reduces the stability of P53 by inhibiting its acetylation, thus alleviating the P53-mediated inhibition of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) by P53. CONCLUSION: Pae inhibits ferroptosis by promoting P53 ubiquitination out of the nucleus, inhibiting P53 acetylation, and modulating the SLC7A11-GPX4 pathway.

Paeoniflorin Promotes Ovarian Development in Mice by Activating Mitophagy and Preventing Oxidative Stress.

During the development of animal organs, various adverse stimuli or toxic environments can induce oxidative stress and delay ovarian development. Paeoniflorin (PF), the main active ingredient of the traditional Chinese herb Paeonia lactiflora Pall., has protective effects on various diseases by preventing oxidative stress. However, the mechanism by which PF attenuates oxidative damage in mouse ovaries remains unclear. We evaluated the protective effects of PF on ovaries in an H2O2-induced mouse oxidative stress model. The H2O2-induced mouse ovarian oxidative stress model was used to explore the protective effect of PF on ovarian development. Histology and follicular development were observed. We then detected related indicators of cell apoptosis, oxidative stress, and autophagy in mouse ovaries. We found that PF inhibited H2O2-induced ovarian cell apoptosis and ferroptosis and promoted granulosa cell proliferation. PF prevented oxidative stress by increasing nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression levels. In addition, the autophagic flux of ovarian cells was activated and was accompanied by increased lysosomal biogenesis. Moreover, PF-mediated autophagy was involved in clearing mitochondria damaged by H2O2. Importantly, PF administration significantly increased the number of primordial follicles, primary follicles, secondary follicles, and antral follicles. PF administration improved ovarian sizes compared with the H2O2 group. The present study suggested that PF administration reversed H2O2-induced ovarian developmental delay and promoted follicle development. PF-activated mitophagy is crucial for preventing oxidative stress and improving mitochondrial quality.

Insecticidal and Repellent Activity of Essential Oils from Copaifera reticulata, Citrus paradisi, Lavandula hybrida and Salvia sclarea Against Immature and Adult Stages of Ctenocephalides felis felis.

PURPOSE: The flea Ctenocephalides felis (Siphonaptera: Pulicidae), parasitizes dogs and cats globally, acting as a vector for various pathogens affecting both animals and humans. Growing interest in environmentally friendly, plant-based products prompted this study. The aim of the study was to determine the chemical composition of essential oils (EOs) from Copaifera reticulata, Citrus paradisi, Lavandula hybrida and Salvia sclarea, assessing their insecticidal and repellent properties, determining lethal concentrations (LC50 and LC90), and evaluating residual efficacy in vitro against Ctenocephalides felis felis. METHODS: Gas Chromatography with Flame Ionization Detector analyzed EO composition. In vitro tests involved preparing EO solutions at various concentrations. Ten specimens from each life stage (egg, larva, pupa, adult) were used for insecticidal activity assessment. Adulticidal activity was assessed using 10 cm2 filter paper strip, each treated with 0.200 mL of the test solution. Immature stages activities were evaluated using 23.76 cm2 discs of the same filter paper, each treated with 0.470 mL of the test solution. Mortality percentage was calculated using (number of dead insects × 100) / number of incubated insects. Probit analysis calculated LC50 values with a 95% confidence interval. RESULTS: Major EO constituents were ß-caryophyllene (EOCR), linalool (EOLH), linalyl acetate (EOSS), and limonene (EOCP). LC50 values were obtained for all stages except for the essential oil of C. paradisi. All oils showed repellent activity at 800 µg/cm2. OECR exhibited greater residual efficacy. CONCLUSION: Each EO demonstrated superior insecticidal activity against specific C. felis felis stages.