A novel mutation in non-constitutive lycopene beta cyclase (CstLcyB2a) from Crocus sativus modulates carotenoid/apocarotenoid content, biomass and stress tolerance in plants.

MAIN CONCLUSION: Mutation at A126 in lycopene-ß-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene without affecting lycopene binding, thereby diverting metabolic flux towards ß-carotene and apocarotenoid biosynthesis. Crocus sativus, commonly known as saffron, has emerged as an important crop for research because of its ability to synthesize unique apocarotenoids such as crocin, picrocrocin and safranal. Metabolic engineering of the carotenoid pathway can prove a beneficial strategy for enhancing the quality of saffron and making it resilient to changing climatic conditions. Here, we demonstrate that introducing a novel mutation at A126 in stigma-specific lycopene-ß-cyclase of Crocus (CstLcyB2a) sterically hinders its binding of δ-carotene, but does not affect lycopene binding, thereby diverting metabolic flux towards ß-carotene formation. Thus, A126L-CstLcyB2a expression in lycopene-accumulating bacterial strains resulted in enhanced production of ß-carotene. Transient expression of A126L-CstLcyB2a in C. sativus stigmas enhanced biosynthesis of crocin. Its stable expression in Nicotiana tabacum enhanced ß-branch carotenoids and phyto-hormones such as abscisic acid (ABA) and gibberellic acids (GA's). N. tabacum transgenic lines showed better growth performance and photosynthetic parameters including maximum quantum efficiency (Fv/Fm) and light-saturated capacity of linear electron transport. Exogenous application of hormones and their inhibitors demonstrated that a higher ratio of GA4/ABA has positive effects on biomass of wild-type and transgenic plants. Thus, these findings provide a platform for the development of new-generation crops with improved productivity, quality and stress tolerance.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Distribution of Main Bioactive Compounds from Saffron Species as a Function of Infusion Temperature and Time in an Oil/Water System.

Most research on saffron has focused on its composition and beneficial effects, while the culinary perspective to enhance its gastronomic potential remains unexplored. This study aims to define the transfer of the main compounds responsible for color, flavor, and aromatic properties, evaluating three critical variables: temperature (60 °C, 80 °C and 100 °C), infusion time (ranging from 10 to 30 min), and the composition of the medium (water, oil, and water/oil). Samples were analyzed using the LC-QTOF MS/MS and ISO 3632-1:2011 methods. The major compounds were crocins, including trans-crocin and picrocrocin. Among the flavonoids, kaempferol 3-O-sophoroside stands out. Regarding extraction conditions, crocins, glycoside flavonoids, and picrocrocin were enhanced in water, the former in 100% water and at low temperatures, while picrocrocin proved to be the most stable compound with extraction favored at high temperatures. The variable with the greatest incidence of picrocrocin isolation seemed to be the concentration of water since water/oil compositions reported higher concentrations. Safranal and kaempferol were enriched in the oil phase and at lower temperatures. This study provides a chemical interpretation for the appropriate gastronomic use of saffron according to its versatility. Finally, the determination of safranal using the ISO method did not correlate with that obtained using chromatography.

Perimenopausal and Menopausal Mammary Glands In A 4-Vinylcyclohexene Diepoxide Mouse Model.

As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments.

HSPiP, Computational, and Thermodynamic Model-Based Optimized Solvents for Subcutaneous Delivery of Tolterodine Tartrate and GastroPlus­Based In Vivo Prediction in Humans: Part II.

In part I, we reported Hansen solubility parameters (HSP, HSPiP program), experimental solubility at varied temperatures for TOTA delivery. Here, we studied dose volume selection, stability, pH, osmolality, dispersion, clarity, and viscosity of the explored combinations (I-VI). Ex vivo permeation and deposition studies were performed to observe relative diffusion rate from the injected site in rat skin. Confocal laser scanning microscopy (CLSM) study was conducted to support ex vivo findings. Moreover, GastroPlus predicted in vivo parameters in humans and the impact of various critical factors on pharmacokinetic parameters (PK). Immediate release product (IR) contained 60% of PEG400 whereas controlled release formulation (CR) contained PEG400 (60%), water (10%) and d-limonene (30%) to deliver 2 mg of TOTA. GastroPlus predicted the plasma drug concentration of weakly basic TOTA as function of pH (from pH 2.0 to 9). The cumulative drug permeation and drug deposition were found to be in the order as B-VI˃ C-VI˃A-VI across rat skin. This finding was further supported with CLSM. Moreover, IR and CR were predicted to achieve Cmax of 0.0038 µg/ mL and 0.00023 µg/mL, respectively, after sub-Q delivery. Added limonene in CR extended the plasma drug concentration over period of 12 h as predicted in GastroPlus. Parameters sensitivity analysis (PSA) assessment predicted that sub-Q blood flow rate is the only factor affecting PK parameters in IR formulation whereas this was insignificant for CR. Thus, sub-Q delivery CR would be promising alternative with ease of delivery to children and aged patient.

Determination of in vitro synergy and antibiofilm activities of antimicrobials and essential oil components.

Using existing adrentimicrobials with essential oil components to prevent antimicrobial resistance is an alternative strategy. This study aimed to evaluate the resistance status, synergistic combinations, and in vitro biofilm formation activities of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), Stenotrophomonas maltophilia and Candida albicans against antimicrobial agents and cinnamaldehyde, carvacrol, eugenol, limonene and eucalyptol. Antimicrobial activities were evaluated by microdilution, cytotoxicity by XTT, synergy by checkerboard and time-kill, and biofilm inhibition by microplate methods. Cinnamaldehyde and carvacrol showed strong antimicrobial activity. Synergistic effects were observed when using all essential oils with antimicrobials. Only two C. albicans isolates showed antagonism with cinnamaldehyde and fluconazole. The constituents showed cytotoxic effects in the L929 cell line (except limonene). A time-kill analysis revealed a bacteriostatic effect on S. maltophilia and MRSA isolates and a fungicidal effect on C. albicans isolates. These results are important for further research to improve antimicrobial efficacy or to develop new agents.

Synthesis and characterization of bagasse cellulose-based quaternary ammonium peroxyphosphotungstate and their catalytic properties for cyclohexene oxidation in the absence of any solvent.

Bagasse cellulose, an industrial waste byproduct of sugar production, was demonstrated to be a viable solid support for a solid-phase ionic oxidation catalyst enabling organic solvent-free aqueous reaction conditions and facile catalyst recovery. Bagasse cellulose-supported quaternary ammonium peroxyphosphotungstate was synthesized from bagasse cellulose-supported quaternary ammonium chloride, phosphotungstic acid, and hydrogen peroxide. The chemical structure of this material was characterized by SEM, XRD, FT-IR, XPS, and 13C NMR, revealing stability of the cellulose matrix to the catalyst loading conditions and effective dispersion of the acicular catalyst crystals throughout the matrix. High catalytic activity of this synthetic complex was demonstrated in the oxidation of cyclohexene to 1,2-cyclohexanediol with hydrogen peroxide in the absence of solvent. Optimized conditions providing trans-1,2-cyclohexanediol with 86.2 % selectivity were 12 wt% catalyst and 4 mL/g 30 % H2O2 (vs. cyclohexene) at 50 °C for 10 h.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

The Protective Role of Magnoliae Flos in Preventing Ovotoxicity and Managing Ovarian Function: An In Vitro and In Vivo Study.

Magnoliae Flos (MF) is a medicinal herb widely employed in traditional medicine for relieving sinusitis, allergic rhinitis, headaches, and toothaches. Here, we investigated the potential preventive effects of MF extract (MFE) against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in ovarian cells and a mouse model of premature ovarian insufficiency (POI). The cytoprotective effects of MFE were assessed using CHO-K1 or COV434 cells. In vivo, B6C3F1 female mice were intraperitoneally injected with VCD for two weeks to induce POI, while MFE was orally administered for four weeks, beginning one week before VCD administration. VCD led to a significant decline in the viabilities of CHO-K1 and COV434 cells and triggered excessive reactive oxygen species (ROS) production and apoptosis specifically in CHO-K1 cells. However, pretreatment with MFE effectively prevented VCD-induced cell death and ROS generation, while also activating the Akt signaling pathway. In vivo, MFE increased relative ovary weights, follicle numbers, and serum estradiol and anti-Müllerian hormone levels versus controls under conditions of ovary failure. Collectively, our results demonstrate that MFE has a preventive effect on VCD-induced ovotoxicity through Akt activation. These results suggest that MFE may have the potential to prevent and manage conditions such as POI and diminished ovarian reserve.

Safranal alleviates pentetrazole-induced epileptic seizures in mice by inhibiting the NF-κB signaling pathway and mitochondrial-dependent apoptosis through GSK-3ß inactivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, a traditional Chinese medicine, is derived from Crocus sativus L. stigmas and has been reported to possess neuroprotective properties and potentially contribute to the inhibition of apoptosis and inflammation. Safranal, a potent monothyral aldehyde, is a main component of saffron that has been reported to have antiepileptic activity. However, the specific mechanism by which safranal suppresses epileptic seizures via its antiapoptotic and anti-inflammatory properties is unclear. AIM: To evaluate the effect of safranal on seizure severity, inflammation, and postictal neuronal apoptosis in a mouse model of pentetrazole (PTZ)-induced seizures and explore the underlying mechanism involved. MATERIALS AND METHODS: The seizure stage and latency of stage 2 and 4 were quantified to assess the efficacy of safranal in mitigating PTZ-induced epileptic seizures in mice. Electroencephalography (EEG) was employed to monitor epileptiform afterdischarges in each experimental group. The cognitive abilities and motor functions of the mice were evaluated using the novel object recognition test and the open field test, respectively. Neurons were quantified using hematoxylin and eosin staining. Additionally, bioinformatics tools were utilized to predict the interactions between safranal and specific target proteins. Glycogen synthase kinase-3ß (GSK-3ß), mitochondrial apoptosis-related proteins, and inflammatory factor levels were analyzed through western blotting. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) concentrations in brain tissue were assessed by ELISA. RESULTS: Safranal decreased the average seizure stage and increased the lantency of stage 2 and 4 seizures in PTZ-induced epileptic mice. Additionally, safranal exhibited neuroprotective effects on hippocampal CA1 and CA3 neurons and reduced hyperactivity caused by postictal hyperexcitability. Bioinformatics analysis revealed that safranal can bind to five specific proteins, including GSK-3ß. By promoting Ser9 phosphorylation and inhibiting GSK-3ß activity, safranal effectively suppressed the NF-κB signaling pathway. Moreover, the findings indicate that safranal treatment can decrease TNF-α and IL-1ß levels in the cerebral tissues of epileptic mice and downregulate mitochondrial apoptosis-related proteins, including Bcl-2, Bax, Bak, Caspase 9, and Caspase 3. CONCLUSION: Safranal can suppress the NF-κB signaling pathway and mitochondrial-dependent apoptosis through GSK-3ß inactivation, suggesting that it is a promising therapeutic agent for epilepsy treatment.

Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells.

BACKGROUND: Cancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative. HYPOTHESIS/PURPOSE: In this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7. STUDY DESIGN: An integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene. METHODS: We applied a systems-based analysis to introduce a drug-target-pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis. RESULTS: The mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22). CONCLUSION: Our data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.

Bu-Shen-Ning-Xin decoction inhibits macrophage activation to ameliorate premature ovarian insufficiency-related osteoimmune disorder via FSH/FSHR pathway.

Limited studies are associated with premature ovarian insufficiency (POI)-related osteoimmune disorder currently. Bu-Shen-Ning-Xin decoction (BSNXD) displayed a favorable role in treating postmenopausal osteoporosis. However, its impact on the POI-related osteoimmune disorder remains unclear. The study primarily utilized animal experiments and network pharmacology to investigate the effects and underlying mechanisms of BSNXD on the POI-related osteoimmune disorder. First, a 4-vinylcyclohexene dioxide (VCD)-induced POI murine model was conducted to explore the therapeutical action of BSNXD. Second, we analyzed the active compounds of BSNXD and predicted their potential mechanisms for POI-related osteoimmune disorder via network pharmacology, further confirmed by molecular biology experiments. The results demonstrated that VCD exposure led to elevated follicle-stimulating hormone (FSH) levels, a 50% reduction in the primordial follicles, bone microstructure changes, and macrophage activation, indicating an osteoimmune disorder. BSNXD inhibited macrophage activation and osteoclast differentiation but did not affect serum FSH and estradiol levels in the VCD-induced POI model. Network pharmacology predicted the potential mechanisms of BSNXD against the POI-related osteoimmune disorder involving tumor necrosis factor α and MAPK signaling pathways, highlighting BSNXD regulated inflammation, hormone, and osteoclast differentiation. Further experiments identified BSNXD treatment suppressed macrophage activation via downregulating FSH receptor (FSHR) expression and inhibiting the phosphorylation of ERK and CCAAT enhancer binding proteins ß. In conclusion, BSNXD regulated POI-related osteoimmune disorder by suppressing the FSH/FSHR pathway to reduce macrophage activation and further inhibiting osteoclastogenesis.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Therapeutic effects of saffron (Crocus sativus L) on female reproductive system disorders: A systematic review.

The effect of Crocus sativus on several disorders has been discussed or even confirmed, but the efficacy of this herb on the female reproductive system has not been well presented. In this regard, this systematic review comprehensively discussed the efficacy of C. sativus and its main phytochemical compounds on the female reproductive system and its disorders for the first time. In this systematic review, scientific databases, including PubMed, Web of Sciences, Google Scholar, Scopus, and Scientific Information Database, were explored profoundly. In vivo, in vitro, and human studies published until the end of July 2023, which had investigated the pharmacological properties of C. sativus, crocin, crocetin, safranal, or picrocrocin on the female reproductive system, were selected. A total of 50 studies conducted on the effect of C. sativus on the female reproductive system were acquired. These studies confirmed the efficacy of C. sativus or its main phytochemical ingredients in several aspects of the female reproductive system, including regulation of sex hormones, folliculogenesis, ovulation, and protection of the ovary and uterus against several oxidative stress. Several retrieved studies indicated that this herb also can alleviate the symptoms of patients suffering from dysmenorrhea, premenstrual syndrome, menopause, polycystic ovary disease (PCOD), and sexual dysfunction. Furthermore, it is a promising candidate for future studies or even trials regarding ovarian and cervical cancers. This review concluded that C. sativus can improve the symptoms of several female reproductive system disorders, which is particularly due to the presence of phytochemical ingredients, such as crocin, crocetin, and safranal.

Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Synergistic interactions of essential oil components with antibiotics against multidrug-resistant Corynebacterium striatum.

AIM: In this study, it was aimed to examine the antibacterial activity of the essential oil components (EOCs), carvacrol (CAR), cinnamaldehyde (CIN), thymol (TH), alpha pinene (α-PN), eucalyptol (EU), limonene (LIM), and the antibiotics, linezolid (LZD), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), clindamycin (CLN), and penicillin (PEN) against 50 multidrug resistant Corynebacterium striatum strains, and the synergistic interactions of CAR and CIN with the antibiotics against 10 randomly selected Coryne. striatum strains to explore synergistic interactions to determine if their combined use could enhance antibiotic activity and potentially reduce resistance. METHODS AND RESULTS: The activity of the EOCs and the antibiotics against Coryne. striatum strains isolated from clinical specimens, was examined by broth microdilution method. The synergistic interactions of the EOCs with the antibiotics against 10 randomly selected Coryne. striatum strains were determined by checkerboard method. EOCs, CIN, and CAR and antibiotics, LZD, VAN, GEN, CIP, and CLN were detected to have antibacterial activity against Coryne. striatum strains alone and either synergistic interactions were observed in combinations of the antibiotics with EOCs. CONCLUSIONS: All Coryne. striatum strains were determined to be susceptible to VAN and LZD and resistant to GEN, PEN, CIP, and CLN. Synergistic interactions were observed in all combinations of antibiotics tested with CAR and CIN.

Dehydroepiandrosterone modulates the PTEN/PI3K/AKT signaling pathway to alleviate 4-vinylcyclohexene diepoxide-induced premature ovarian insufficiency in rats.

Dehydroepiandrosterone (DHEA) is frequently integrated as an adjuvant in over a quarter of controlled ovarian hyperstimulation (COH) protocols, despite the ongoing debate regarding its impact. This study aimed to evaluate the efficacy and mechanism of action of DHEA on ovarian follicular development and ovarian response in rats with varying ovarian reserves. The study involved 75 rats categorized into 15 distinct groups. The ovarian tissues of rats in both the normal ovarian reserve group and the premature ovarian insufficiency (POI) group, induced by 4-vinylcyclohexene diepoxide (VCD) injection, were subjected to histomorphological and biochemical analyses following the administration of DHEA, either alone or in combination with COH. Follicle counting was performed on histological sections obtained from various tissues. Serum concentrations of anti-Müllerian hormone (AMH) and the quantification of specific proteins in ovarian tissue, including phosphatase and tensin homolog of chromosome 10 (PTEN), phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (pAKT), cyclooxygenase 2 (COX-2), caspase-3, as well as assessments of total antioxidant status and total oxidant status, were conducted employing the ELISA method. The impact of DHEA exhibited variability based on ovarian reserve. In the POI model, DHEA augmented follicular development and ovarian response to the COH protocol by upregulating the PTEN/PI3K/AKT signaling pathway, mitigating apoptosis, inflammation, and oxidative stress, contrary to its effects in the normal ovarian reserve group. In conclusion, it has been determined that DHEA may exert beneficial effects on ovarian stimulation response by enhancing the initiation of primordial follicles and supporting antral follicle populations.

PDCD4 deficiency improved 4-vinylcyclohexene dioxide-induced mouse premature ovarian insufficiency.

RESEARCH QUESTION: What role does programmed cell death 4 (PDCD4) play in premature ovarian insufficiency (POI)? DESIGN: A PDCD4 gene knockout (PDCD4-/-) mouse model was constructed, a POI mouse model was established similar to human POI with 4-vinylcyclohexene dioxide (VCD), a PDCD4-overexpressed adenovirus was designed and the regulatory role in POI in vitro and in vivo was investigated. RESULTS: PDCD4 expression was significantly increased in the ovarian granulosa cells of patients with POI (P ≤ 0.002 protein and mRNA) and mice with VCD-induced POI (P < 0.001 protein expression in both mouse ovaries and granulosa cells). In POI-induced mice model, PDCD4 knockouts significantly increased anti-Müllerian hormone, oestrodiol and numbers of developing follicles, and the PI3K-AKT-Bcl2/Bax signalling pathway is involved in it. CONCLUSION: The expression and regulation of PDCD4 significantly affects the POI pathology in a mouse model. This effect is closely related to the regulation of Bcl2/Bax and the activation of the PI3K-AKT signalling pathway.

Identification of novel Nrf2-activating neuroprotective agents: Elucidation of structural congeners of (-)-galiellalactone and congener-based novel Nrf2 activators.

Herein, (-)-galiellalactone 1 congeners responsible for the nuclear factor erythroid 2-related factor 2 (Nrf2)-activating neuroprotective effects were elucidated. Additionally, novel congener-based Nrf2 activators were identified using a drug repositioning strategy. (-)-Galiellalactone, which comprises a tricyclic lactone skeleton, significantly activates antioxidant response element (ARE)-mediated transcription in neuroblastoma SH-SY5Y cells. Interestingly, two cyclohexene-truncated [3.3] bicyclic lactone analogs, which possess an exocyclic α-methylene-γ-butyrolactone moiety, exhibited higher Nrf2/ARE transcriptional activities than the parent (-)-galiellalactone. We confirmed that the cyclohexene moiety embedding the [3.3] bicyclic lactone congener does not play the essential role of (-)-galiellalactone for Nrf2/ARE activation. Nrf2/ARE activation by novel analogs resulted in the upregulation of downstream antioxidative and phase II detoxifying enzymes, heme oxygenase-1, and NAD(P)H quinone oxidoreductase 1, which are closely related to the cytoprotective effects on neurodegenerative diseases. (-)-Galiellalactone and its [3.3] bicyclic variants 3l and 3p increased the expression of antioxidant genes and exhibited neuroprotective effects against 6-hydroxydopamine-mediated neurotoxicity in the neuroblastoma SH-SY5Y cell line.

Design of Two New Sulfur Derivatives of Perezone: In Silico Study Simulation Targeting PARP-1 and In Vitro Study Validation Using Cancer Cell Lines.

Poly-ADP-Ribose Polymerase (PARP-1) is an overexpressed enzyme in several carcinomas; consequently, the design of PARP-1 inhibitors has acquired special attention. Hence, in the present study, three compounds (8-10) were produced through a Michael addition protocol, using phenylmethanethiol, 5-fluoro-2-mercaptobenzyl alcohol, and 4-mercaptophenylacetic acid, respectively, as nucleophiles and perezone as the substrate, expecting them to be convenient candidates that inhibit PARP-1. It is convenient to note that in the first stage of the whole study, the molecular dynamics (MD) simulations and the quantum chemistry studies of four secondary metabolites, i.e., perezone (1), perezone angelate (2), hydroxyperezone (3), and hydroxyperezone monoangelate (4), were performed, to investigate their interactions in the active site of PARP-1. Complementarily, a docking study of a set of eleven sulfur derivatives of perezone (5-15) was projected to explore novel compounds, with remarkable affinity to PARP-1. The molecules 8-10 provided the most adequate results; therefore, they were evaluated in vitro to determine their activity towards PARP-1, with 9 having the best IC50 (0.317 µM) value. Additionally, theoretical calculations were carried out using the density functional theory (DFT) with the hybrid method B3LYP with a set of base functions 6-311++G(d,p), and the reactivity properties were compared between the natural derivatives of perezone and the three synthesized compounds, and the obtained results exhibited that 9 has the best properties to bind with PARP-1. Finally, it is important to mention that 9 displays significant inhibitory activity against MDA-MB-231 and MCF-7 cells, i.e., 145.01 and 83.17 µM, respectively.