Quantifying Peripheral Modulation of Olfaction by Trigeminal Agonists.

In the mammalian nose, two chemosensory systems, the trigeminal and the olfactory mediate the detection of volatile chemicals. Most odorants are able to activate the trigeminal system, and vice versa, most trigeminal agonists activate the olfactory system as well. Although these two systems constitute two separate sensory modalities, trigeminal activation modulates the neural representation of an odor. The mechanisms behind the modulation of olfactory response by trigeminal activation are still poorly understood. We addressed this question by looking at the olfactory epithelium (OE), where olfactory sensory neurons (OSNs) and trigeminal sensory fibers co-localize and where the olfactory signal is generated. Our study was conducted in a mouse model. Both sexes, males and females, were included. We characterize the trigeminal activation in response to five different odorants by measuring intracellular Ca2+ changes from primary cultures of trigeminal neurons (TGNs). We also measured responses from mice lacking TRPA1 and TRPV1 channels known to mediate some trigeminal responses. Next, we tested how trigeminal activation affects the olfactory response in the olfactory epithelium using electro-olfactogram (EOG) recordings from wild-type (WT) and TRPA1/V1-knock out (KO) mice. The trigeminal modulation of the olfactory response was determined by measuring responses to the odorant, 2-phenylethanol (PEA), an odorant with little trigeminal potency after stimulation with a trigeminal agonist. Trigeminal agonists induced a decrease in the EOG response to PEA, which depended on the level of TRPA1 and TRPV1 activation induced by the trigeminal agonist. This suggests that trigeminal activation can alter odorant responses even at the earliest stage of the olfactory sensory transduction.SIGNIFICANCE STATEMENT Most odorants reaching the olfactory epithelium (OE) can simultaneously activate olfactory and trigeminal systems. Although these two systems constitute two separate sensory modalities, trigeminal activation can alter odor perception. Here, we analyzed the trigeminal activity induced by different odorants proposing an objective quantification of their trigeminal potency independent from human perception. We show that trigeminal activation by odorants reduces the olfactory response in the olfactory epithelium and that such modulation correlates with the trigeminal potency of the trigeminal agonist. These results show that the trigeminal system impacts the olfactory response from its earliest stage.

Natural variations in the Sl-AKR9 aldo/keto reductase gene impact fruit flavor volatile and sugar contents.

The unique flavors of different fruits depend upon complex blends of soluble sugars, organic acids, and volatile organic compounds. 2-Phenylethanol and phenylacetaldehyde are major contributors to flavor in many foods, including tomato. In the tomato fruit, glucose, and fructose are the chemicals that most positively contribute to human flavor preferences. We identified a gene encoding a tomato aldo/keto reductase, Sl-AKR9, that is associated with phenylacetaldehyde and 2-phenylethanol contents in fruits. Two distinct haplotypes were identified; one encodes a chloroplast-targeted protein while the other encodes a transit peptide-less protein that accumulates in the cytoplasm. Sl-AKR9 effectively catalyzes reduction of phenylacetaldehyde to 2-phenylethanol. The enzyme can also metabolize sugar-derived reactive carbonyls, including glyceraldehyde and methylglyoxal. CRISPR-Cas9-induced loss-of-function mutations in Sl-AKR9 significantly increased phenylacetaldehyde and lowered 2-phenylethanol content in ripe fruit. Reduced fruit weight and increased soluble solids, glucose, and fructose contents were observed in the loss-of-function fruits. These results reveal a previously unidentified mechanism affecting two flavor-associated phenylalanine-derived volatile organic compounds, sugar content, and fruit weight. Modern varieties of tomato almost universally contain the haplotype associated with larger fruit, lower sugar content, and lower phenylacetaldehyde and 2-phenylethanol, likely leading to flavor deterioration in modern varieties.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Olfactory-trigeminal integration in the primary olfactory cortex.

Humans naturally integrate signals from the olfactory and intranasal trigeminal systems. A tight interplay has been demonstrated between these two systems, and yet the neural circuitry mediating olfactory-trigeminal (OT) integration remains poorly understood. Using functional magnetic resonance imaging (fMRI), combined with psychophysics, this study investigated the neural mechanisms underlying OT integration. Fifteen participants with normal olfactory function performed a localization task with air-puff stimuli, phenylethyl alcohol (PEA; rose odor), or a combination thereof while being scanned. The ability to localize PEA to either nostril was at chance. Yet, its presence significantly improved the localization accuracy of weak, but not strong, air-puffs, when both stimuli were delivered concurrently to the same nostril, but not when different nostrils received the two stimuli. This enhancement in localization accuracy, exemplifying the principles of spatial coincidence and inverse effectiveness in multisensory integration, was associated with multisensory integrative activity in the primary olfactory (POC), orbitofrontal (OFC), superior temporal (STC), inferior parietal (IPC) and cingulate cortices, and in the cerebellum. Multisensory enhancement in most of these regions correlated with behavioral multisensory enhancement, as did increases in connectivity between some of these regions. We interpret these findings as indicating that the POC is part of a distributed brain network mediating integration between the olfactory and trigeminal systems. PRACTITIONER POINTS: Psychophysical and neuroimaging study of olfactory-trigeminal (OT) integration. Behavior, cortical activity, and network connectivity show OT integration. OT integration obeys principles of inverse effectiveness and spatial coincidence. Behavioral and neural measures of OT integration are correlated.

Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.

BACKGROUND: The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported. METHODS: We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway. RESULTS: CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1ß, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1. CONCLUSION: CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.

Protective effect of synbiotic combination of Lactobacillus plantarum SC-5 and olive oil extract tyrosol in a murine model of ulcerative colitis.

BACKGROUND: Ulcerative colitisis (UC) classified as a form of inflammatory bowel diseases (IBD) characterized by chronic, nonspecific, and recurrent symptoms with a poor prognosis. Common clinical manifestations of UC include diarrhea, fecal bleeding, and abdominal pain. Even though anti-inflammatory drugs can help alleviate symptoms of IBD, their long-term use is limited due to potential side effects. Therefore, alternative approaches for the treatment and prevention of inflammation in UC are crucial. METHODS: This study investigated the synergistic mechanism of Lactobacillus plantarum SC-5 (SC-5) and tyrosol (TY) combination (TS) in murine colitis, specifically exploring their regulatory activity on the dextran sulfate sodium (DSS)-induced inflammatory pathways (NF-κB and MAPK) and key molecular targets (tight junction protein). The effectiveness of 1 week of treatment with SC-5, TY, or TS was evaluated in a DSS-induced colitis mice model by assessing colitis morbidity and colonic mucosal injury (n = 9). To validate these findings, fecal microbiota transplantation (FMT) was performed by inoculating DSS-treated mice with the microbiota of TS-administered mice (n = 9). RESULTS: The results demonstrated that all three treatments effectively reduced colitis morbidity and protected against DSS-induced UC. The combination treatment, TS, exhibited inhibitory effects on the DSS-induced activation of mitogen-activated protein kinase (MAPK) and negatively regulated NF-κB. Furthermore, TS maintained the integrity of the tight junction (TJ) structure by regulating the expression of zona-occludin-1 (ZO-1), Occludin, and Claudin-3 (p < 0.05). Analysis of the intestinal microbiota revealed significant differences, including a decrease in Proteus and an increase in Lactobacillus, Bifidobacterium, and Akkermansia, which supported the protective effect of TS (p < 0.05). An increase in the number of Aspergillus bacteria can cause inflammation in the intestines and lead to the formation of ulcers. Bifidobacterium and Lactobacillus can regulate the micro-ecological balance of the intestinal tract, replenish normal physiological bacteria and inhibit harmful intestinal bacteria, which can alleviate the symptoms of UC. The relative abundance of Akkermansia has been shown to be negatively associated with IBD. The FMT group exhibited alleviated colitis, excellent anti-inflammatory effects, improved colonic barrier integrity, and enrichment of bacteria such as Akkermansia (p < 0.05). These results further supported the gut microbiota-dependent mechanism of TS in ameliorating colonic inflammation. CONCLUSION: In conclusion, the TS demonstrated a remission of colitis and amelioration of colonic inflammation in a gut microbiota-dependent manner. The findings suggest that TS could be a potential natural medicine for the protection of UC health. The above results suggest that TS can be used as a potential therapeutic agent for the clinical regulation of UC.

Interaction of olive oil-based propolis and caffeic acid phenethyl ester with methylprednisolone used in the treatment of human acute myeloid leukemia.

BACKGROUND: Methylprednisolone (MP) is a pharmaceutical agent employed in the management of Leukemia, which is a systemic malignancy that arises from abnormalities in the hematological system. Numerous investigations in the field of cancer research have directed their attention towards propolis, a natural substance with significant potential as a treatment-supportive agent. Its utilization aims to mitigate the potential adverse effects associated with chemotherapy medications. The objective of this study was to examine the impact of olive oil-based propolis (OEP) and caffeic acid phenethyl ester (CAPE) on the treatment of acute myeloid leukemia, as well as to determine if they exhibit a synergistic effect when combined with the therapeutic support product methylprednisolone. METHODS AND RESULTS: The proliferation of HL-60 cells was quantified using the WST-8 kit. The PI Staining technique was employed to do cell cycle analysis of DNA in cells subjected to OEP, CAPE, and MP, with subsequent measurement by flow cytometry. The apoptotic status of cells was determined by analyzing them using flow cytometry after staining with the Annexin V-APC kit. The quantification of apoptotic gene expression levels was conducted in HL-60 cells. In HL-60 cells, the IC50 dosages of CAPE and MP were determined to be 1 × 10- 6 M and 5 × 10- 4 M, respectively. The HL-60 cells were subjected to apoptosis and halted in the G0/G1 and G2/M phases of the cell cycle after being treated with MP, CAPE, and OEP. CONCLUSIONS: Propolis and its constituents have the potential to serve as effective adjunctive therapies in chemotherapy.

The effect of tyrosol on diclofenac sodium-induced acute nephrotoxicity in rats.

Although diclofenac (DCF) is a nonsteroidal anti-inflammatory drug that is considered safe, its chronic use and overdose may show some toxic effects. The protective effect of tyrosol (Tyr) pretreatment against DCF-induced renal damage was investigated in this study. The 32 rats used in the study were randomly divided into four groups of eight rats each. According to the data obtained, it was determined that creatinine, urea, and blood urea nitrogen (BUN) levels increased in serum samples of the DCF group. Besides, the levels of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity decreased and the malondialdehyde (MDA) level increased in the kidney tissue. However, no change was observed in catalase (CAT) activity. Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), and tumor necrosis factor-alpha (Tnf-α) levels increased and nuclear factor erythroid 2-related factor 2 (Nrf-2) levels decreased. No change was detected in the level of interleukin 1 beta (IL-1ß). When the DCF+Tyr group and the DCF group were compared, it was assessed that Tyr had a curative effect on all biochemical parameters. Also, kidney damages, such as degeneration and necrosis of tubular epithelium and congestion of veins, were obviated by treatment with tyrosol in histopathological examinations. It was determined that Tyr pretreatment provided a protective effect against nephrotoxicity induced by DCF with its anti-inflammatory and antioxidant properties.

Nrf2 activation by hydroxytyrosol and dimethyl fumarate ameliorates skin tissue repair in high-fat diet-fed mice by promoting M2 macrophage polarization and normalizing inflammatory response and oxidative damage.

Hydroxytyrosol (HT) or dimethyl fumarate (DMF), activators of nuclear factor erythroid 2-related factor 2 (Nrf2), may reduce obesity in high-fat diet (HFD)-fed animals; nevertheless, the role of these activators on skin tissue repair of HFD-fed animals was not reported. This study investigated whether HT or DMF could improve skin wound healing of HFD-fed obese animals. Mice were fed with an HFD, treated with HT or DMF, and full-thickness skin wounds were created. Macrophages isolated from control and obese animals were treated in vitro with HT. DMF, but not HT, reduced the body weight of HFD-fed mice. Collagen deposition and wound closure were improved by HT or DMF in HFD-fed animals. HT or DMF increased anti-inflammatory macrophage phenotype and protein Nrf2 levels in wounds of HFD-fed mice. Lipid peroxidation and protein tumor necrosis factor-α levels were reduced by HT or DMF in wounds of HFD-fed animals. In in vitro, HT stimulated Nrf2 activation in mouse macrophages isolated from obese animals. In conclusion, HT or DMF improves skin wound healing of HFD-fed mice by reducing oxidative damage and inflammatory response. HT or DMF may be used as a therapeutic strategy to improve the skin healing process in individuals with obesity.

Comprehensive investigations of 2-phenylethanol production by the filamentous fungus Annulohypoxylon stygium.

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain "Jinjiling" (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. KEY POINTS: • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Hydroxytyrosol Alleviates Methotrexate-Induced Pulmonary Fibrosis in Rats: Involvement of TGF-ß1, Tissue Factor, and VEGF.

Methotrexate (MTX) is an indispensable drug used for the treatment of many autoimmune and cancerous diseases. However, its clinical use is associated with serious side effects, such as lung fibrosis. The main objective of this study is to test the hypothesis that hydroxytyrosol (HT) can mitigate MTX-induced lung fibrosis in rats while synergizing MTX anticancer effects. Pulmonary fibrosis was induced in the rats using MTX (14 mg/kg/week, per os (p.o.)). The rats were treated with or without HT (10, 20, and 40 mg/kg/d p.o.) or dexamethasone (DEX; 0.5 mg/kg/d, intraperitoneally (i.p.)) for two weeks concomitantly with MTX. Transforming growth factor beta 1 (TGF-ß1), interleukin-4 (IL-4), thromboxane A2 (TXA2), vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxy-guanosine (8-OHdG), tissue factor (TF) and fibrin were assessed using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and RT-PCR. Pulmonary fibrosis was manifested by an excessive extracellular matrix (ECM) deposition and a marked increase in TGF-ß1 and IL-4 in lung tissues. Furthermore, cotreatment with HT or dexamethasone (DEX) significantly attenuated MTX-induced ECM deposition, TGF-ß1, and IL-4 expression. Similarly, HT or DEX notably reduced hydroxyproline contents, TXA2, fibrin, and TF expression in lung tissues. Moreover, using HT or DEX downregulated the gene expression of TF. A significant decrease in lung contents of VEGF, IL-8, and 8-OHdG was also observed in HT + MTX- or DEX + MTX -treated animals in a dose-dependent manner. Collectively, the results of our study suggest that HT might represent a potential protective agent against MTX-induced pulmonary fibrosis.

Caffeic acid phenethyl ester mediates apoptosis in serum-starved HT29 colon cancer cells through modulation of heat shock proteins and MAPK pathways.

Colorectal cancer (CRC) is among the most prevalent gastrointestinal cancers of epithelial origin worldwide, with over 2 million cases detected every year. Emerging evidence suggests a significant increase in the levels of inflammatory and stress-related markers in patients with CRC, indicating that oxidative stress and lipid peroxidation may influence signalling cascades involved in the progression of the disease. However, the precise molecular and cellular basis underlying CRC and their modulations during bioactive compound exposure have not yet been deciphered. This study examines the effect of caffeic acid phenethyl ester (CAPE), a natural bioactive compound, in HT29 CRC cells grown under serum-supplemented and serum-deprived conditions. We found that CAPE inhibited cell cycle progression in the G2/M phase and induced apoptosis. Migration assay confirmed that CAPE repressed cancer invasiveness. Protein localisation by immunofluorescence microscopy and protein expression by western blot analysis reveal increased expressions of key inflammatory signalling mediators such as p38α, Jun N-terminal kinase and extracellular signal-regulated kinase (ERK) proteins. Molecular docking data demonstrates that CAPE shows a higher docking score of -5.35 versus -4.59 to known p38 inhibitor SB203580 as well as a docking score of -4.17 versus -3.86 to known ERK1/2 inhibitor AZD0364. Co-immunoprecipitation data reveals that CAPE treatment effectively downregulates heat shock protein (HSP) expression in both sera-supplemented and limited conditions through its interaction with mitogen-activated protein kinase 14 (MAPK14). These results suggest that stress induction via serum starvation in HT29 CRC cells leads to the induction of apoptosis and co-ordinated activation of MAPK-HSP pathways. Molecular docking studies support that CAPE could serve as an effective inhibitor to target p38 and MAPK compared to their currently known inhibitors.

The anticancer impacts of free and liposomal caffeic acid phenethyl ester (CAPE) on melanoma cell line (A375).

The deadliest type of skin cancer, malignant melanoma, is also the reason for the majority of skin cancer-related deaths. The objective of this article was to investigate the efficiency of free caffeic acid phenethyl ester (CAPE) and liposomal CAPE in inducing apoptosis in melanoma cells (A375) in in vitro. CAPE was loaded into liposomes made up of hydrogenated soybean phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], and their physicochemical properties were assessed. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed for comparing the cytotoxicity of free CAPE and liposomal CAPE at dosages of 10, 15, 25, 50, 75 and the highest dose of 100 µg/mL for period of 24 and 48 h on A375 cell line to calculate IC50. Apoptosis and necrosis were evaluated in A375 melanoma cancer cells using flow cytometry. Atomic force microscopy was utilized to determine the nanomechanical attributes of the membrane structure of A375 cells. To determine whether there were any effects on apoptosis, the expression of PI3K/AKT1 and BAX/BCL2 genes was analyzed using the real-time polymerase chain reaction technique. According to our results, the maximum amount of drug release from nanoliposomes was determined to be 91% and the encapsulation efficiency of CAPE in liposomes was 85.24%. Also, the release of free CAPE was assessed to be 97%. Compared with liposomal CAPE, free CAPE showed a greater effect on reducing the cancer cell survival after 24 and 48 h. Therefore, IC50 values of A375 cells treated with free and liposomal CAPE were calculated as 47.34 and 63.39 µg/mL for 24 h. After 48 h of incubation of A375 cells with free and liposomal CAPE, IC50 values were determined as 30.55 and 44.83 µg/mL, respectively. The flow cytometry analysis revealed that the apoptosis induced in A375 cancer cells was greater when treated with free CAPE than when treated with liposomal CAPE. The highest nanomechanical changes in the amount of cell adhesion forces, and elastic modulus value were seen in free CAPE. Subsequently, the greatest decrease in PI3K/AKT1 gene expression ratio occurred in free CAPE.

Functional differentiation of olive PLP_deC genes: insights into metabolite biosynthesis and genetic improvement at the whole-genome level.

KEY MESSAGE: This study identified 16 pyridoxal phosphate-dependent decarboxylases in olive at the whole-genome level, conducted analyses on their physicochemical properties, evolutionary relationships and characterized their activity. Group II pyridoxal phosphate-dependent decarboxylases (PLP_deC II) mediate the biosynthesis of characteristic olive metabolites, such as oleuropein and hydroxytyrosol. However, there have been no report on the functional differentiation of this gene family at the whole-genome level. This study conducted an exploration of the family members of PLP_deC II at the whole-genome level, identified 16 PLP_deC II genes, and analyzed their gene structure, physicochemical properties, cis-acting elements, phylogenetic evolution, and gene expression patterns. Prokaryotic expression and enzyme activity assays revealed that OeAAD2 and OeAAD4 could catalyze the decarboxylation reaction of tyrosine and dopa, resulting in the formation of their respective amine compounds, but it did not catalyze phenylalanine and tryptophan. Which is an important step in the synthetic pathway of hydroxytyrosol and oleuropein. This finding established the foundational data at the molecular level for studying the functional aspects of the olive PLP_deC II gene family and provided essential gene information for genetic improvement of olive.

Evaluation of the effects of hydroxytyrosol on human sperm parameters during cryopreservation.

Human sperm cryopreservation is a routine procedure in assisted reproductive technology, but it has detrimental effects on different sperm parameters due to oxidative stress. Our objective was to assess the impacts of hydroxytyrosol (HT), as an antioxidant, on human sperm parameters following cryopreservation. In the first phase, 20 normal human semen samples were cryopreserved using the rapid freezing method with different concentrations of HT including 0, 50, 100, 150, and 200 µg/mL. In the second phase, 20 normal semen samples were collected and cryopreserved with 50 and 100 µg/mL HT. The beneficial effects of HT were determined by evaluation of motility (computer-assisted sperm analysis; CASA), viability (Eosin-nigrosine stain), DNA integrity (sperm chromatic dispersion test, SCD), reactive oxygen species (DCF and DHE staining by flowcytometry) lipid peroxidation (malondialdehyde, MDA test) and mitochondrial membrane potential (JC1 staining by flowcytometry) of sperm after cryopreservation. After thawing, sperm motility had an increasing trend in 50 and 100 µg/mL HT groups in comparison with other groups, althought the difference was not significant. However, sperm viability was significantly increased at 50 and 100 µg/mL HT. Our data also showed that sperm DNA fragmentation was significantly decreased after thawing at 100 µg/mL in comparison with 0 and 50 µg/mL HT. However, the level of intracellular reactive oxygen species, lipid peroxidation and mitochondrial membrane potential were not significantly different between groups. Our results showed that HT may have protective effects on the viability and DNA integrity of human sperm during the freezing-thawing process.

Highly efficient biosynthesis of salidroside by a UDP-glucosyltransferase-catalyzed cascade reaction.

OBJECTIVE: Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). RESULTS: The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana, which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. CONCLUSION: This is the highest salidroside titer ever reported so far using biocatalytic approach.

The neuroprotective effects of caffeic acid phenethyl ester against methamphetamine-induced neurotoxicity.

Methamphetamine (METH) is a highly abused substance on a global scale and has the capacity to elicit toxicity within the central nervous system. The neurotoxicity induced by METH encompasses neuronal degeneration and cellular demise within the substantia nigra-striatum and hippocampus. Caffeic acid phenethyl ester (CAPE), a constituent of propolis, is a diminutive compound that demonstrates antioxidative and anti-inflammatory characteristics. Numerous investigations have demonstrated the safeguarding effects of CAPE in various neurodegenerative ailments. Our hypothesis posits that CAPE may exert a neuroprotective influence on METH-induced neurotoxicity via specific mechanisms. In order to validate the hypothesis, a series of experimental techniques including behavioral tests, immunofluorescence labeling, RNA sequencing, and western blotting were employed to investigate the neurotoxic effects of METH and the potential protective effects of CAPE. The results of our study demonstrate that CAPE effectively ameliorates cognitive memory deficits and anxiety symptoms induced by METH in mice. Furthermore, CAPE has been observed to attenuate the upregulation of neurotoxicity-associated proteins that are induced by METH exposure and also reduced the loss of hippocampal neurons in mice. Moreover, transcriptomics analysis was conducted to determine alterations in gene expression within the hippocampus of mice. Subsequently, bioinformatics analysis was employed to investigate the divergent outcomes and identify potential key genes. Interferon-stimulated gene 15 (ISG15) was successfully identified and confirmed through RT-qPCR, western blotting, and immunofluorescence techniques. Our research findings unequivocally demonstrated the neuroprotective effect of CAPE against METH-induced neurotoxicity, with ISG15 may have an important role in the underlying protective mechanism. These results offer novel perspectives on the treatment of METH-induced neurotoxicity.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Caffeic acid phenethyl ester restores mitochondrial homeostasis against peritoneal fibrosis induced by peritoneal dialysis through the AMPK/SIRT1 pathway.

Increasing evidence suggests that peritoneal fibrosis induced by peritoneal dialysis (PD) is linked to oxidative stress. However, there are currently no effective interventions for peritoneal fibrosis. In the present study, we explored whether adding caffeic acid phenethyl ester (CAPE) to peritoneal dialysis fluid (PDF) improved peritoneal fibrosis caused by PD and explored the molecular mechanism. We established a peritoneal fibrosis model in Sprague-Dawley rats through intraperitoneal injection of PDF and lipopolysaccharide (LPS). Rats in the PD group showed increased peritoneal thickness, submesothelial collagen deposition, and the expression of TGFß1 and α-SMA. Adding CAPE to PDF significantly inhibited PD-induced submesothelial thickening, reduced TGFß1 and α-SMA expression, alleviated peritoneal fibrosis, and improved the peritoneal ultrafiltration function. In vitro, peritoneal mesothelial cells (PMCs) treated with PDF showed inhibition of the AMPK/SIRT1 pathway, mitochondrial membrane potential depolarization, overproduction of mitochondrial reactive oxygen species (ROS), decreased ATP synthesis, and induction of mesothelial-mesenchymal transition (MMT). CAPE activated the AMPK/SIRT1 pathway, thereby inhibiting mitochondrial membrane potential depolarization, reducing mitochondrial ROS generation, and maintaining ATP synthesis. However, the beneficial effects of CAPE were counteracted by an AMPK inhibitor and siSIRT1. Our results suggest that CAPE maintains mitochondrial homeostasis by upregulating the AMPK/SIRT1 pathway, which alleviates oxidative stress and MMT, thereby mitigating the damage to the peritoneal structure and function caused by PD. These findings suggest that adding CAPE to PDF may prevent and treat peritoneal fibrosis.

Chemical Constituents and Anticancer Activities of the Extracts from Phlomis × commixta Rech. f. (P. cretica × P. lanata).

The present work is the first report on the ingredients of the P. × commixta hybrid, a plant of the genus Phlomis. So far, thirty substances have been isolated by various chromatographic techniques and identified by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS. The compounds are classified as flavonoids: naringenin, eriodyctiol, eriodyctiol-7-O-ß-D-glucoside, luteolin, luteolin-7-O-ß-D-glucoside, apigenin, apigenin-7-O-ß-D-glucoside, diosmetin-7-O-ß-D-glucoside, quercetin, hesperetin and quercetin-3-O-ß-D-glucoside; phenylpropanoids: martynoside, verbascoside, forsythoside B, echinacoside and allysonoside; chromene: 5,7-dihydroxychromone; phenolic acids: caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, chlorogenic acid methyl ester, gallic acid, p-coumaric acid and vanillic acid; aliphatic hydrocarbon: docos-1-ene; steroids: brassicasterol and stigmasterol; a glucoside of allylic alcohol, 3-O-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranosyl-oct-1-ene-3-ol, was fully characterized as a natural product for the first time. Two tyrosol esters were also isolated: tyrosol lignocerate and tyrosol methyl ether palmitate, the latter one being isolated as a natural product for the first time. Moreover, the biological activities of the extracts from the different polarities of the roots, leaves and flowers were estimated for their cytotoxic potency. All root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines.