Cell Wall Damage Research Articles

Antioxidant and anti-staphylococcal activity of polyphenolic-rich extracts from Ataulfo mango seed

In recent years, the search for new antioxidant and antimicrobial agents is one of the first subjects of scientific research. The polyphenolic compounds of food byproducts have attracted scientists across the world in the search for new natural substances. In this study, antioxidant (DPPH, ABTS, and TAA) and anti-Staphylococcus aureus (ATCC and clinical isolates strains) activity of mango seed extracts was investigated. The extracts were obtained by Microwave-Assisted Extraction (MAE), Fermentation-Assisted Extraction (FAE), and Solvent-Assisted Extraction (SAE). Chemical profile as well as influence of applied extraction technique were also investigated. The extracts showed potent antioxidant activity with EC50 values (MAE: 27.1 ± 0.15 μg/mL, SAE:14.3 ± 0.37 μg/mL and FAE:12.5 ± 0.41 μg/mL) higher than Trolox (for DPPH). Higher antibacterial activity was observed (MIC: 7.31–750 μg/mL and CMB: 156.3–1500 μg/mL) compared to synthetic antibiotics. Cell wall damage and cell lysis were clearly visualized under Scanning Electron Microscopy (SEM). HPLC–MS allowed the detection of 6 (SAE), 8 (MAE), and 11 (FAE) polyphenolic compounds. These findings suggest that Ataulfo mango seed extracts may be utilized in the developed natural antioxidant and antibacterial agents.

Involvement of a Multidrug Efflux Pump and Alterations in Cell Surface Structure in the Synergistic Antifungal Activity of Nagilactone E and Anethole against Budding Yeast Saccharomyces cerevisiae.

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi, inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae. Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.

OsPG1 Encodes a Polygalacturonase that Determines Cell Wall Architecture and Affects Resistance to Bacterial Blight Pathogen in Rice

BackgroundPlant cell walls are the main physical barrier encountered by pathogens colonizing plant tissues. Alteration of cell wall integrity (CWI) can activate specific defenses by impairing proteins involved in cell wall biosynthesis, degradation and remodeling, or cell wall damage due to biotic or abiotic stress. Polygalacturonase (PG) depolymerize pectin by hydrolysis, thereby altering pectin composition and structures and activating cell wall defense. Although many studies of CWI have been reported, the mechanism of how PGs regulate cell wall immune response is not well understood.ResultsNecrosis appeared in leaf tips at the tillering stage, finally resulting in 3–5 cm of dark brown necrotic tissue. ltn-212 showed obvious cell death and accumulation of H2O2 in leaf tips. The defense responses were activated in ltn-212 to resist bacterial blight pathogen of rice. Map based cloning revealed that a single base substitution (G-A) in the first intron caused incorrect splicing of OsPG1, resulting in a necrotic phenotype. OsPG1 is constitutively expressed in all organs, and the wild-type phenotype was restored in complementation individuals and knockout of wild-type lines resulted in necrosis as in ltn-212. Transmission electron microscopy showed that thicknesses of cell walls were significantly reduced and cell size and shape were significantly diminished in ltn-212.ConclusionThese results demonstrate that OsPG1 encodes a PG in response to the leaf tip necrosis phenotype of ltn-212. Loss-of-function mutation of ltn-212 destroyed CWI, resulting in spontaneous cell death and an auto-activated defense response including reactive oxygen species (ROS) burst and pathogenesis-related (PR) gene expression, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). These findings promote our understanding of the CWI mediated defense response.

Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity.

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskişehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform ınfrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), ınductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl4 concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5–15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20–40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.

Escherichia coli Colonization of Intestinal Epithelial Layers In Vitro in the Presence of Encapsulated Bifidobacterium breve for Its Protection against Gastrointestinal Fluids and Antibiotics.

Encapsulation of probiotic bacteria can enhance their functionality when used in combination with antibiotics for treating intestinal tract infections. The interaction strength of encapsulating shells, however, varies among the encapsulation methods and impacts encapsulation. Here, we compared the protection offered by encapsulating shells with different interaction strengths toward probiotic Bifidobacterium breve against simulated gastric fluid and tetracycline, including protamine-assisted SiO2 nanoparticle yolk–shell packing (weak interaction across a void), alginate gelation (intermediate interaction due to hydrogen binding), and ZIF-8 mineralization (strong interaction due to coordinate covalent binding). The presence of encapsulating shells was demonstrated using X-ray-photoelectron spectroscopy, particulate microelectrophoresis, and dynamic light scattering. Strong interaction upon ZIF-8 encapsulation caused demonstrable cell wall damage to B. breve and slightly reduced bacterial viability, delaying the growth of encapsulated bacteria. Cell wall damage and reduced viability did not occur upon encapsulation with weakly interacting yolk–shells. Only alginate-hydrogel-based shells yielded protection against simulated gastric acid and tetracycline. Accordingly, only alginate-hydrogel-encapsulated B. breve operated synergistically with tetracycline in killing tetracycline-resistant Escherichia coli adhering to intestinal epithelial layers and maintained surface coverage of transwell membranes by epithelial cell layers and their barrier integrity. This synergy between alginate-hydrogel-encapsulated B. breve and an antibiotic warrants further studies for treating antibiotic-resistant E. coli infections in the gastrointestinal tract.

Cooking of short, medium and long-grain rice in limited and excess water: Effects on microstructural characteristics and gastro-small intestinal starch digestion in vitro

The purpose of present study was to investigate the impact of cooking methods on starch hydrolysis of rice grain using an in vitro digestion model. Three varieties of short, medium and long-grain rice were cooked in two different ways: in limited water method (LWM) using a rice cooker and excess water method (EWM) using a pan. The water absorption of raw rice grain was found to differ among the different rice varieties. The moisture, crude protein, total starch and resistant starch contents of the grain were affected by the cooking method. Starch hydrolysis for medium and long-grain rice at 210 min was higher for rice cooked through LWM (75.1 and 87.5%, respectively) than those cooked using the EWM (65.8 and 64.5%, respectively). Microscopic observations of grain cooked through LWM and EWM showed that the former had bigger voids present throughout the grain and had more cell wall damage than the latter, confirming that the microstructural characteristics were responsible for better enzyme accessibility and higher starch hydrolysis. These results revealed that the starch digestibility of rice grain cooked through different methods was affected by the disruption of the tissue structure that was dependent on the cooking method.

Review of Cell wall damage impairs root hair cell patterning and tissue morphogenesis mediated by the Arabidopsis receptor kinase STRUBBELIG

Review of Cell wall damage impairs root hair cell patterning and tissue morphogenesis mediated by the Arabidopsis receptor kinase STRUBBELIG

S. boulardii Fails to Hold Its Cell Wall Integrity against Nonpathogenic E. coli: Are Probiotic Yeasts Losing the Battle?

Probiotic yeast Saccharomyces boulardii exerts direct probiotic action on pathogenic E. coli by trapping them on surfaces and inactivating toxic lipopolysaccharides. Using optical dark-field microscopy, we show that nonpathogenic E. coli cells also readily bind probiotic S. boulardii. More importantly, the adhered nonpathogenic E. coli progressively damage S. boulardii cell walls and lyse them. Co-cultured methylene blue-supplemented agar-plate assay indicates that rough lipopolysaccharides might be playing a key role in S. boulardii cell wall damage. When experiments are repeated with lipopolysaccharide-depleted E. coli and also lipopolysaccharide-deficient E. coli, adhesion decreases substantially. The co-cultured assay further reveals that free lipopolysaccharides, released from E. coli, are also causing damage to S. boulardii walls like adhered E. coli. These new findings contradict the known S. boulardii-E. coli interaction mechanisms. We confirm that E. coli cells do not bind or damage human erythrocyte cell walls; therefore, they have not developed pathogenicity. The combined results demonstrate the first example of nonpathogenic E. coli being harmful to probiotic yeast S. boulardii. This finding is important because gut microbial flora contain large numbers of nonpathogenic E. coli. If they bind or damage probiotic S. boulardii cell walls, then the probiotic efficiency toward pathogenic E. coli will be compromised.

Responses of Sporothrix globosa to the cell wall perturbing agents Congo Red and Calcofluor White.

It is well documented that disturbance of cell surface by some agents triggers compensatory responses aimed to maintain the cell wall integrity in fungi and other organisms. Here, the thermodimorphic fungus Sporothrix globosa, a member of the pathogenic clade of the Sporothrix complex, was propagated in yeast-peptone-dextrose medium under conditions to obtain the mycelium (pH 4.5, 27-28°C) or the yeast (pH 7.8, 32-34°C) morphotypes in the absence and presence of the wall-interacting dyes Congo Red (CR) and Calcofluor White (CFW) either alone or in combination. After different periods of time, growth, cell morphology and activity of glucosamine-6-phosphate synthase (GlcN-6-P synthase), an ubiquitous enzyme that plays a crucial role in cell wall biogenesis, were determined. CR and to a lower extent CFW affected growth and morphology of both fungal morphotypes and significantly increased enzyme activity. Notoriously, CR or CR in combination with CFW induced the transient conversion of yeasts into conidia-forming filamentous cells even under culture conditions adjusted for yeast development, most likely as a strategy to evade the noxious effect of the dye. After sometime, hypha returned to yeast cells. An hypothetical model to explain the effect of CR on morphology and enzyme activity based on the possible role of membrane-spanning proteins known as mechanosensors is proposed. Results are discussed in terms of the fungal responses to cell wall damage.

Study on the Inhibitory Activity and Possible Mechanism of Myriocin on Clinically Relevant Drug-Resistant Candida albicans and Its Biofilms.

In order to prevent and control the infection of Candida albicans, the antifungal activity, possible mechanism of myriocin against C. albicans and its biofilm were studied. The antifungal activity of myriocin was investigated by microdilution method. The effect of myriocin on fungal cell wall or membrane was evaluated by adding sorbitol, ergosterol or phytosphingosine (PHS). The damage to the cell membrane was investigated with propidium iodide (PI) staining and visualized by scanning electron microscope (SEM). The effects on biofilms and extracellular polysaccharides (EPS) were observed by crystal violet staining method and phenol-sulfuric acid method respectively. The adhesion of C. albicans cells to hydrocarbons was tested to evaluate cell surface hydrophobic (CSH). The combined effects of myriocin and antifungal drugs commonly used in clinical practice were investigated by using the checkerboard microdilution method. Minimal inhibitory concentrations (MICs) were found to be 0.125-4 µg/mL. Myriocin was found to affect both cell wall and cell membrane. After exposure to myriocin, biofilm and EPS were found to be inhibited and removed, and the CSH was decreased. The combined fungistasis of myriocin and voriconazole (VCZ) or amphotericin B (AMB) were additive. Myriocin had significant antifungal activity against C. albicans, and the antifungal mechanisms might be cell wall and membrane damage. Myriocin effectively inhibited and eliminated biofilms, and its mechanism may be related to the inhibition of EPS and CSH.

Antifungal promising agents of zinc(II) and copper(II) derivatives based on azole drug

A series of new metal complexes, [Zn(KTZ)2(Ac)2]·H2O (1), [Zn(KTZ)2Cl2]·0.4CH3OH (2), [Zn(KTZ)2(H2O)(NO3)](NO3) (3), [Cu(KTZ)2(Ac)2]·H2O (4), [Cu(KTZ)2Cl2]·3.2H2O (5), [Cu(KTZ)2(H2O)(NO3)](NO3)·H2O (6), were synthesized by a reaction of ketoconazole (KTZ) with their respective zinc or copper salts under mild conditions. Similarly, six corresponding metal-CTZ (clotrimazole) complexes [Zn(CTZ)2(Ac)2]·4H2O (7), [Zn(CTZ)2Cl2] (8), [Zn(CTZ)2(H2O)(NO3)](NO3)·4H2O (9), [Cu(CTZ)2(Ac)2]·H2O (10), [Cu(CTZ)2Cl2]·2H2O (11), [Cu(CTZ)2(H2O)(NO3)](NO3)·2H2O (12), were obtained. These metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV/Vis, and infrared spectroscopies. Further, the crystal structure for complexes 7 and 10 was determined by single-crystal X-ray diffraction. The antifungal activity of these metal complexes was evaluated against three fungal species of medical relevance: Candida albicans, Cryptococcus neoformans, and Sporothrix brasiliensis. Complexes 1 and 3 exhibited the greatest antifungal activity with a broad spectrum of action at low concentrations and high selectivity. Some morphological changes induced by these metal complexes in S. brasiliensis cells included yeast-hyphae conversion, an increase in cell size and cell wall damage. The strategy of coordination of clinic drugs (KTZ and CTZ) to zinc and copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activities displayed by Zn-KTZ complexes make them potential candidates for the development of an alternative drug to treat mycoses.

Host Cell Wall Damage during Pathogen Infection: Mechanisms of Perception and Role in Plant-Pathogen Interactions.

The plant cell wall (CW) is a complex structure that acts as a mechanical barrier, restricting the access to most microbes. Phytopathogenic microorganisms can deploy an arsenal of CW-degrading enzymes (CWDEs) that are required for virulence. In turn, plants have evolved proteins able to inhibit the activity of specific microbial CWDEs, reducing CW damage and favoring the accumulation of CW-derived fragments that act as damage-associated molecular patterns (DAMPs) and trigger an immune response in the host. CW-derived DAMPs might be a component of the complex system of surveillance of CW integrity (CWI), that plants have evolved to detect changes in CW properties. Microbial CWDEs can activate the plant CWI maintenance system and induce compensatory responses to reinforce CWs during infection. Recent evidence indicates that the CWI surveillance system interacts in a complex way with the innate immune system to fine-tune downstream responses and strike a balance between defense and growth.

Microbiological evaluation of an experimental denture cleanser containing essential oil of Lippia sidoides

The antimicrobial activity of an experimental solution containing essential oil of Lippia sidoides for denture cleaning was evaluated by (1) minimum inhibitory (MIC) and fungicidal/bactericidal concentration (MFC/MBC) tests against Candida albicans, Staphylococcus aureus, and Pseudomona aeruginosa; (2) the metabolic activity of C. albicans biofilm formed on flat-bottom microplates and denture base specimens based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); and (3) scanning electron microscopy, to evaluate the fungal biofilm morphology. The solution showed antimicrobial action against the pathogens tested (C. albicans - MIC and MFC: 19.53 µg ml−1, S. aureus - MIC and MBC: 78.12 µg ml−1, P. aeruginosa - MIC: 625 µg ml−1, MBC: 2,500 µg ml−1), reduced the metabolic activity of C. albicans biofilm up to 97%, and caused cell wall damage at low concentrations (195.3–390.6 µg ml−1) and in short time periods (20 min). Therefore, the experimental solution has the potential to be used as an alternative in the prevention and treatment of denture-induced infections.

Effective Treatment of Cutaneous Mold Infections by Antimicrobial Blue Light That Is Potentiated by Quinine.

Cutaneous mold infections commonly result from an array of traumatic injuries that involve direct inoculation of contaminated soil into wounds. Here, we explored the use of antimicrobial blue light (aBL; 405 nm wavelength) and the combination of aBL with quinine hydrochloride (aBL + Q-HCL) for the treatment of cutaneous mold infections. Efficacy of aBL and aBL + Q-HCL in killing clinically important pathogenic molds (Aspergillus fumigatus, Aspergillus flavus, and Fusarium oxyprorum) was investigated. Ultraperformance liquid chromatography identified and quantified endogenous porphyrins in the mold conidia. Finally, a mouse model of dermabrasion wound infected with a bioluminescent variant of A. fumigatus was developed to investigate the efficacy of aBL in treating cutaneous mold infections. We demonstrated that mold conidia are tolerant to aBL, but Q-HCL enhances efficacy. Transmission electron microscopy revealed intracellular damage by aBL. aBL + Q-HCL resulted in intracellular and cell wall damage. Porphyrins were observed in all mold strains, with A. fumigatus having the highest concentration. aBL and aBL + Q-HCL effectively reduced the burden of A. fumigatus within an established dermabrasion infection and limited recurrence posttreatment. aBL and aBL + Q-HCL may offer a novel approach for the treatment of mold infections.

Harmful effects of metal(loid) oxide nanoparticles.

The incorporation of nanomaterials (NMs), including metal(loid) oxide (MOx) nanoparticles (NPs), in the most diversified consumer products, has grown enormously in recent decades. Consequently, the contact between humans and these materials increased, as well as their presence in the environment. This fact has raised concerns and uncertainties about the possible risks of NMs to human health and the adverse effects on the environment. These concerns underline the need and importance of assessing its nanosecurity. The present review focuses on the main mechanisms underlying the MOx NPs toxicity, illustrated with different biological models: release of toxic ions, cellular uptake of NPs, oxidative stress, shading effect on photosynthetic microorganisms, physical restrain and damage of cell wall. Additionally, the biological models used to evaluate the potential hazardous of nanomaterials are briefly presented, with particular emphasis on the yeast Saccharomyces cerevisiae, as an alternative model in nanotoxicology. An overview containing recent scientific advances on cellular responses (toxic symptoms exhibited by yeasts) resulting from the interaction with MOx NPs (inhibition of cell proliferation, cell wall damage, alteration of function and morphology of organelles, presence of oxidative stress bio-indicators, gene expression changes, genotoxicity and cell dead) is critically presented. The elucidation of the toxic modes of action of MOx NPs in yeast cells can be very useful in providing additional clues about the impact of NPs on the physiology and metabolism of the eukaryotic cell. Current and future trends of MOx NPs toxicity, regarding their possible impacts on the environment and human health, are discussed.Key points• The potential hazardous effects of MOx NPs are critically reviewed.• An overview of the main mechanisms associated with MOx NPs toxicity is presented.• Scientific advances about yeast cell responses to MOx NPs are updated and discussed.

Efg1 and Cas5 Orchestrate Cell Wall Damage Response to Caspofungin in Candida albicans.

Echinocandins are recommended as the first-line drugs for the treatment of systemic candidiasis. Cas5 is a key transcription factor involved in the response to cell wall damage induced by echinocandins. In this study, through a genetic screen, we identified a second transcription factor, Efg1, that is also crucial for proper transcriptional responses to echinocandins. Like CAS5, deletion of EFG1 confers hypersensitivity to caspofungin. Efg1 is required for the induction of CAS5 in response to caspofungin. However, ectopically expressed CAS5 cannot rescue the growth defect of efg1 mutant in caspofungin-containing medium. Deleting EFG1 in the cas5 mutant exacerbates the cell wall stress upon caspofungin addition and renders caspofungin-resistant Candida albicans responsive to treatment. Genome-wide transcription profiling of efg1/efg1 and cas5/cas5 using transcriptome sequencing (RNA-Seq) indicates that Efg1 and Cas5 coregulate caspofungin-responsive gene expression, but they also independently control induction of some genes. We further show that Efg1 interacts with Cas5 by yeast two-hybrid and in vivo immunoprecipitation in the presence or absence of caspofungin. Importantly, Efg1 and Cas5 bind to some caspofungin-responsive gene promoters to coordinately activate their expression. Thus, we demonstrate that Efg1, together with Cas5, controls the transcriptional response to cell wall stress induced by caspofungin.

A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to β-lactam antibiotics.

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level β-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism's ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.

A physico-chemical study of the interaction of ethanolic extracts of propolis with bacterial cells

In the present study, an effort has been made to understand the interaction mode of propolis, a natural substance produced by honey bees, with gram-positive and gram-negative bacterial cells by measuring alterations in cell surface physico-chemical properties following the incubation of the cells with different sub-inhibitory concentrations of this antimicrobial agent. Electrophoretic mobility and surface hydrophobicity measurements revealed for the first time that propolis induced substantial changes in the volumetric charge density, electrophoretic softness and degree of hydrophobicity characterizing the outermost surface layer of cells. These changes, which appear to be dose-dependent, seem to be consistent with the increasing accumulation and penetration of the propolis antimicrobial components through the cells extracellular layer. Moreover, electron microscopy observation and the determination of the cell constituents’ release demonstrated that propolis at sub-bactericidal concentrations already provoked (at least localized) cell wall damage and/or perturbations. These findings thus suggest that the initial mechanism of action of propolis is most likely structural, resulting from sufficient interaction between the different propolis components and bacterial cell wall structures.

Widely targeted metabolomics analysis reveals new biomarkers and mechanistic insights on chestnut (Castanea mollissima Bl.) calcification process

Chestnut calcification is a quality deterioration due to fast water loss, which has been of deep concern for chestnut quality control because its mechanism is unclear. In order to find out the different key metabolites and metabolic pathways related to the occurrence of chestnut calcification, in this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) based widely targeted metabolomics analysis was performed on chestnuts that were stored at 50%–55% (low relative humidity, LRH) at 25 °C and 85%–90% (high relative humidity, HRH) at 25 °C. A total of 611 metabolites were detected, and 55 differentially accumulated metabolites were identified as key metabolites involved in chestnut calcification process. The decrease in some monosaccharides accompanied with the increase in some unsaturated fatty acids indicated the degradation of chestnut cell wall and cell membrane during calcification process. As a stress response, amino acid metabolism related to membrane stability was significantly activated. In addition, the enhancement of phenylpropanoid biosynthesis pathway and flavonoid biosynthesis pathway characterized by the accumulation of lignin precursors and antioxidants suggested that lignification process was triggered in calcified chestnut. Therefore, the degradation and hardening of the cell wall and membrane damage were proposed to be associated with the calcification occurrence of chestnut. The metabolic profile of chestnut characterized in this study provided new insights into chestnut calcification process and laid a foundation for further chestnut quality control.

In-vitro Antibacterial and Antibiofilm Activity of Cinnamomum verum Leaf Oil against Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae.

Phytomedicines are becoming more popular in treatment of infectious diseases worldwide. Cinnamomum verum essential oil (EO) has been used as a therapeutic alternative for various diseases. This study aimed to evaluate the antibacterial and antibiofilm activity of the C. verum leaf EO against Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Effect of EO vapor on planktonic cells was determined using microatmosphere technique. CLSI M7-A10 method was employed in Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) experiments. Effect of EO on established biofilms was quantified and visualized using XTT and Scanning Electron Microscope (SEM). In-vitro toxicity was evaluated using Human Keratinocytes (HaCaT). Chemical analysis of EO was done using Gas Chromatography- Mass Spectrometry (GC-MS). All tested strains were sensitive to cinnamon oil vapor. EO exhibited 0.5 and 1.0 mg/mL MIC and MBC against all test strains. Minimum Biofilm Inhibitory and Biofilm Eradication Concentrations (MBIC50 and MBEC) were 1.0 and 4.0 mg/mL. SEM indicated cellular shrinkages, cell wall damages, and decreased biofilm densities. Cinnamon oil didn't show any toxicity on HaCaT cell at any concentration tested. Eugenol was the most abundant compound in C. verum oil. C. verum EO shows an antibacterial and antibiofilm activity with minimal toxicity on host.