Inhibitory effect and mechanism of oregano essential oil on Listeria monocytogenes cells, toxins and biofilms.

Listeria monocytogenes (L. monocytogenes) is a prevalent foodborne pathogen with a remarkable capacity to form biofilms on utensil surfaces. The Listeriolysin O (LLO) exhibits hemolytic activity, which is responsible for causing human infections. In this study, we investigated the inhibitory effect and mechanism of oregano essential oil (OEO) on L. monocytogenes, evaluated the effects on its biofilm removal and hemolytic activity. The minimum inhibitory concentration (MIC) of OEO against L. monocytogenes was 0.03% (v/v). L. monocytogenes was treated with OEO at 3/2 MIC for 30 min the bacteria was decreased below the detection limit (10 CFU/mL) in PBS and TSB (the initial bacterial load was about 6.5 log CFU/mL). The level of L. monocytogenes in minced pork co-cultured with OEO (15 MIC) about 2.5 log CFU/g lower than that in the untreated group. The inhibitory mechanisms of OEO against planktonic L. monocytogenes encompassed perturbation of cellular morphology, elevation in reactive oxygen species levels, augmentation of lipid oxidation extent, hyperpolarization of membrane potential, and reduction in intracellular ATP concentration. In addition, OEO reduced biofilm coverage on the surface of glass slides by 62.03% compared with the untreated group. Meanwhile, OEO (1/8 MIC) treatment reduced the hemolytic activity of L. monocytogenes to 24.6% compared with the positive control. Molecular docking suggested carvacrol and thymol might reduce the hemolytic activity of L. monocytogenes. The results of this study demonstrate that OEO exhibits inhibitory effects against L. monocytogenes, biofilms and LLO, which had potential as natural antimicrobial for the inhibition of L. monocytogenes.

Mitochondrial calcium uniporter promotes mitophagy by regulating the PINK1/Parkin pathway in caerulein­treated pancreatic ductal epithelial cells in vitro.

The mitochondrial calcium uniporter (MCU) is a major protein for the uptake of mitochondrial calcium to regulate intracellular energy metabolism, including processes such as mitophagy. The present study investigated the effect of the MCU on mitophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP) in vitro. The normal human PDECs (HPDE6-C7) were treated with caerulein (CAE) to induce AP-like changes, with or without ruthenium red to inhibit the MCU. The mitochondrial membrane potentials (MMPs) and mitochondrial Ca2+ levels were analyzed by fluorescence. The expression levels of MCU, LC3, p62, and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), putative kinase 1 (PINK1), and Parkin were measured by western blotting and immunofluorescence. Mitophagy was observed by confocal fluorescence microscopy and transmission electron microscopy. The results showed that CAE increased the MCU protein expression, mitochondrial Ca2+ levels, MMP depolarization and the protein expression of mitophagy markers including the LC3II/I ratio, PINK1, and Parkin. CAE decreased the protein expression of p62 and TOMM20, and promoted the formation of mitophagosomes in HPDE6-C7 cells. Notably, changes in these markers were reversed by inhibiting the MCU. In conclusion, an activated MCU may promote mitophagy by regulating the PINK1/Parkin pathway in PDECs in AP.

LARP1 knockdown inhibits cultured gastric carcinoma cell cycle progression and metastatic behavior.

This study aimed to clarify the role of la-related protein 1 (LARP1) in cell cycle progression and metastatic behavior of cultured gastric carcinoma (GC) cells. To do that, LARP1 expression was detected in clinical GC tissues and cell lines using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The cell viability, apoptosis, cell cycle, migration, invasion, and cell growth were examined using a Cell Counting Kit-8, Annexin V-FITC staining, propidium iodide staining, Transwell migration and invasion assays, and colony formation assays after LARP1 knockdown. Phosphatidyl inositol 3-kinase (PI3K) and AKT1 mRNA and protein expression levels of PI3K, p-AKT1, AKT1, p-BAD, p-mTOR, and p21 in si-LARP1 transfected GC cells were determined using qRT-PCR and western blotting. Here, we've shown that LARP1 expression was upregulated in human GC tissues and KATO III cells. LARP1 knockdown inhibited GC cell proliferation, cell cycle progression, migration, invasion, and colony formation and promoted apoptosis. In si-LARP1-transfected KATO III cells, the mRNA expression levels of PI3K and AKT1, PI3K protein expression, and the p-AKT1/AKT1 ratio were significantly suppressed. p-mTOR and p-BAD were significantly decreased, whereas p21 was significantly increased in si-LARP1-transfected KATO III cells. In conclusion LARP1 knockdown induces apoptosis and inhibits cell cycle progression and metastatic behavior via PI3K/AKT1 signaling in GC cells.

Antimicrobial Activity of Eugenol Against Bacillus cereus and Its Application in Skim Milk.

Bacillus cereus is a foodborne pathogen widely distributed in the large-scale catering industry and produces spores. The study explored the antibacterial activity, potential mechanism of eugenol against B. cereus, and spores with germination rate. The minimum inhibitory concentration (MIC; 0.6 mg/mL) of eugenol to six B. cereus strains was compared with the control; B. cereus treated with eugenol had a longer lag phase. Eugenol at a concentration of more than 1/2MIC decreased viable B. cereus (∼5.7 log colony-forming unit [CFU]/mL) counts below detectable limits within 2 h, and eugenol of 3MIC reduced B. cereus (∼5.9 log CFU/mL) in skim milk below detectable limits within 30 min. The pH values of skim milk were unaffected by the addition of eugenol. The ΔE values below 2 show that the color variations of skim milk were not visible to the human eye. For sensory evaluation, eugenol did not significantly affect the color or structural integrity of the skim milk. It had a negative impact on the flavor and general sensory acceptance of the treated milk. Eugenol hyperpolarized B. cereus cell membrane, decreased intracellular ATP concentration, and increased intracellular reactive oxygen species contents and extracellular malondialdehyde contents, resulting in the cell membrane of B. cereus being damaged and permeabilized, and cell morphology being changed. In addition, according to the viable count, confocal laser scanning microscopy, and spore morphology changes, eugenol reduced the germination rate of B. cereus spores. These findings suggest that eugenol can be used as a new natural antibacterial agent to control B. cereus and spores in the food production chain.

Inhibition of Cronobacter sakazakii Biofilm Formation and Expression of Virulence Factors by Coenzyme Q0.

In this study, we investigated the inhibitory effects of coenzyme Q0 (CoQ0) on biofilm formation and the expression of virulence genes by Cronobacter sakazakii. We found that the minimum inhibitory concentration of CoQ0 against C. sakazakii strains ATCC29544 and ATCC29004 was 100 µg/mL, while growth curve assays showed that subinhibitory concentrations (SICs) of CoQ0 for both strains were 6.4, 3.2, 1.6 and 0.8 µg/mL. Assays exploring the inhibition of specific biofilm formation showed that SICs of CoQ0 inhibited biofilm formation by C. sakazakii in a dose-dependent manner, which was confirmed by scanning electron microscopy and confocal laser scanning microscopy analyses. CoQ0 inhibited the swimming and swarming motility of C. sakazakii and reduced its ability to adhere to and invade HT-29 cells. In addition, CoQ0 impeded the ability of C. sakazakii to survive and replicate within RAW 264.7 cells. Finally, real-time polymerase chain reaction analysis confirmed that nine C. sakazakii genes associated with biofilm formation and virulence were downregulated in response to CoQ0 treatment. Overall, our findings suggest that CoQ0 is a promising antibiofilm agent and provide new insights for the prevention and control of infections caused by C. sakazakii.

Antimicrobial and Antibiofilm Efficacy and Mechanism of Oregano Essential Oil Against Shigella flexneri.

The aim of this study was to assess the antimicrobial activity of oregano essential oil (OEO) against Shigella flexneri and eradication efficacy of OEO on biofilm. The results showed that the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of OEO against S. flexneri were 0.02% (v/v) and 0.04% (v/v), respectively. OEO effectively killed S. flexneri in Luria-Bertani (LB) broth and contaminated minced pork (the initial population of S. flexneri was about 7.0 log CFU/mL or 7.2 log CFU/g), and after treatment with OEO at 2 MIC in LB broth or at 15 MIC in minced pork, the population of S. flexneri decreased to an undetectable level after 2 or 9 h, respectively. OEO increased intracellular reactive oxygen species concentration, destroyed cell membrane, changed cell morphology, decreased intracellular ATP concentration, caused cell membrane depolarization, and destroyed proteins or inhibited proteins synthesis of S. flexneri. In addition, OEO effectively eradicated the biofilm of S. flexneri by effectively inactivating S. flexneri in mature biofilm, destroying the three-dimensional structure, and reducing exopolysaccharide biomass of S. flexneri. In conclusion, OEO exerts its antimicrobial action effectively and also has a valid scavenging effect on the biofilm of S. flexneri. These findings suggest that OEO has the potential to be used as a natural antibacterial and antibiofilm material in the control of S. flexneri in meat product supply chain, thereby preventing meat-associated infections.

Synergistic antibacterial and anti-biofilm mechanisms of ultrasound combined with citral nanoemulsion against Staphylococcus aureus 29213.

This study investigated the antibacterial and antibiofilm mechanism of ultrasound (US) combined with citral nanoemulsion (CLNE) against Staphylococcus aureus and mature biofilm. Combined treatments resulted in greater reductions in bacterial numbers compared to ultrasound or CLNE treatments alone. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein nucleic acid leakage, and N-phenyl-l-naphthylamine (NPN) uptake analysis showed that the combined treatment disrupted cell membrane integrity and permeability. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+CLNE exacerbated cellular oxidative stress and membrane lipid peroxidation. Field emission scanning electron microscopy (FESEM) revealed that the synergistic processing of ultrasound and CLNE resulted in cell rupture and collapse. In addition, US+CLNE showed a more pronounced removal effect than both alone in the biofilm on the stainless steel sheet. US+CLNE reduced biomass, the number of viable cells in the biofilm, cell viability and EPS polysaccharide contents. The results of CLSM also showed that US+CLNE disrupted the structure of the biofilm. This research elucidates the synergistic antibacterial and anti-biofilm mechanism of ultrasound combined citral nanoemulsion, which provides a safe and efficient sterilization method for the food industry.

Molecular recognition of SARS-CoV-2 spike protein with three essential partners: exploring possible immune escape mechanisms of viral mutants.

OBJECTIVE: The COVID-19 epidemic is raging around the world, with the emergence of viral mutant strains such as Delta and Omicron, posing severe challenges to people's health and quality of life. A full understanding life cycle of the virus in host cells helps to reveal inactivation mechanism of antibody and provide inspiration for the development of a new-generation vaccines. METHODS: In this work, molecular recognitions and conformational changes of SARS-CoV-2 spike protein mutants (i.e., Delta, Mu, and Omicron) and three essential partners (i.e., membrane receptor hACE2, protease TMPRSS2, and antibody C121) both were compared and analyzed using molecular simulations. RESULTS: Water basin and binding free energy calculations both show that the three mutants possess higher affinity for hACE2 than WT, exhibiting stronger virus transmission. The descending order of cleavage ability by TMPRSS2 is Mu, Delta, Omicron, and WT, which is related to the new S1/S2 cutting site induced by transposition effect. The inefficient utilization of TMPRSS2 by Omicron is consistent with its primary entry into cells via the endosomal pathway. In addition, RBD-directed antibody C121 showed obvious resistance to Omicron, which may have originated from high fluctuation of approaching angles, high flexibility of I472-F490 loop, and reduced binding ability. CONCLUSIONS: According to the overall characteristics of the three mutants, high infectivity, high immune escape, and low virulence may be the future evolutionary selection of SARS-CoV-2. In a word, this work not only proposes the possible resistance mechanism of SARS-CoV-2 mutants, but also provides theoretical guidance for the subsequent drug design against COVID-19 based on S protein structure.

Inhibitory Effects of Trans-Cinnamaldehyde Against Pseudomonas aeruginosa Biofilm Formation.

Pseudomonas aeruginosa biofilm formation has been considered to be an important determinant of its pathogenicity in most infections. The antibiofilm activity of trans-cinnamaldehyde (TC) against P. aeruginosa was investigated in this study. Results demonstrated that the minimum inhibitory concentration (MIC) of TC against P. aeruginosa was 0.8 mg/mL, and subinhibitory concentrations (SICs) was 0.2 mg/mL and below. Crystal violet staining showed that TC at 0.05-0.2 mg/mL reduced biofilm biomass in 48 h in a concentration-dependent mode. The formation area of TC-treated biofilms was significantly declined (p < 0.01) on the glass slides observed by light microscopy. Field-emission scanning electron microscopy further demonstrated that TC destroyed the biofilm morphology and structure. Confocal laser scanning microscopic observed the dispersion of biofilms and the reduction of exopolysaccharides after TC treatment stained with concanavalin A (Con-A)-fluorescein isothiocyanate conjugate and Hoechst 33258. Meanwhile, TC caused a significant decrease (p < 0.01) in the component of polysaccharides, proteins, and DNA in extracellular polymeric substance. The swimming and swarming motility and quorum sensing of P. aeruginosa was also found to be significantly inhibited (p < 0.01) by TC at SICs. Furthermore, SICs of TC repressed the several genes transcription associated with biofilm formation as determined by real-time quantitative polymerase chain reaction. Overall, our findings suggest that TC could be applied as natural and safe antibiofilm agent to inhibit the biofilm formation of P. aeruginosa.

Antibacterial Activity of Thymoquinone Against Shigella flexneri and Its Effect on Biofilm Formation.

Thymoquinone (TQ) has been demonstrated to have anti-cancer, anti-inflammatory, antioxidant, and anti-diabetic activities. Shigella flexneri is the main pathogen causing shigellosis in developing countries. In this study, the antibacterial activity of TQ against S. flexneri and its possible antibacterial mechanism were studied. In addition, the inhibitory effect of TQ on the formation of S. flexneri biofilm was also investigated. The results showed that both the minimum inhibitory concentration and the minimum bactericidal concentration of TQ against S. flexneri ATCC 12022 were 0.2 mg/mL. After treatment with TQ at 0.4 mg/mL in Luria-Bertani broth for 3 h, or treatment with 0.2 mg/mL TQ in phosphate-buffered saline for 60 min, the number of S. flexneri (initial number is 6.5 log colony-forming units/mL) dropped below the detection limit. TQ also displayed good antibacterial activity in contaminated lettuce juice. TQ caused an increase in intracellular reactive oxygen species level, a decrease in intracellular adenosine triphosphate (ATP) concentration, a change in the intracellular protein, damage to cell membrane integrity and changes in cell morphology. In addition, TQ showed the ability to inhibit the formation of S. flexneri biofilm; treatment resulted in a decrease in the amount of biofilm and extracellular polysaccharides, and the destruction of biofilm structure. These findings indicated that TQ had strong antimicrobial and antibiofilm activities and a potential to be applied in the fruit and vegetable processing industry or other food industries to control S. flexneri.

Antibacterial Mechanism of Eugenol Against Shigella sonnei and Its Antibacterial Application in Lettuce Juice.

Shigella sonnei is a species of Shigella, and the infection rate of S. sonnei is increasing year by year. Eugenol is an active ingredient in clove essential oil and is a generally recognized as safe (GRAS)-certified food ingredient. The mechanism of inhibition of S. sonnei by eugenol has been investigated in this study. The minimum inhibitory concentration of eugenol against both S. sonnei ATCC 25931 and S. sonnei CMCC 51592 was 0.5 mg/mL and minimum bactericidal concentration (MBC) for both strains was 0.8 mg/mL. The inhibition effect of eugenol against S. sonnei was due to increased levels of reactive oxygen species in cells, changed cell membrane permeability, and induced cell membrane dysfunction, for instance, cell membrane hyperpolarization and intracellular ATP concentration drops. The results of confocal laser scanning microscope and field emission scanning electron microscopy showed that eugenol leads to decreased cell membrane integrity, resulting in changed cell morphology. Moreover, eugenol inactivated S. sonnei in Luria-Bertani (LB) broth and lettuce juice. These results indicated that eugenol could inactivate S. sonnei and has the potential to control S. sonnei in the food industry.

Inhibitory effect of protocatechualdehyde on Yersinia enterocolitica and its critical virulence factors.

Yersinia enterocolitica (Y. enterocolitica) is a gastrointestinal pathogen that is distributed worldwide, involved in systemic, extraintestinal and invasive infections in immunocompromised patients. Establishment of antibiotic resistance in the pathogen has produced a need for new antibacterial agents. The purpose of this study was to elucidate antibacterial mechanism of protocatechualdehyde (PCA) extracted from the roots of Salvia miltiorrhiza towards Y. enterocolitica, and to investigate effects of PCA on key virulence factors associated with human infection. Present results indicated that PCA exerted its antibacterial activity against Y. enterocolitica mainly by the rapid rise of intracellular reactive oxygen species, leading to change in permeability and integrity of cell membrane, and ultimately decline of membrane potential and intracellular ATP. Furthermore, scanning electron microscopic analysis revealed that Y. enterocolitica presented gradually shrinkage in length and partial wrinkles upon PCA treatment. PCA also effectively decreased motility, biofilm formation, quorum sensing in a dose-dependent manner without affecting bacterial growth. Further, at SICs, PCA substantially suppressed the adhesion and invasion of Y. enterocolitica to HT-29 cells and the downregulation of essential virulence factor-encoding genes unveiled impaired virulence. Overall, the findings revealed the potential of PCA as an alternative antibacterial agent to combat Y. enterocolitica contamination and infections.

Effects of cinnamaldehyde against planktonic bacteria and biofilm formation of Shigella flexneri.

Cinnamaldehyde (CA) has demonstrated anti-inflammatory, anti-tumor and anti-cancer activities; Its antimicrobial and antibiofilm actions against Shigella flexneri, on the other hand, have not been investigated. Sh. flexneri is a gram-negative foodborne pathogen that can be widely found in nature and some industrial production environments. In this current research, our aim was to examine the influences of CA on planktonic bacteria and biofilm formation. The minimum inhibitory concentration (MIC) of CA against Sh. flexneri strain was 100 µg/mL, while bacteria treated with CA showed a longer lag phase compared with the untreated control. CA effectively inactivated the Sh. flexneri in LB broth and fresh lettuce juice. CA treatment resulted in cell membrane permeability changes and dysfunction, as proven by cell membrane depolarization, decreased intracellular ATP concentration. In addition, CA was also discovered to increase the level of reactive oxygen species (ROS) in cells, and induce morphological changes in cells. Crystal violet staining showed that the biomass of biofilm was decreased significantly with CA in 24 h. Light microscopy and field emission scanning electron microscopy (FESEM) observations demonstrated decreased biofilm adhesion and destruction of biofilm architecture after treatment with CA. These findings indicated that CA acts as a natural bacteriostatic agent to control Sh. flexneri in food processing and production.

Efficacy of 405 nm Light-Emitting Diode Illumination and Citral Used Alone and in Combination for Inactivation of Vibrio parahaemolyticus on Shrimp.

Vibrio parahaemolyticus is a widely distributed pathogen, which is frequently the lead cause of infections related to seafood consumption. The objective of the present study was to investigate the antimicrobial effect of the combination of 405 nm light-emitting diode (LED) and citral on V. parahaemolyticus. The antimicrobial effect of LED illumination and citral was evaluated on V. parahaemolyticus not only in phosphate-buffered saline (PBS) but also on shrimp. Quality changes of shrimp were determined by sensory evaluation. Changes in bacteria cell membrane morphology, cell membrane permeability, cell lipid oxidation level, and DNA degradation were examined to provide insights into the antimicrobial mechanism. The combination of LED treatments and citral had better antimicrobial effects than either treatment alone. LED combined with 0.1 mg/mL of citral effectively reduced V. parahaemolyticus from 6.5 log CFU/mL to below the detection limit in PBS. Combined treatment caused a 3.5 log reduction of the pathogen on shrimp within 20 min and a 6 log reduction within 2 h without significant changes in the sensory score. Furthermore, combined LED and citral treatment affected V. parahaemolyticus cellular morphology and outer membrane integrity. The profile of the comet assay and DNA fragmentation analysis revealed that combination treatment did not cause a breakdown of bacterial genomic DNA. In conclusion, LED may act synergistically with citral. They have the potential to be developed as novel microbial intervention strategies.

Comparison and Optimization of Continuous Flow Reactors for Aerobic Granule Sludge Cultivation from the Perspective of Hydrodynamic Behavior.

Improving treatment efficiency and reducing investment and operating costs make aerobic granular sludge technology (AGS) a promising technology for treating aquaculture wastewater. The development of continuous flow reactors (CFRs) has become a new direction in the research of AGS. This study clarifies the granulation effect, hydrodynamic behavior and particle separation of three different CFRs (R1 to R3). The established CFD model was able to explain the hydrodynamic behavior in all three CFRs; in particular, R3 performed the best from the perspective of hydrodynamic behavior due to its abundant turbulence. In addition, the optimal baffle distance and baffle angle of R3 were simulated to be 40 mm and 60°, respectively, due to them providing the best turbulent flow and particle separation effect. However, an overlarge baffle angle could weaken the turbulent pattern in the reactor. The retention time distribution further confirmed the reasonability of these optimal parameters with the highest effective volume ratio of 0.82. In short, this study gives an instruction for exploring the rapid formation mechanism of AGS in a CFR to promote its engineering application.

Omicron-included mutation-induced changes in epitopes of SARS-CoV-2 spike protein and effectiveness assessments of current antibodies.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading globally and continues to rage, posing a serious threat to human health and life quality. Antibody therapy and vaccines both have shown great efficacy in the prevention and treatment of COVID-19, whose development progress and adaptation range have attracted wide attention. However, with the emergence of variant strains of SARS-CoV-2, the neutralization activity of therapeutic or vaccine-induced antibodies may be reduced, requiring long-term virus monitoring and drug upgrade in response to its evolution. In this paper, conformational changes including continuous epitopes (CPs), discontinuous epitopes (DPs) and recognition interfaces of the three representative SARS-CoV-2 spike protein (SP) mutants (i.e., the Delta (B.1.617.2), Mu (B.1.621) and Omicron (B.1.1.529) strains), were analyzed to evaluate the effectiveness of current mainstream antibodies. The results showed that the conformation of SP wild type (WT) and mutants both remained stable, while the local antigenic epitopes underwent significant changes. Sufficient flexibility of SP CPs is critical for effective antibody recognition. The DPs of Delta, Mu and Omicron variants have showed stronger binding to human angiotensin converting enzyme-2 (hACE2) than WT; the possible drug resistance mechanisms of antibodies against three different epitopes (i.e., NTD_DP, RBD1_DP and RBD2_DP) were also proposed, respectively; the RBD2 of Delta, NTD of Mu, NTD and RBD2 of Omicron are deserve more attention in the subsequent design of next-generation vaccines. The simulation results not only revealed structural characteristics of SP antigenic epitopes, but also provided guidance for antibody modification, vaccine design and effectiveness evaluation.

Research Progress in Human AP Endonuclease 1: Structure, Catalytic Mechanism, and Inhibitors.

The high stability of phosphodiester bonds is considered to be one of the important reasons for the genetic role of nucleic acids, and their cleavage is also the core of many key biochemical processes, including DNA replication/ repair, and RNA processing/ degradation. As an important part of the base excision repair (BER) pathway, human apurinic/ apyrimidinic endonuclease 1 (APE1) is indispensable for the repair of abasic sites and other DNA damage, including ionizing radiation, DNA covalently bonding induced by cytotoxic antitumor drugs, etc. For tumor cells, the DNA repair activity of APE1 may lead to the occurrence of radiotherapy and chemotherapy resistance. The overexpression of APE1 often poses a serious threat to the effectiveness of tumor treatment, indicating a longer time, a much larger dose, less effective chemotherapy, and poor prognosis. It is of great urgency to design novel APE1 inhibitors. Rational design and modification of inhibitor molecules are closely related to the research progress of both structural biology and catalytic mechanism. In this review, the structure, catalytic mechanism, inhibitors, and other important biochemical information regarding APE1 are summarized, which will help in the design and modification of drug molecules targeting APE1.

Citral mitigates inflammation of Caco-2 cells induced by Cronobacter sakazakii.

The aim of this study was to explore the anti-inflammatory effect and mechanism of citral in Cronobacter sakazakii-stimulated Caco-2 cells. Safe doses of citral were first determined in Caco-2 cells. Then, the effect of citral on the adhesion and invasion of C. sakazakii into Caco-2 cells and the translocation of C. sakazakii through Caco-2 monolayers were investigated. The release of nitric oxide (NO), interleukin (IL)-1ß, IL-6, and TNF-α, transcription of inflammatory genes, and expression of proteins associated with inflammatory signaling pathways were determined. Subsequently, activation of caspase-3, -8, and -9 and apoptosis induced by C. sakazakii were assessed. The results showed that up to 10 µg mL-1 citral had no cytotoxicity in Caco-2 cells. Citral protected Caco-2 cells by affecting the adhesion and invasion of C. sakazakii into Caco-2 cells and the translocation of C. sakazakii across Caco-2 monolayers. Additionally, inflammation induced by C. sakazakii was effectively inhibited by citral via suppression of inflammatory factors that included NO, IL-1ß, IL-6, and TNF-α, transcription of related genes, and expression of proteins associated with inflammatory signaling pathways. Moreover, the activation of caspase-3, -8, and -9, and apoptosis caused by C. sakazakii were suppressed by pretreatment with citral. These findings suggest that citral mitigates the inflammatory response of Caco-2 cells. Citral has the potential to prevent the inflammation of Caco-2 associated with C. sakazakii.

Gelsolin impairs barrier function in pancreatic ductal epithelial cells by actin filament depolymerization in hypertriglyceridemia-induced pancreatitis in vitro.

Gelsolin (GSN) is a calcium-regulated actin-binding protein that can sever actin filaments. Notably, actin dynamics affect the structure and function of epithelial barriers. The present study investigated the role of GSN in the barrier function of pancreatic ductal epithelial cells (PDECs) in hypertriglyceridemia-induced pancreatitis (HTGP). The human PDEC cell line HPDE6-C7 underwent GSN knockdown and was treated with caerulein (CAE) + triglycerides (TG). Intracellular calcium levels and the actin filament network were analyzed under a fluorescence microscope. The expression levels of GSN, E-cadherin, nectin-2, ZO-1 and occludin were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. Ultrastructural changes in tight junctions were observed by transmission electron microscopy. Furthermore, the permeability of PDECs was analyzed by fluorescein isothiocyanate-dextran fluorescence. The results revealed that CAE + TG increased intracellular calcium levels, actin filament depolymerization and GSN expression, and increased PDEC permeability by decreasing the expression levels of E-cadherin, nectin-2, ZO-1 and occludin compared with the control. Moreover, changes in these markers, with the exception of intracellular calcium levels, were reversed by silencing GSN. In conclusion, GSN may disrupt barrier function in PDECs by causing actin filament depolymerization in HTGP in vitro.

Efficacy of 405-nm LED illumination and citral used alone and in combination for the inactivation of Cronobacter sakazakii in reconstituted powdered infant formula.

Cronobacter sakazakii, a foodborne opportunistic pathogen, mainly affects neonates and infants, with mortality rates of 26.9%. Most outbreaks arise from powdered infant formula (PIF). The aim of this study was to investigate the efficacy and mechanism of 405-nm light-emitting diode (LED) and citral treatment used in combination against C. sakazakii in reconstituted PIF. LED-illumination combined with citral showed better antimicrobial effects than either treatment alone. In reconstituted PIF, the abundance of C. sakazakii cells was reduced by 6.5 log 10 CFU/mL following combined LED and 9 µL/mL citral treatment for 90 min compared with untreated controls, respectively. Combined LED and 6 µL/mL citral treatment destroyed C. sakazakii cellular morphology and membrane integrity, prolonged the recovery time of sublethally-injured cells, and induced lipid peroxidation. Besides, LED treatment decreased the amount of lipid peroxidation caused by citral treatment alone. Neither LED illumination nor citral treatment resulted in breakdown of C. sakazakii genomic DNA. Because of its safe, environmentally-friendly, economical, and high-performance characteristics, the combination of LED-illumination and citral treatment has the potential to be developed into a strategy to control C. sakazakii contamination in stored and reconstituted PIF.