Antibacterial effect of ultrasound combined with Litsea cubeba essential oil nanoemulsion on Salmonella Typhimurium in kiwifruit juice.

This study investigated the antibacterial effect of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella Typhimurium in kiwifruit juice and effect on the quality and sensory properties of kiwifruit juice. In this study, LEON prepared by ultrasonic emulsification method had a good particle size distribution and high stability. The US+LEON treatment significantly (P < 0.05) improved antibacterial efficacy, compared to the control, and would not destroy the nutritional components containing ascorbic acid, flavonoids, total phenol and total soluble solids. Meanwhile, US+LEON treatment enhanced 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) radical scavenging capacity and ferric ion reducing antioxidant power (FRAP). In terms of sensory properties, US and LEON had a significant (P < 0.05) effect on the odor and overall morphology of kiwifruit juice. The enhance of antibacterial efficacy and the retention of nutrients by combined treatments shows that US+LEON is a promising antibacterial method that will provide new ideas for the processing and safety of fruit juices, and the US parameters and LEON concentration should be adjusted to reduce the effect on food sensory properties in future studies.

Protocol to generate traceable CAR T cells for syngeneic mouse cancer models.

The efficacy of chimeric antigen receptor (CAR) T cell immunotherapy is limited by insufficient infiltration and activation of T cells due to the immunosuppressive tumor microenvironment. Preclinical studies with optimized mouse CAR T cells in immunocompetent mouse cancer models will help define the mechanisms underlying immunotherapy resistance. Here, we present a protocol for preparing mouse T cells and generating CAR T cells. We then detail procedures for testing their therapeutic efficacy and tracking them in a syngeneic mouse glioma model. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems.

The viable but nonculturable (VBNC) state is adopted by many foodborne pathogenic bacteria to survive in adverse conditions. This study found that lactic acid, a widely used food preservative, can induce Yersinia enterocolitica to enter a VBNC state. Y. enterocolitica treated with 2 mg/mL lactic acid completely lost culturability within 20 min, and 10.137 ± 1.693 % of the cells entered a VBNC state. VBNC state cells could be recovered (resuscitated) in tryptic soy broth (TSB), 5 % (v/v) Tween80-TSB, and 2 mg/mL sodium pyruvate-TSB. In the VBNC state of Y. enterocolitica induced by lactic acid, the intracellular adenosine triphosphate (ATP) concentration and various enzyme activities were decreased, and the reactive oxygen species (ROS) level was elevated, compared with uninduced cells. The VBNC state cells were significantly more resistant to heat and simulated gastric fluid than uninduced cells, but their ability to survive in a high-osmotic-pressure environment was significantly less than that of uninduced cells. The VBNC state cells induced by lactic acid changed from long rod-like to short rod-like, with small vacuoles at the cell edges; the genetic material was loosened and the density of cytoplasm was increased. The VBNC state cells had decreased ability to adhere to and invade Caco-2 (human colorectal adenocarcinoma) cells. The transcription levels of genes related to adhesion, invasion, motility, and resistance to adverse environmental stress were downregulated in VBNC state cells relative to uninduced cells. In meat-based broth, all nine tested strains of Y. enterocolitica entered the VBNC state after lactic acid treatment; among these strains, only VBNC state cells of Y. enterocolitica CMCC 52207 and Isolate 36 could not be recovered. Therefore, this study is a wake-up call for food safety problems caused by VBNC state pathogens induced by lactic acid.

Inactivation of Salmonella using ultrasound in combination with Litsea cubeba essential oil nanoemulsion and its bactericidal application on cherry tomatoes.

The presence of Salmonella in nature poses a significant and unacceptable threat to the human public health domain. In this study, the antibacterial effect and mechanism of ultrasound (US) combined with Litsea cubeba essential oil nanoemulsion (LEON) on Salmonella. LEON + US treatment has a significant bactericidal effect on Salmonella. Reactive oxygen species (ROS), malondialdehyde (MDA) detection, N-phenyl-l-naphthylamine (NPN) uptake and nucleic acid release assays showed that LEON + US exacerbated cell membrane lipid peroxidation and increased the permeability of the cell membrane. The results of field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) showed that LEON + US treatment was able to alter cell morphology. It can be observed by flow cytometry (FCM) that LEON + US treatment can cause cell apoptosis. In addition, bacterial counts of cherry tomatoes treated with LEON (0.08 µL/mL) + US (345 W/cm2) for 9 min were reduced by 6.50 ± 0.20 log CFU/mL. This study demonstrates that LEON + US treatment can be an effective way to improve the safety of fruits and vegetables in the food industry.

Antibacterial Activity and Possible Mechanism of Litsea cubeba Essential Oil Against Shigella sonnei and Its Application in Lettuce.

Shigella sonnei, the causative agents of bacillary dysentery, remains a significant threat to public health. Litsea cubeba essential oil (LC-EO), one of the natural essential oils, exhibited promising biological activities. In this study, the antibacterial effects and possible mechanisms of LC-EO on S. sonnei and its application in lettuce medium were investigated. The minimum inhibitory concentration (MIC) of LC-EO against S. sonnei ATCC 25931 and CMCC 51592 was 4 and 6 µL/mL, respectively. The LC-EO could inhibit the growth of S. sonnei, and decreased S. sonnei to undetectable levels with 4 µL/mL for 1 h in Luria-Bertani broth. The antibacterial mechanism indicated that after the treatment of LC-EO, the production of reactive oxygen species and the activity of superoxide dismutase were significantly elevated in S. sonnei cells, and eventually led to the lipid oxidation product, the malondialdehyde content that significantly increased. Moreover, LC-EO at 2 MIC could destroy 96.51% of bacterial cell membrane integrity, and made S. sonnei cells to appear wrinkled with a rough surface, so that the intracellular adenosine triphosphate leakage was about 0.352-0.030 µmol/L. Finally, the results of application evaluation indicated that the addition of LC-EO at 4 µL/mL in lettuce leaves and 6 µL/mL in lettuce juice could decrease the number of S. sonnei to undetectable levels without remarkable influence on the lettuce leaf sensory quality. In summary, LC-EO exerted strong antibacterial activity and has the potential to control S. sonnei in food industry.

Inhibition of Cronobacter sakazakii by Litsea cubeba Essential Oil and the Antibacterial Mechanism.

Litsea cubeba essential oil (LC-EO) has anti-insecticidal, antioxidant, and anticancer proper-ties; however, its antimicrobial activity toward Cronobacter sakazakii has not yet been researched extensively. The objective of this study was to investigate the antimicrobial and antibiofilm effects of LC-EO toward C. sakazakii, along with the underlying mechanisms. The minimum inhibitory concentrations of LC-EO toward eight different C. sakazakii strains ranged from 1.5 to 4.0 µL/mL, and LC-EO exposure showed a longer lag phase and lower specific growth compared to untreated bacteria. LC-EO increased reactive oxygen species production, decreased the integrity of the cell membrane, caused cell membrane depolarization, and decreased the ATP concentration in the cell, showing that LC-EO caused cellular damage associated with membrane permeability. LC-EO induced morphological changes in the cells. LC-EO inhibited C. sakazakii in reconstituted infant milk formula at 50 °C, and showed effective inactivation of C. sakazakii biofilms on stainless steel surfaces. Confocal laser scanning and attenuated total reflection-Fourier-transform infrared spectrometry indicated that the biofilms were disrupted by LC-EO. These findings suggest a potential for applying LC-EO in the prevention and control of C. sakazakii in the dairy industry as a natural antimicrobial and antibiofilm agent.

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.

Antibacterial Activity and Mechanism of Linalool against Shigella sonnei and Its Application in Lettuce.

Shigella sonnei (S. sonnei) infection accounted for approximately 75% of annual outbreaks of shigellosis, with the vast majority of outbreaks due to the consumption of contaminated foods (e.g., fresh vegetables, potato salad, fish, beef, etc.). Thus, we investigated the antibacterial effect and mechanism of linalool on S. sonnei and evaluated the effect of linalool on the sensory quality of lettuce. The minimum inhibitory concentration (MIC) of linalool against S. sonnei ATCC 25931 was 1.5 mg/mL. S. sonnei was treated with linalool at 1× MIC for 30 min and the amount of bacteria was decreased below the detection limit (1 CFU/mL) in phosphate-buffered saline (PBS) and Luria-Bertani (LB) medium. The bacterial content of the lettuce surface was reduced by 4.33 log CFU/cm2 after soaking with linalool at 2× MIC. Treatment with linalool led to increased intracellular reactive oxygen species (ROS) levels, decreased intracellular adenosine-triphosphate (ATP) content, increased membrane lipid oxidation, damaged cell membrane integrity, and hyperpolarized cell membrane potential in S. sonnei. The application of linalool to lettuce had no effect on the color of lettuce compared to the control. The sensory evaluation results showed that linalool had an acceptable effect on the sensory quality of lettuce. These findings indicate that linalool played an antibacterial effect against S. sonnei and had potential as a natural antimicrobial for the inhibition of this foodborne pathogen.

Nesfatin-1 inhibits myocardial ischaemia/reperfusion injury through activating Akt/ERK pathway-dependent attenuation of endoplasmic reticulum stress.

Nesfatin-1 (encoded by NUCB2) is a cardiac peptide possessing protective activities against myocardial ischaemia/reperfusion (MI/R) injury. However, the regulation of NUCB2/nesfatin-1 and the molecular mechanisms underlying its roles in MI/R injury are not clear. Here, by investigating a mouse MI/R injury model developed with transient myocardial ischaemia followed by reperfusion, we found that the levels of NUCB2 transcript and nesfatin-1 amount in the heart were both decreased, suggesting a transcriptional repression of NUCB2/nesfatin-1 in response to MI/R injury. Moreover, cardiac nesfatin-1 restoration reduced infarct size, troponin T (cTnT) level and myocardial apoptosis, supporting its cardioprotection against MI/R injury in vivo. Mechanistically, the Akt/ERK pathway was activated, and in contrast, endoplasmic reticulum (ER) stress was attenuated by nesfatin-1 following MI/R injury. In an in vitro system, similar results were obtained in nesfatin-1-treated H9c2 cardiomyocytes with hypoxia/reoxygenation (H/R) injury. More importantly, the treatment of wortmannin, an inhibitor of Akt/ERK pathway, abrogated nesfatin-1 effects on attenuating ER stress and H/R injury in H9c2 cells. Furthermore, nesfatin-1-mediated protection against H/R injury also vanished in the presence of tunicamycin (TM), an ER stress inducer. Lastly, Akt/ERK inhibition reversed nesfatin-1 effects on mouse ER stress and MI/R injury in vivo. Taken together, these findings demonstrate that NUCB2/nesfatin-1 inhibits MI/R injury through attenuating ER stress, which relies on Akt/ERK pathway activation. Hence, our study provides a molecular basis for understanding how NUCB2/nesfatin-1 reduces MI/R injury.

Identification of the putative specific pathogenic genes of Porphyromonas gingivalis with type II fimbriae.

Porphyromonas gingivalis, the key etiologic agent of periodontitis, can be classified into six types (I to V and Ib) based on the fimA genes that encode FimA (a subunit of fimbriae). Accumulated evidence indicates that P. gingivalis expressing Type II fimbriae (Pg-II) is the most frequent isolate from severe periodontitis cases and is more virulent than other types of P. gingivalis. However, during the Pg-II infection process, which specific virulence factors play the key role is still unclear. In this study, we examined the capabilities of three Pg-II strains to invade and modulate the inflammatory cytokine expression of human gingival epithelial cells (GECs) compared to two Pg-I strains. P. gingivalis oligo microarrays were used to compare gene expression profiles of Pg-II strains that invade GECs with Pg-I strains. The differential gene expression of Pg-II was confirmed by quantitative reverse transcription-polymerase chain reaction. Our results showed that all of the Pg-II strains could induce interleukin (IL)-1ß and IL-6 secretion significantly when compared to Pg-I strains. Thirty-seven genes that were specifically expressed during the pathogenic process of Pg-II were identified by a microarray assay. These findings provide a new insight at the molecular level to explain the specific pathogenic mechanism of Pg-II strains.