APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

CRIF1 siRNA-Encapsulated PLGA Nanoparticles Suppress Tumor Growth in MCF-7 Human Breast Cancer Cells.

Mitochondrial oxidative phosphorylation (OXPHOS) system dysfunction in cancer cells has been exploited as a target for anti-cancer therapeutic intervention. The downregulation of CR6-interacting factor 1 (CRIF1), an essential mito-ribosomal factor, can impair mitochondrial function in various cell types. In this study, we investigated whether CRIF1 deficiency induced by siRNA and siRNA nanoparticles could suppress MCF-7 breast cancer growth and tumor development, respectively. Our results showed that CRIF1 silencing decreased the assembly of mitochondrial OXPHOS complexes I and II, which induced mitochondrial dysfunction, mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential depolarization, and excessive mitochondrial fission. CRIF1 inhibition reduced p53-induced glycolysis and apoptosis regulator (TIGAR) expression, as well as NADPH synthesis, leading to additional increases in ROS production. The downregulation of CRIF1 suppressed cell proliferation and inhibited cell migration through the induction of G0/G1 phase cell cycle arrest in MCF-7 breast cancer cells. Similarly, the intratumoral injection of CRIF1 siRNA-encapsulated PLGA nanoparticles inhibited tumor growth, downregulated the assembly of mitochondrial OXPHOS complexes I and II, and induced the expression of cell cycle protein markers (p53, p21, and p16) in MCF-7 xenograft mice. Thus, the inhibition of mitochondrial OXPHOS protein synthesis through CRIF1 deletion destroyed mitochondrial function, leading to elevated ROS levels and inducing antitumor effects in MCF-7 cells.

Dual Function of Secreted APE1/Ref-1 in TNBC Tumorigenesis: An Apoptotic Initiator and a Regulator of Chronic Inflammatory Signaling.

The simultaneous regulation of cancer cells and inflammatory immune cells in the tumor microenvironment (TME) can be an effective strategy in treating aggressive breast cancer types, such as triple-negative breast cancer (TNBC). Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multi-functional nuclear protein that can be stimulated and then secreted. The extracellular APE1/Ref-1 causes a reduction in disulfide bonds in cytokine receptors, resulting in their conformational changes, thereby inhibiting inflammatory signaling. Furthermore, the secreted APE1/Ref-1 in response to acetylation has been shown to bind to a receptor for the advanced glycation end product (RAGE), initiating the apoptotic cell death of TNBC in vitro and in vivo. This study used PPTLS-APE1/Ref-1 in an adenovirus vector (Ad-PPTLS-APE1/Ref-1) for the constant expression of extracellular APE1/Ref-1, and our results demonstrated its dual function as an apoptotic initiator and inflammation regulator. Injecting MDA-MB 231 orthotopic xenografts with the Ad-PPTLS-APE1/Ref-1 inhibited tumor growth and development in response to acetylation. Moreover, Ad-PPTLS-APE1/Ref-1 generated reactive oxygen species (ROS), and tumor tissues derived from these xenografts exhibited apoptotic bodies. Compared to normal mice, a comparable ratio of anti- and pro-inflammatory cytokines was observed in the plasma of Ad-PPTLS-APE1/Ref-1-injected mice. Mechanistically, the disturbed cytokine receptor by reducing activity of PPTLS-APE1/Ref-1 inhibited inflammatory signaling leading to the inactivation of the p21-activated kinase 1-mediated signal transducer and activator of transcription 3/nuclear factor-κB axis in tumor tissues. These results suggest that the regulation of inflammatory signaling with adenoviral-mediated PPTLS-APE1/Ref-1 in tumors modulates the secretion of pro-inflammatory cytokines in TME, thereby inhibiting aggressive cancer cell progression, and could be considered as a promising and safe therapeutic strategy for treating TNBCs.

Changes in the Plasma Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1(APE1/Ref-1) Level during Cancer Surgery: An Observational Study.

Background and Objectives: Propofol-based total intravenous anesthesia (TIVA) is presumed to have more favorable effects on the prognosis of patients with cancer compared with volatile inhaled anesthesia (VIA). We hypothesized that these anesthetics target plasma apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1) as a possible mechanism of action. Materials and Methods: The plasma APE1/Ref-1 level was evaluated three times during surgery for cancer, i.e., before anesthesia, immediately after cancer resection, and finally, in the recovery room. Blood (3 cc) was drawn from the radial artery catheter, and plasma APE1/Ref-1 levels were compared according to measurement time and between the two groups. Spearman's Rho correlation analysis was performed to determine relationships among body mass index, American Society of Anesthesiologists classification, age, sex, cancer type, and tumor-node-metastasis (TNM) stage. A total of 166 patients (VIA: 129; TIVA: 37) were enrolled. Results: Plasma APE1/Ref-1 level increased significantly (p = 0.028) after cancer resection compared with before surgery, but no significant difference was observed between anesthetics (p = 0.134). The post-resection plasma APE1/Ref-1 level showed a positive correlation with the NM stages, but not the T stage. Conclusions: The plasma APE1/Ref-1 level increased during surgery with more severe lymph node invasion, but there were no significant differences according to the anesthetics used.

Effective inhibition of Salmonella Typhimurium in fresh produce by a phage cocktail targeting multiple host receptors.

Salmonella contamination of fresh produce is the primary bacterial cause of a significant number of foodborne outbreaks and infections. Bacteriophages can be used as natural antibacterial agents to control foodborne pathogens. However, the rapid development of bacterial resistance to phage infection is a significant barrier to practical phage application. To overcome this problem, we developed a novel phage cocktail consisting of the three phages (BSPM4, BSP101 and BSP22A) that target different host receptors, including flagella, O-antigen and BtuB, respectively. Whole genome sequence analysis of the phages revealed that three phages do not harbor genes involved in lysogen formation or toxin production, suggesting they are safe for use as biocontrol agents in foods. In vitro treatment of the phage cocktail resulted in a significant reduction in the development of bacterial resistance. Phage cocktail treatments achieved 4.7-5.5 log CFU/cm2 reduction of viable cell number in iceberg lettuce and 4.8-5.8 log CFU/cm2 reduction in cucumber after 12 h at room temperature (25 °C). The phage cocktail exhibited good antimicrobial efficiency, suggesting that it could reduce S. Typhimurium contamination of fresh produce. The strategy of developing cocktails of phages that target multiple host receptors can be used to develop novel biocontrol agents of S. Typhimurium.

Frequent Implication of Multistress-Tolerant Campylobacter jejuni in Human Infections.

Campylobacter jejuni, a major cause of bacterial foodborne illnesses, is considered highly susceptible to environmental stresses. In this study, we extensively investigated the stress tolerance of 121 clinical strains of C. jejuni against 5 stress conditions (aerobic stress, disinfectant exposure, freeze-thaw, heat treatment, and osmotic stress) that this pathogenic bacterium might encounter during foodborne transmission to humans. In contrast to our current perception about high stress sensitivity of C. jejuni, a number of clinical strains of C. jejuni were highly tolerant to multiple stresses. We performed population genetics analysis by using comparative genomic fingerprinting and showed that multistress-tolerant strains of C. jejuni constituted distinct clades. The comparative genomic fingerprinting subtypes belonging to multistress-tolerant clades were more frequently implicated in human infections than those in stress-sensitive clades. We identified unique stress-tolerant C. jejuni clones and showed the role of stress tolerance in human campylobacteriosis.

Expansion of Shiga Toxin-Producing Escherichia coli by Use of Bovine Antibiotic Growth Promoters.

Antibiotics are routinely used in food-producing animals to promote growth and prevent infectious diseases. We investigated the effects of bovine antibiotic growth promoters (bAGPs) on the propagation and spread of Shiga toxin (Stx)-encoding phages in Escherichia coli. Co-culture of E. coli O157:H7 and other E. coli isolated from cattle in the presence of sublethal concentrations of bAGPs significantly increased the emergence of non-O157, Stx-producing E. coli by triggering the SOS response system in E. coli O157:H7. The most substantial mediation of Stx phage transmission was induced by oxytetracyline and chlortetracycline, which are commonly used in agriculture. bAGPs may therefore contribute to the expansion of pathogenic Stx-producing E. coli.

Novel adjuvant strategy to potentiate bacitracin against MDR MRSA.

OBJECTIVES: Bacitracin is an antimicrobial peptide that is frequently used as an active ingredient in antimicrobial ointments. However, bacitracin resistance is highly prevalent in community-associated MRSA (CA-MRSA) strains and significantly compromises the effectiveness of existing antimicrobial ointments. In this study, we aimed to develop novel adjuvants to enhance the antimicrobial activity of bacitracin by using alkyl gallates. METHODS: The growth of MRSA USA300, the predominant CA-MRSA strain in the USA, was determined in the presence of bacitracin and alkyl gallates at various concentrations. The viability of USA300 and MDR clinical isolates of MRSA was measured after exposure to various combinations of bacitracin and alkyl gallates. RESULTS: Whereas 100 U/mL bacitracin did not inhibit USA300, 1 U/mL bacitracin in combination with as low as 2 mg/L octyl gallate (OG) and 8 mg/L dodecyl gallate (DG), respectively, completely inhibited the growth of USA300. Among the tested alkyl gallates, OG most significantly enhanced the bactericidal activity of bacitracin. For example, 10(-3) U/mL bacitracin with 5 mg/L OG effectively killed USA300, which is an ∼200 000-fold decrease in the MBC of bacitracin for USA300. Furthermore, bacitracin/OG combinations demonstrated similar levels of antimicrobial activity against human clinical isolates of MRSA resistant to multiple antibiotics of clinical importance. CONCLUSIONS: Some alkyl gallates, particularly OG, significantly increased the antimicrobial activity of bacitracin against MDR MRSA.

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

UNLABELLED: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE: The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.

Enhanced transmission of antibiotic resistance in Campylobacter jejuni biofilms by natural transformation.

Campylobacter jejuni is a leading food-borne pathogen, and its antibiotic resistance is of serious concern to public health worldwide. C. jejuni is naturally competent for DNA transformation and freely takes up foreign DNA harboring genetic information responsible for antibiotic resistance. In this study, we demonstrate that C. jejuni transfers antibiotic resistance genes more frequently in biofilms than in planktonic cells by natural transformation.

Identification of a novel membrane transporter mediating resistance to organic arsenic in Campylobacter jejuni.

Although bacterial mechanisms involved in the resistance to inorganic arsenic are well understood, the molecular basis for organic arsenic resistance has not been described. Campylobacter jejuni, a major food-borne pathogen causing gastroenteritis in humans, is highly prevalent in poultry and is reportedly resistant to the arsenic compound roxarsone (4-hydroxy-3-nitrobenzenearsonic acid), which has been used as a feed additive in the poultry industry for growth promotion. In this study, we report the identification of a novel membrane transporter (named ArsP) that contributes to organic arsenic resistance in Campylobacter. ArsP is predicted to be a membrane permease containing eight transmembrane helices, distinct from other known arsenic transporters. Analysis of multiple C. jejuni isolates from various animal species revealed that the presence of an intact arsP gene is associated with elevated resistance to roxarsone. In addition, inactivation of arsP in C. jejuni resulted in 4- and 8-fold reductions in the MICs of roxarsone and nitarsone, respectively, compared to that for the wild-type strain. Furthermore, cloning of arsP into a C. jejuni strain lacking a functional arsP gene led to 16- and 64-fold increases in the MICs of roxarsone and nitarsone, respectively. Neither mutation nor overexpression of arsP affected the MICs of inorganic arsenic, including arsenite and arsenate, in Campylobacter. Moreover, acquisition of arsP in NCTC 11168 led to accumulation of less roxarsone than the wild-type strain lacking arsP. Together, these results indicate that ArsP functions as an efflux transporter specific for extrusion of organic arsenic and contributes to the resistance to these compounds in C. jejuni.

Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.

OBJECTIVES: CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA). METHODS: Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility. RESULTS: While antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting. CONCLUSIONS: The cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.

Comparative proteomic label-free analysis of Campylobacter jejuni NCTC 11168 cultured with porcine mucin.

Campylobacter jejuni is a major gastrointestinal pathogen in humans. Poultry is a primary reservoir for C. jejuni, and C. jejuni appears to be highly adapted to the gastrointestinal tracts of avian species. We determined the protein expression profiles of C. jejuni NCTC 11168 cultured in medium containing porcine mucin. Differentially expressed proteins in the presence and absence of porcine mucin were identified using the label-free method. We identified 52 proteins with expression that was either upregulated (32 proteins) or downregulated (20 proteins) by porcine mucin. These proteins are involved in diverse cellular functions, such as motility, cell wall synthesis, iron transport, energy production, and amino acid metabolism. In particular, the upregulated proteins were involved in chemotaxis (CheV and CetA), motility (FlaA), colonization and adherence (CadF, FrdA, CfrA, MapA, and HydA), and stress tolerance (TrxB and ClpB). These results suggest that C. jejuni changes its protein expression in response to porcine mucin and that this change in expression may contribute to host adaptation of C. jejuni NCTC 11168.

Cold tolerance in Campylobacter jejuni and its impact on food safety.

Campylobacter jejuni is a major cause of foodborne illnesses worldwide and is primarily transmitted to humans through contaminated poultry meat. To control this pathogen, it is critical to understand its cold tolerance because poultry products are usually distributed in the cold chain. However, there is limited information regarding how this thermotolerant, microaerophilic pathogen can survive in cold and aerobic environments in the poultry cold chain. In this study, we investigated the cold tolerance of C. jejuni by measuring the viability of 90 C. jejuni strains isolated from retail raw chicken at 4 °C under aerobic and microaerobic conditions. Despite the microaerophilic nature of C. jejuni, under aerobic conditions, C. jejuni exhibited higher viability at 4 °C and required an extended inactivation time compared to microaerobic conditions. Some strains were highly tolerant to refrigeration temperatures and exhibited increased survival at 4 °C. These cold-tolerant strains mostly belonged to multilocus sequence typing (MLST) clonal complex (CC)-21 and CC-443, indicating that cold tolerance is associated with the phylogeny of C. jejuni. Notably, cold-tolerant strains had an increased probability of illness and were more likely to cause human infections due to their extended survival on refrigerated chicken meat compared to those sensitive to cold stress. Furthermore, the majority of cold-tolerant strains exhibited elevated aerotolerance, indicating that cold tolerance is related to aerotolerance. These findings suggest that refrigeration of chicken meat under aerobic conditions may not be effective at controlling C. jejuni and that cold-tolerant C. jejuni can pose an increased risk to food safety.

Synergistic effects of anti-CmeA and anti-CmeB peptide nucleic acids on sensitizing Campylobacter jejuni to antibiotics.

The CmeABC efflux pump in Campylobacter jejuni confers resistance to structurally divergent antimicrobials, and inhibition of CmeABC represents a promising strategy to control antibiotic-resistant Campylobacter. Antisense peptide nucleic acids (PNAs) targeting the three components of CmeABC were evaluated for inhibition of CmeABC expression. The result revealed a synergistic effect of the PNAs targeting CmeA and CmeB on sensitizing C. jejuni to antibiotics. This finding further demonstrates the feasibility of using PNAs to potentiate antibiotics against antibiotic-resistant Campylobacter.

Enhancement of the Antibiofilm Activity of Nisin against Listeria monocytogenes Using Food Plant Extracts.

Listeria monocytogenes is a foodborne pathogen exhibiting a high mortality rate. In addition to the robust tolerance to environmental stress, the ability of L. monocytogenes to develop biofilms increases the risk of contaminating food processing facilities and ultimately foods. This study aims to develop a synergistic approach to better control Listeria biofilms using nisin, the only bacteriocin approved as a food preservative, in combination with gallic-acid-rich food plant extracts. Biofilm assays in the presence of nisin and gallic acid or its derivatives revealed that gallic acid significantly decreased the level of biofilm formation in L. monocytogenes, whereas ethyl gallate, propyl gallate, and lauryl gallate enhanced biofilm production. As gallic acid is widely distributed in plants, we examined whether extracts from gallic-acid-rich food plants, such as clove, chestnut, oregano, and sage, may generate similar antibiofilm effects. Remarkably, sage extracts enhanced the antibiofilm activity of nisin against L. monocytogenes; however, the other tested extracts increased biofilm formation, particularly at high concentrations. Moreover, sage extracts and nisin combinations significantly reduced the biofilm formation of L. monocytogenes on stainless steel. Sage is a common food spice and has various beneficial health effects, including antioxidation and anti-cancer properties. The findings in this study demonstrate that sage extracts can be potentially combined with nisin to prevent biofilm production in L. monocytogenes.

Stimulation of Surface Polysaccharide Production under Aerobic Conditions Confers Aerotolerance in Campylobacter jejuni.

The ability of a foodborne pathogen to tolerate environmental stress critically affects food safety by increasing the risk of pathogen survival and transmission in the food supply chain. Campylobacter jejuni, a leading bacterial cause of foodborne illnesses, is an obligate microaerophile and is sensitive to atmospheric levels of oxygen. Currently, the molecular mechanisms of how C. jejuni withstands oxygen toxicity under aerobic conditions have not yet been fully elucidated. Here, we show that when exposed to aerobic conditions, C. jejuni develops a thick layer of bacterial capsules, which in turn protect C. jejuni under aerobic conditions. The presence of both capsular polysaccharides and lipooligosaccharides is required to protect C. jejuni from excess oxygen in oxygen-rich environments by alleviating oxidative stress. Under aerobic conditions, C. jejuni undergoes substantial transcriptomic changes, particularly in the genes of carbon metabolisms involved in amino acid uptake, the tricarboxylic acid (TCA) cycle, and the Embden-Meyerhof-Parnas (EMP) pathway despite the inability of C. jejuni to grow aerobically. Moreover, the stimulation of carbon metabolism by aerobiosis increases the level of glucose-6-phosphate, the EMP pathway intermediate required for the synthesis of surface polysaccharides. The disruption of the TCA cycle eliminates aerobiosis-mediated stimulation of surface polysaccharide production and markedly compromises aerotolerance in C. jejuni. These results in this study provide novel insights into how an oxygen-sensitive microaerophilic pathogen survives in oxygen-rich environments by adapting its metabolism and physiology. IMPORTANCE Oxygen-sensitive foodborne pathogens must withstand oxygen toxicity in aerobic environments during transmission to humans. C. jejuni is a major cause of gastroenteritis, accounting for 400 million to 500 million infection cases worldwide per year. As an obligate microaerophile, C. jejuni is sensitive to air-level oxygen. However, it has not been fully explained how this oxygen-sensitive zoonotic pathogen survives in aerobic environments and is transmitted to humans. Here, we show that under aerobic conditions, C. jejuni boosts its carbon metabolism to produce a thick layer of bacterial capsules, which in turn act as a protective barrier conferring aerotolerance. The new findings in this study improve our understanding of how oxygen-sensitive C. jejuni can survive in aerobic environments.

Characterization of the lytic phage MSP1 for the inhibition of multidrug-resistant Salmonella enterica serovars Thompson and its biofilm.

The increasing prevalence of multidrug-resistant (MDR) Salmonella is a serious public health threat. Intervention strategies available to control Salmonella mostly target Salmonella enterica serovars Typhimurium and Enteritidis, and little has been investigated to control serovars in serogroup C, such as S. enterica serovar Thompson, despite their increasing prevalence. Here, we isolated phages targeting MDR S. Thompson and characterized the antimicrobial activities of MSP1 phage, a virulent phage with a broad host range. MSP1 phage strongly infected S. Thompson and S. Mbandaka isolates from retail chicken and also other serovars, including Dublin, Enteritidis, Heidelberg, Paratyphi, and Typhimurium. MSP1 phage was able to inhibit the biofilm formation on stainless steel and glass formation by around 42.7-47.9 %. MSP1 phage was robust to withstand wide ranges of pH (4-12) and temperature (30-60 °C), and no genes associated with antibiotic resistance and virulence were found in the phage genome, suggesting that this phage is suitable for food application. When MSP1 phage was tested on foods (chicken meat and milk), MSP1 phage significantly reduced the level of MDR S. Thompson below the detection limit. Our findings suggest that MSP1 phage is a promising antimicrobial agent for the control of food contamination by MDR S. Thompson.

IDH2 Deficiency Promotes Endothelial Senescence by Eliciting miR-34b/c-Mediated Suppression of Mitophagy and Increased ROS Production.

Endothelial senescence impairs vascular function and thus is a primary event of age-related vasculature diseases. Isocitrate dehydrogenase 2 (IDH2) plays an important role in inducing alpha-ketoglutarate (α-KG) production and preserving mitochondrial function. However, the mechanism and regulation of IDH2 in endothelial senescence have not been elucidated. We demonstrated that downregulation of IDH2 induced accumulation of miR-34b/c, which impaired mitophagy and elevated mitochondrial reactive oxygen species (ROS) levels by inhibiting mitophagy-related markers (PTEN-induced putative kinase 1 (PINK1), Parkin, LC-II/LC3-I, and p62) and attenuating Sirtuin deacetylation 3 (Sirt3) expression. The mitochondrial dysfunction induced by IDH2 deficiency disrupted cell homeostasis and the cell cycle and led to endothelial senescence. However, miR-34b/c inhibition or α-KG supplementation restored Sirt3, PINK1, Parkin, LC-II/LC3-I, p62, and mitochondrial ROS levels, subsequently alleviating endothelial senescence. We showed that IDH2 played a crucial role in regulating endothelial senescence via induction of miR-34b/c in endothelial cells.

Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni.

CosR is an essential response regulator in Campylobacter jejuni, a major food-borne pathogen causing enteritis worldwide. A transcriptomic analysis performed in this study discovered 93 genes whose transcriptional levels were changed >2-fold due to the repression of CosR expression by antisense peptide nucleic acid. The identified CosR-regulated genes are involved in various cellular functions, such as energy production, protein synthesis and folding, flagellum biogenesis, and lipid metabolism. Interestingly, 17 of the 93 CosR-regulated genes (18.3%) are predicted essential genes, indicating that CosR may participate in the regulation of vital biological processes in C. jejuni. In particular, CosR knockdown increased the transcriptional levels of cmeA, cmeB, and cmeC genes, whose protein product (CmeABC) is an important determinant conferring multidrug resistance in Campylobacter. Negative regulation of cmeABC by CosR was verified by quantitative real-time PCR (qRT-PCR) and P(cmeABC)::lacZ assay. The results of electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays demonstrated that CosR directly binds to the cmeABC promoter. Another notable finding is that CosR regulates the transcription of katA, the sole catalase gene in C. jejuni. Further characterization with qRT-PCR, the catalase enzyme assay, EMSA, and DNase I footprinting assays successfully demonstrated that CosR affects the katA transcription and the catalase activity by direct interactions with the katA promoter. The findings in this study clearly demonstrated that CosR regulates resistance mechanisms in C. jejuni by controlling the expression of genes involved in oxidative stress defense and extrusion of toxic compounds out of the cell.