Escherichia coli to survive in the strongly acidic environment of the human stomach (pH < 2.5). Among these, the glutamic acid decarboxylase (GAD) system is the most effective. However, the precise mechanism of GAD induction is unknown. We previously reported that a tolC mutant lacking the TolC outer membrane channel was defective in GAD induction. Here, we show that indole, a substrate of TolC-dependent efflux pumps and produced by the tryptophanase encoded by the tnaA gene, negatively regulates GAD expression. GAD expression was restored by deleting tnaA in the tolC mutant; in wild-type E. coli, it was suppressed by adding indole to the growth medium. RNA-sequencing revealed that tnaA mRNA levels drastically decreased upon exposure to moderately acidic conditions (pH 5.5). This decrease was suppressed by RNase E deficiency. Collectively, our results demonstrate that the RNase E-dependent degradation of tnaA mRNA is accelerated upon acid exposure, which decreases intracellular indole concentrations and triggers GAD induction.Acid-resistance systems are essential for pathogenic  Escherichia coli strains cause severe illness, such as enteric and diarrheal diseases2. As they have low infectious doses and are transmitted through ubiquitous media, including food and water, such outbreaks are serious public health concerns in both developing and developed countries1. In developing countries in sub-Saharan Africa and South Asia, pathogenic E. coli is a major contributor to diarrheal disease, particularly in children under 5 years of age3. In Japan, the largest outbreak of enterohemorrhagic E. coli O157:H7, which occurred in primary schools in 1996, caused >7,500 reported infections and three deaths5. The most serious outbreak in Germany in 2011 was caused by Shiga toxin-producing E. coli O104:H4, which caused 3,816 infection cases and 54 deaths6.Pathogenic E. coli are classified into several categories based on unique features of their interactions with host cells7, infection is universally established by colonization in the intestine, mainly through oral transmission8. Pathogenic E. coli are inevitably exposed to the extremely acidic stomach environment, which acts as a primary bactericidal barrier10. To overcome this acidic stress, E. coli (both pathogenic and non-pathogenic strains) possess sophisticated acid-resistance (AR) systems. These systems and their complex regulation have been extensively described in recent reviews13. Five primary systems, designated AR1–AR5, have been defined to date. The AR1 system is effective at pH 2.5 in minimal medium14 and is induced when E. coli cells are grown to stationary phase in complex medium buffered at pH 5.5 under oxidative conditions. Although AR1 induction was suppressed by glucose and requires the alternative sigma factor σS (RpoS) and cyclic AMP receptor protein (CRP)15, its precise regulatory mechanisms are unclear. The AR2–AR5 systems are composed of specific amino acid decarboxylases and antiporters which are induced at low pH. The amino acids required for AR2, AR3, AR4, and AR5 are glutamic acid, arginine, lysine, and ornithine, respectively17. The AR2 and AR3 systems primarily contribute to survival in extremely acidic environments (~pH 2.5), whereas AR4 and AR5 contribute to survival in moderately acidic environments (~pH 4.5)16. AR2 has been shown to be the most effective system for promoting survival under extreme acidic stress19.Although pathogenic +-consuming conversion of glutamic acid to γ-aminobutyric acid (GABA)21. GABA is released extracellularly by the GadC antiporter in exchange for the uptake of external glutamic acid23. The GAD system increases intracellular pH by consumption of cytoplasmic H+ and reversion of the electrical membrane potential, making the charge inside of the cell positive24. The positive internal charge seems to slow proton entry into the cell; the same strategy is present in multiple acidophiles that grow in extremely low pH environments25. The effectiveness of the GAD system for survival in the gastrointestinal tract has also been demonstrated in vivo, as the number of colonies isolated from calf faeces was significantly decreased relative to the wild-type strain when the GAD system-deficient E. coli strain was orally inoculated into calves26. A recent study suggests that genes encoding GAD constitute a significant proportion of human gut flora, which could affect human health via the gut-brain axis by producing GABA, a major inhibitory neurotransmitter of the mammalian central nervous system27.The AR2 system is also known as the glutamic acid decarboxylase (GAD) system. It is composed of GadA and GadB, which are isozymes of pyridoxal-5′-phosphate (PLP)-dependent GADs, as well as the GadC antiporter. GadA and GadB catalyse the H70, σS, SspA, TrmE, TopA, TorS/R, and YdeO, as well as 4 small non-coding RNAs (sRNAs), ArrS, DsrA, GadY, and GcvB, are known to regulate this system28. They form a complex, hierarchical regulatory cascade. Among them, a transcription factor, GadE, acts as a central activator of the GAD system. It directly induces gadA, gadB, and gadC transcription under moderately acidic conditions (~pH 5.5) and the stationary growth phase29, while other protein factors regulate gadE expression, directly or indirectly. Induction of the GAD system has been extensively investigated by genetic analyses16. However, the first event that initiates induction of the GAD system after exposure to acidic environments remains unknown. Elucidating this initial step would help us to better understand this network and is likely to be important in developing clinical preventive strategies against pathogenic E. coli infections.Induction of the GAD system is controlled by an extraordinarily complex regulatory network. Nearly 20 proteins, including CRP, EvgA/S, GadE, GadX, GadW, H-NS, Lon, PhoP/Q, RNase E, σE. coli tolC mutant lacking the TolC outer membrane channel showed increased acid sensitivity, because GadA and GadB were not induced30. The TolC protein forms tripartite trans-periplasmic channels with inner membrane efflux pump proteins and their cognate periplasmic proteins32. This TolC-dependent efflux system contributes to exporting intracellular metabolites including porphyrin33, enterobactin34, and indole35, and multiple xenobiotics including antibiotics, dyes, organic solvents, and detergents39. Substrates of the TolC-dependent efflux system are thought to accumulate intracellularly when it does not function properly. Therefore, we hypothesised that a regulatory signal which represses expression of the GAD system would accumulate in tolC-mutant cells.Previously, we reported that the gadA, B, C, E, and X) and reduce acid resistance in E. coli41. Indole is produced from tryptophan by the tryptophanase encoded by the tnaA gene. It has been reported that the production of indole is induced at high pH, probably owing to increased expression of TnaA, while it is repressed at low pH43. Although indole is a characteristic compound of E. coli44, high indole concentrations inhibit its growth45. Therefore, E. coli need to export indole; moreover, the TolC–AcrEF efflux pump can function as an indole exporter35. Based on these findings, we hypothesised that accumulated indole impairs GAD induction in tolC-mutant cells.One of the substrates of the TolC-dependent efflux system, indole, is reported to suppress expression of GAD-related genes , were grown in Luria–Bertani (LB) broth buffered with 3-morpholinopropanesulfonic acid (MOPS) at pH 8.0 or 2-morpholinoethanesulfonic acid (MES) at pH 5.5 for 30 min. Intracellular indole concentrations were determined using Kovacs’ reagent. Intracellular indole levels in wild-type cells decreased when the pH was shifted to 5.5 .To test our hypothesis, we first measured the intracellular concentrations of indole in the wild-type and 5.5 Fig. . DecreastnaA mutation on the expression of the GAD system in the tolC mutant. MG1655 and its derivatives, MG1655T (tolC::Tn10), TK20 (ΔtnaA::kan), and TK12 (tolC::Tn10 ΔtnaA::kan), were grown in LB broth buffered with MES (pH 5.5) for 16 h. Both were challenged at pH 2.5 in M9-glucose minimal medium supplemented with 1.5 mM l-glutamic acid. As reported previously30, the survival of the tolC mutant after acid challenge was markedly lower than that of wild-type E. coli. When the tnaA mutation was introduced into the tolC mutant, the survival rate was comparable to that of wild-type  or MES (pH 5.5); gadA-mRNA levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The gadA mRNA levels at pH 5.5 were 12-fold higher than those at pH 8.0 in wild-type cells. Although gadA-mRNA levels increased even in the tolC mutant after exposure to pH 5.5, they were clearly lower than those in wild-type. However, they were restored to levels comparable to wild-type cells by introducing the tnaA mutation into the tolC mutant . Considering these data, we hypothesised that higher indole levels in tolC-mutant cells at pH 5.5 suppressed induction of the GAD system. Consistent with this hypothesis, adding 50 – 600 μM indole to LB medium suppressed the induction of GAD activity in wild-type cells  cultured at pH 8.0 or pH 5.5 for 30 min , Δhfq mutant (TK50), and rne-105 mutant  E. coli, after culture for 30 min at pH 8.0 or 5.5. The amounts of tnaA mRNA were measured by qRT-PCR. In wild-type cells, tnaA mRNA expression was decreased by more than 100-fold by the acidic shift. Under this condition, tnaA mRNA expression in both the Δhfq and rne-105 mutants decreased to almost the same level as that of wild-type cells  of the tnaA mRNA by RNase E under neutral and acidic conditions.Acid-induced degradation was limited to tes Fig. , suggestE. coli  the remaining indole is excreted by the TolC-dependent efflux pump, and (4) consequently, intracellular levels of indole decrease, which triggers induction of the GAD system.Based on the results presented above, we propose the following model to explain the induction of GAD in oli Fig. : (1) exptnaA gene restored expression of the GAD system in the tolC mutant and that indole addition suppressed GAD induction in wild-type cells . When necessary, the pH was adjusted by adding 0.5 ml of either 1 M MOPS buffer (pH 8.0) or 1 M MES buffer (pH 5.5) per 4.5 ml of LB broth. For the acid-resistance assays, we used M9-glucose minimal medium  supplemented with 1.5 mM l-glutamic acid and adjusted to pH 2.5 with HCl.The E. coli were aerobically grown at 37 °C in 4.5 ml of LB broth (pH 7.0). When the OD660 reached ~0.5, 0.5 ml of 1 M MOPS (pH 8.0) or 1 M MES (pH 5.5) was added to the culture. After 30 min of incubation, cells were harvested by centrifugation, quickly rinsed, and resuspended in 0.25 ml of 50 mM sodium phosphate buffer (pH 7.0). Cells were then sonicated with a Bioruptor UCD-250 . A 0.2-ml sample was mixed immediately with 0.2 ml of Kovacs’ indole reagent  and incubated for 2 min at room temperature. The reaction mixture was centrifuged at 15,800 × g for 5 min and the upper layer was collected. The concentrations of indole were determined spectrophotometrically by measuring absorbance at 540 nm. To obtain intracellular indole concentrations, these measurements were normalized to total protein concentrations. Values are presented as means ± SEM from three independent experiments and were statistically analysed using the one-way ANOVA with Holm’s post-hoc test (*p < 0.05).E. coli grown in 5 ml of LB broth buffered with MES (pH 5.5) at 37 °C for 16 h were challenged with acid by diluting the cells 1000-fold into 5 ml of M9-glucose minimal medium supplemented with 1.5 mM l-glutamic acid (pH 2.5). After acid exposure for 30 or 60 min at 37 °C, the cells were plated on LB plates (1.5% agar) after appropriate dilution. The plates were incubated at 37 °C overnight and percent survival was calculated by counting colony-forming units (CFUs) after acid challenge, relative to those of unexposed control cells. After acid exposure, the pH of the culture was checked to ensure that it was within 0.2 pH units of that of the original uninoculated medium. Survival values are presented as means ± SEM from three independent experiments and were statistically analysed using one-way ANOVA with the Bonferroni post-hoc test .l-glutamic acid, 0.05 g/l bromocresol green, 90 g/l NaCl, and 3 ml/l Triton X-100) as described previously52 with minor modifications. E. coli were aerobically grown at 37 °C in 4.5 ml of LB broth (pH 7.0). When the OD660 reached ~0.5, 0.5 ml of 1 M MES (pH 5.5) was added and growth was continued for 30 min. Cells were harvested by centrifugation and washed with 1 ml of 0.85% NaCl to adjust cell concentrations. An aliquot of each cell suspension (6.0 × 108 cells) was transferred to 200 μl of GAD reagent. The reaction mixtures were incubated at 35 °C for 6 h and then evaluated qualitatively for decarboxylase activity, based on a colour change from yellow to blue. To examine the effects of indole addition on GAD expression, E. coli cells were grown in the absence or presence of the indicated concentrations of indole (50–600 μM) in 4.5 mL of LB broth (pH 7.0), then GAD activities were evaluated as described above.Activity was assessed using GAD reagent  or MES (pH 5.5) was added to the cultures. In the case of rne-1-mutant strains, cells were grown in LB broth at 30 °C, and the growth temperature was shifted to 42 °C just before adding MOPS (pH 8.0) or MES (pH 5.5). After a 30-min incubation, the cells were harvested by centrifugation and suspended in RNA Protect Bacterial Reagent  to stabilize cellular RNA. Total cellular RNA was isolated with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was treated with DNase I at room temperature for 1 h. In the rifampicin-chase experiments, cells were treated with 150 μg/ml rifampicin immediately after adding MOPS or MES buffer, then total cellular RNA was isolated 0, 5, or 10 min after rifampicin addition.E. coli str. K-12 substr. MG1655 (accession number: NC_000913.3) using CLC Genomics Workbench version 10.0.1. (Qiagen) with the following parameters: length fraction: 0.7; similarity fraction: 0.9; maximum number of hits for a read: 1. The RNA level for each gene was calculated by counting the number of reads mapped to each gene, normalized by calculating reads per kilobase of transcript per million mapped reads (RPKM) values, and shown as mean from two independent experiments. Original sequence reads have been deposited in the DRA/SRA database under accession numbers DRR162758–DRR162761.One microgram of total RNA was used to create complementary DNA (cDNA) libraries. Ribosomal RNA was removed using the RiboZero Bacteria Kit , per the manufacturer’s protocol. Sequencing libraries were prepared using the NEBNext mRNA Library Prep Kit for Illumina , with the following modifications. Random hexamer primers were used for reverse transcription. After second-strand synthesis, the double-stranded cDNA was fragmented to an average length of 300 base pairs using a Covaris S2 sonication system . One hundred cycles of paired-end sequencing were carried out using the HiSeq. 2500 system, according to the manufacturer’s specifications (Illumina). After the sequencing reactions were complete, the Illumina analysis pipeline (CASAVA 1.8.0) was used to process the raw sequencing data. The reads were trimmed using CLC Genomics Workbench ver. 10.0.1. with the following parameters: Phred quality score >30; removing the terminal 15 nucleotides from the 5′ end and two nucleotides from the 3′ end; and removing truncated reads of <30 nucleotides. Trimmed reads were mapped to all genes in tnaA mRNA was produced using the PCR DIG Probe Synthesis Kit (Roche Diagnostics). To generate the probe, specific DNA fragments were amplified using PCR with the primers 5′-AAACATCTCCCTGAACCGTTCCGCATTC-3′ and 5′-TCGACCAGATACTGTACCTGCGCGATAC-3′.Total RNA (6.8 μg) was separated by electrophoresis using a Formaldehyde-Free RNA Gel Kit (1.2% agarose) . The gels were blotted onto positively charged nylon membranes  using the capillary transfer method. A digoxigenin (DIG)-labelled antisense DNA probe targeting the entire coding region of tnaA mRNA were measured on the basis of the band intensities using ImageJ software.Prehybridization (30 min) and hybridization (16 h) were performed in DIG Easy Hyb (Roche Diagnostics) at 50 °C. The membrane was washed using DIG Wash and Block Buffer Set (Roche Diagnostics) according to the manufacturer’s instructions. Hybridization was detected using the CDP-Star reagent (Roche Diagnostics) with an exposure time of 5 min. The amounts of The mRNA levels were quantified by qRT-PCR using an Eco Real-Time PCR System (Illumina) and the QuantiFast SYBR Green RT-PCR Kit (Qiagen). Twenty nanograms of each total RNA sample were used for qRT-PCR with each primer pair. Each target-gene mRNA level was normalised to a reference gene transcript (16S rRNA) from the same RNA sample. The cycle threshold for each sample was generated according to the procedures described in the Eco Real-Time PCR System User Guide. The sequences of the primers used are shown in Supplementary Table Supplementary Information.


Pyropia yezoensis, a red alga, is popular and harvested a lot in East Asia and is famous for its medicinal properties attributable to its bioactive compounds including amino acids , polysaccharides, phytosterols, and pigments, but its anti-inflammatory effect and mechanism of anti-atopic dermatitis (AD) have not been elucidated. In this study, we investigate the anti-AD effect of P. yezoensis extract (PYE) on mRNA and protein levels of the pro-inflammatory chemokines, thymus, and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), in human HaCaT keratinocyte cells treated to interferon (IFN)-γ or tumor necrosis factor (TNF)-α (10 ng/mL each). The effect of the PYE on extracellular signal-regulated kinase (ERK) and other mitogen-activated protein kinases (MAPKs) was related to its suppression of TARC and MDC production by blocking NF-κB activation in HaCaT cells. Furthermore, astaxanthin and xanthophyll from P. yezoensis were identified as anti-AD candidate compounds. These results suggest that the PYE may improve AD and contained two carotenoids by regulating pro-inflammatory chemokines. Atopic dermatitis (AD) is a chronic inflammatory skin disease. It has a complex pathogenesis involving genetic, immunological, and environmental factors that lead to a dysfunctional skin barrier and dysregulation of the immune system . MillionSeveral studies have demonstrated that cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) stimulate epidermal keratinocytes that lead to the activation of signaling pathways involved in pro-inflammatory responses ,3. ThereThe increased understanding of signal transduction mechanisms and gene regulation involved in the immune response has provided us with opportunities to discover novel natural resource therapeutic compounds that are useful for treating inflammatory disorders. One of the most studied signaling routes is the mitogen-activated protein kinase (MAPK) signaling pathway, which plays a crucial role in many aspects of immune-mediated inflammatory responses ,9. For tPyropia yezoensis (P. yezoensis) is a red alga mainly found in Korea, Japan, and China and is consumed as food. Edible marine algae contain a range of components with potential health benefits [P. yezoensis is known to afford protective effects against UV and have antioxidant [benefits ,11. P. yioxidant ,13,14, aioxidant , anti-inioxidant ,17, antiioxidant , and tisioxidant ,20,21. IP. yezoensis extract has been shown to have various biological activities, its effect as an anti-inflammatory agent for AD remains poorly understood. This study is designed to evaluate the anti-inflammatory effects of P. yezoensis extract on IFN-γ- and TNF-α-induced immune responses in an HaCaT human keratinocyte model.Although P. yezoensis was a kind gift from Professor Taejun Han . Astaxanthin and xanthophyll were purchased from Sigma-Aldrich . For extract preparation, the P. yezoensis was washed with tap water to remove the salts and a dried sample (100 g) was pulverized, followed by extraction with 80% methanol  for 30 min by sonication. Then, it was filtered by Whatman filter paper and the solvents of filtrates were evaporated by the vacuum rotary evaporator. In this study, the water fraction of the P. yezoensis extract (PYE) was isolated as the supernatant and then lyophilized using a freeze-dryer . The PYE, astaxanthin, and xanthophyll were dissolved in dimethyl sulfoxide (DMSO) before use in the experiments.The 2 in a humidified incubator. The medium was changed every 3 days. HaCaT cells were seeded at a density of 1.5 × 105 cells/dish in complete medium in a 100 mm cell culture dish. After 24 h, the medium was changed to serum-free DMEM containing the indicated concentrations of PYE  and 10 ng/mL of TNF-α  or 10 ng/mL of IFN-γ  were added to the cells. After 24 h, the cells were collected for western blot analysis and real-time RT-PCR. The HaCaT cell line was purchased from American Type Culture Collection  and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and penicillin-streptomycin  at 37 °C exposed to 5% CO4 cells/dish for 24 h and treated with 10–2000 μg/mL PYE for 24 h. Then, the cells were incubated with 3--2,5-diphenyl-2H-tetrazolium bromide  (0.5 mg/mL) for 3 h. Following this, the medium was removed, and 100 μL DMSO was added to each well to dissolve the formazan crystals and the MTT metabolite. After thoroughly mixing the plate, the optical density was read at 560 nm, which directly correlates with the cell number.For the cytotoxicity assay, HaCaT cells were plated in a 96-well plate at a density of 1.5 × 10TM , according to the manufacturer’s recommendations, and resuspended in diethyl pyrocarbonate (DEPC)-water. The quantity and purity of the RNA was verified at 260/280 nm using a NanoDrop® spectrophotometer . Purification was performed using an RNeasy mini kit . RNA samples were stored at −70 °C until used. The complementary DNA was synthesized from 500 ng total RNA using a Toyobo cDNA kit . Total RNA was extracted using TRIzol reagentTM real-time PCR detection system . The PCR reaction mixture was prepared using SYBR Green Real-time PCR Master Mix , according to the manufacturer's instructions. The system operates using a thermal cycler and a laser directed via fiber optics to each of the 96 sample wells. The thermal cycling conditions were 60 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The relative expression of the target genes was calculated using the CFX ConnectTM Real-Time PCR detection system employing the ΔΔCq method. TARC, MDC, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) oligonucleotide primers were purchased from Bioneer . The primer sequences used are shown in The real-time PCR analysis was performed using an Applied CFX Connect5 cells/dish) were seeded in a 100 mm dish. After 24 h, the cells were incubated with IFN-γ or TNF-α (10 ng/mL each) in the presence or absence of PYE at the indicated concentrations for 24 h. The culture supernatants were collected 24 h after treatment and stored at −70 °C. They were analyzed for MDC and TARC using ELISA , according to the manufacturer’s instructions. The optical density was measured at 450 nm using a microplate reader . The HaCaT cells (1.5 × 105 cells/dish) were washed with cold PBS containing phosphatase inhibitors and collected. To obtain whole cell lysates, cells were lysed in 300 μL complete lysis buffer, mixed completely, and centrifuged at 14,000 × g for 20 min at 4 °C. Supernatants were then stored at −80 °C until use. For nuclear extracts, after washing the cells, they were lysed in hypotonic buffer and centrifuged at 14,000 × g for 30 s at 4 °C as previously described. After separating the supernatant, the pellets were resuspended in 50 μL of complete lysis buffer and centrifuged at 14,000 × g for 10 min at 4 °C, and the supernatants were stored at −80 °C until use.Whole cell lysates and nuclear extracts were separated as described previously . HaCaT cThe protein concentration was determined using the bicinchoninic acid (BCA) protein assay, and equal amounts of protein in the whole cell lysates were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Then, total protein was transferred to the polyvinylidene fluoride (PVDF) membrane  prior to blocking using 5% skim milk or 2–5% bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 (TBS-T) solution . The pri18 column  with the temperature maintained at 30 °C, and the injection volume was 10 μL. The mobile phase consisted of A: 0.05% Trifluoracetic Acid H2O and B: acetonitrile, and the flow rate was 1.0 mL/min. A gradient of 0–20 min from 10–0% and 20–30 min at 100% B was used. The PYE and astaxanthin  were used as the sample and standard at a concentration of 10 mg/mL and 0.1 μg/mL of each of astaxanthin and xanthophyll  was used for HPLC analysis to identify the peaks.Chromatographic analyses were performed on an Agilent HPLC system, 1200 series  equipped with double-beam photometer (Variable Wavelength Dector). UV absorbance was monitored at 470 nm. Quantification was analyzed by integration of the peak areas at 470 nm. The separation was performed using a Kromasil 100-5Cp < 0.05, ** p < 0.01, and *** p < 0.001 were considered to be statistically significant.The data analysis used the GraphPad Prism version 5.0 . The results are expressed as the mean ± standard error of the mean (S.E.M) or standard deviation (SD) of triplicate experiments. Statistical comparisons between of different treatments were performed using one-way ANOVAs with Tukey’s multiple comparison post-hoc tests. * To determine the effects of cytokine (IFN-γ or TNF-α) stimulation and varying concentrations of PYE on the HaCaT cell viability, the MTT assay was performed. As shown in To investigate the effect of PYE on pro-inflammatory chemokine (TARC and MDC) production by TNF-α/IFN-γ-stimulation, HaCaT cells were treated with PYE and IFN-γ or TNF-α for 24 h, and the mRNA expression of MDC and TARC chemokines was analyzed using real-time qPCR. As shown in p < 0.001) and MDC from a basal level of 52.64 ± 4.47 to 516.1 ± 13.19 pg/mL (p < 0.01). TNF-α stimulation increased the secretion of TARC from a basal level of 1 pg/mL to 116.41 ± 70.56 pg/mL (p < 0.05). Additionally, as a result of mRNA expression, the secretion of these chemokines in HaCaT cells was significantly inhibited by PYE compared to the IFN-γ- or TNF-α-stimulated group (p < 0.001). The results showed that PYE inhibited the release of IFN-γ- and TNF-α-stimulated pro-inflammatory chemokines TARC and MDC. We investigated the effect of PYE on the production of TARC and MDC chemokines induced by IFN-γ or TNF-α. HaCaT cells were incubated with the medium alone or IFN-γ or TNF-α in the presence or absence of PYE at indicated concentrations. The levels of TARC and MDC in the culture medium supernatant were determined by ELISA . Even atTo investigate the molecular mechanism of the anti-inflammatory effect of PYE in IFN-γ- or TNF-α-stimulated HaCaT cells, we examined whether PYE inhibits the activation of MAPKs. HaCaT cells were treated with PYE  and IFN-γ or TNF-α for 24 h and cell lysates were collected for an analysis of the activities of MAPKs, such as ERK, JNK, and p38, by Western blotting. As shown in NF-κB is a transcription factor that is critically involved in AD-related signaling in IFN-γ- or TNF-α-induced HaCaT cells. Therefore, the regulation of NF-κB expression is important for the management of skin disease. As shown in We analyzed prior studies and the component analysis to identify which components exhibit anti-inflammatory activity. As a primary study, the total flavonoid, polyphenol, and carotenoid content was measured and compared to other seaweeds . We founIt was investigated whether the astaxanthin (AXT) and xanthophyll (X) contained in PYE influence the NF-κB / IκB-α and MAPK signaling pathways in mechanisms similar to PYE.As shown in In the xanthophyll-treated group, the ratio of ERK and p38 phosphorylation in IFN-γ-stimulated HaCaT cells increased to 172.3% and 110.3%, respectively, compared to the control group. However, after treatment with xanthophyll (0.08 μg/mL), the p-ERK and p-p38 were reduced 110.2% and 78.1%, respectively D. In addPyropia yezoensis (P. yezoensis) is used as a traditional medicine in the treatment of various diseases. P. yezoensis has numerous biological functions, including anti-inflammation, anti-oxidant, anti-cancer, anti-photo aging, and anti-hypertensive effects [P. yezoensis, but studies on atopic dermatitis (AD) from P. yezoensis extract (PYE) have not been reported until now. In this study, we revealed that PYE treatment reduced the production of inflammatory-mediated chemokines in IFN-γ/TNF-α-induced HaCaT cells through the inactivation of NF-κB and MAPK pathway.In Asia,  effects ,24,25,26Keratinocytes produce pro-inflammatory cytokines and chemokines, which are affected in the development of inflammatory skin disorders, such as AD. AD is a Th2-type skin disease characterized by inflammation, in which lymphocytes infiltrate into the dermis. TARC and MDC are Th2 chemokines that bind to and attract CCR4+ Th2 cells to sites of inflammatory tissue ,28,29. PIn keratinocytes, IFN-γ/TNF-α activates several intracellular signaling pathways, including those of mitogen-activated protein kinases (MAPKs) . The MAPElsenia bicyclis inhibited pro-inflammatory cytokines and chemokines production in HaCaT cells by inhibiting NF-κB activation [Ecklonia cava suppressed MDC and TARC production by blocking MAPKs and NF-κB signaling pathways in HaCaT cells [Previous reports have shown that the NF-κB signaling pathway regulates the production of TARC and MDC in HaCaT cells ,43. In ttivation . Cho et aT cells . In a previous study, we first measured the total carotenoid, polyphenol, and flavonoid content in PYE to identify substances with anti-inflammatory effects and compared them to other red-macro algae . We founP. yezoensis and found that carotenoids include both α-carotene, β-carotene. The α-cryptoxanthin and zeinoxanthin, which are monohydroxy-carotenoids, are produced as intermediates when lutein synthesis in α-carotene of P. yezoensis. Carotenoid compounds contribute to light harvesting and photo-protection and have potent antioxidant, anti-inflammatory, and neuroprotective properties [Phaffia rhodozyma and Haematococcus pluvialis. In many previous studies, xanthophyll is known to exist in P. yezoensis. However, it has been reported that their content might be different depending on environmental factors such as an inhabited area and temperature stress [According to previous literature, we investigated the intermediate carotenoids of operties . Astaxanoperties ,52,53. Te stress . Furthere stress ,56.Here, we confirm that astaxanthin and xanthophyll were detected in PYE among carotenoid components through HPLC analysis. Additionally, there was 0.08 μg/mL astaxanthin and 0.05 μg/mL xanthophyll in 10 mg/mL PYE. On the basis of these results, we observed the same signal transduction pathways compared to PYE and investigated the bioactive roles of astaxanthin and xanthophylls contained in PYE. We demonstrated that MAPK and NF-κB/ IκB-α signaling pathways in stimulated HaCaT cells treated with individual astaxanthin and xanthophylls similarly inhibited PYE. Therefore, we strongly expect that, due to the results, astaxanthin and xanthophyll are the main components that have anti-inflammatory effects on PYE.P. yezoensis extract regarding its effect on anti-AD.Although our results suggest that the anti-AD effect of PYE on HaCaT cells may be attributed to astaxanthin and xanthophyll, other compounds present in PYE may also have an effect on the overall ability to inhibit pro-inflammatory chemokines. Through further study, it is expected that researchers will be able to identify more complex derivatives of carotenoids with inflammatory effects. Even though this analyzed data is preliminary, it is crucial because there has been no report on In conclusion, this study investigated the basic mechanism of anti-inflammatory activity of PYE in HaCaT cells, as an in vitro model of human keratinocytes in inflammatory skin disease. In IFN-γ/TNF-α-induced HaCaT cells, PYE reduced the production of Th2 chemokines, such as TARC and MDC. The mechanism underlying these effects included the suppression of the phosphorylation of ERK, JNK, and p38 expression, and then the nuclear translocation of NF-κB/p65, by blocking IκB-α degradation . Moreove


Addition of a serving of mushrooms also decreased the % below EAR for copper, phosphorus, and riboflavin for those 9–18 years and for copper, phosphorus, selenium, zinc, thiamine, riboflavin, niacin, folate, and vitamin B6 for those 19+ years and increased the % above AI for potassium for both age groups. Addition of oyster mushrooms additionally increased 12%–13% vitamin D, and 12%–15% choline in the NHANES 2011–2016 diets. Addition of mushrooms exposed to UV light to increase vitamin D levels to 5 µg/serving also almost doubled vitamin D intake (98%–104%) and decreased inadequacy. Addition of a serving of mushrooms would also add 2.2 mg ergothioneine and 3.5 mg glutathione to the diet. Addition of a mushroom serving to the diet would increase several micronutrients including shortfall nutrients, without having any impact on energy, sodium, or fat.To evaluate the nutritional impact of adding a serving of mushrooms on usual intakes and population adequacy of nutrients the National Health and Nutrition Examination Survey (NHANES) 2011–2016 dietary data for 9–18 years and 19+ years and a composite of commonly consumed raw mushrooms as well as oyster mushrooms (nutrient profiles from USDA data) were used for modeling. Usual intakes of nutrients and the percent population below the Estimated Average Requirement (EAR) or above the Adequate Intake (AI) were estimated before and after addition of mushrooms. Means with nonoverlapping 95th percentile confidence levels were used to assess meaningful differences. Addition of a serving (84 g) of mushrooms to the diet resulted in an increase in dietary fiber (5%–6%), copper (24%–32%), phosphorus (6%), potassium (12%–14%), selenium (13%–14%), zinc (5%–6%), riboflavin (13%–15%), niacin (13%–14%), and choline (5%–6%) in both adolescents and adults; and in iron (2.32%), thiamin (4.07%), folate (3.66%), and vitamin B Mushrooms are fungi and have long been part of the human diet. This modeling study evaluated the nutritional impact of adding mushrooms to current dietary intakes. Addition of mushrooms to NHANES 2011‐2016 increased several micronutrients including “underconsumed” and “shortfall nutrients” and had a minimal or no impact on calories, sodium, or fat. These results suggest that inclusion of mushrooms in the diets may be one effective approach to help Americans to reach dietary goals identified by Dietary Guidelines for Americans. The surUsing NHANES 2001–2010 data, we earlier reported that mushroom intake was associated with higher intakes of several key nutrients and better diet quality; however, their intake was low at 2.3 g per day per capita or 20.6 g per day among consumers  Ethics Review Board and all participants or proxies provided a signed written informed consent. Energy and nutrient intake were obtained from the relevant USDA Nutrient Database for Standard Reference Releases in conjunction with the respective USDA Food and Nutrient Database for Dietary Studies (FNDDS)  software. The data were adjusted for the complex sampling design of NHANES, using day 1 dietary weights, strata, and primary sampling units. Means with nonoverlapping 95th percentile confidence levels were used to assess meaningful differences in usual intakes of energy and nutrients, and the percentage of the population below the EAR or above AI of nutrients before and after addition of mushrooms.3Addition of a serving (84 g) of commonly consumed mushrooms to the NHANES 2011–2016 diets resulted in an increase (nonoverlapping 95% CI) in dietary fiber (6.16%), copper (31.6%), phosphorus (6.46%), potassium (14.1%), selenium (13.6%), zinc (5.71%), riboflavin (14.6%), niacin (14.2%), and choline (6.11%), but had no impact on energy, carbohydrate, protein, fat, or sodium for adolescents 9–18 years; and an increase in dietary fiber (4.57%), protein (2.64%), copper (23.6%), iron (2.70%), phosphorus (6.34%), potassium (11.8%), selenium (12.9%), zinc (5.31%), thiamin (4.32%), riboflavin (13.4%), niacin (12.5%), folate (3.72%), vitamin B6 (4.61%), and choline (5.03%) for adults 19+ years. However, addition of commonly consumed mushrooms did not have any impact on energy, carbohydrate, fat, or sodium in NHANES 2011–2016 adult diets  vitamin D  and choline  in the NHANES 2011–2016 diets  vitamin D by 98.0% from 5.10  to 10.1  µg for those 9–18 years and by 104% from 4.68  to 9.53  µg for those 19+ years. Similar to commonly consumed mushrooms, addition of oyster mushrooms to the diets also increased (nonoverlapping 95% CI) fiber, copper, iron, phosphorus, potassium, zinc, thiamine, riboflavin, niacin, folate, and vitamin B6, for age 19 + years (Table Addition of a serving of commonly consumed mushrooms to NHANES 2011–2016 diets decreased (nonoverlapping 95% CI) the population inadequacy (% below EAR) for copper, phosphorus, and riboflavin for age 9–18 years, and for copper, phosphorus, selenium, zinc, thiamine, riboflavin, niacin, folate, and vitamin Brs Table . Populat4Results of this NHANES 2011–2016 modeling study indicate that addition of mushrooms to the diet increased the intake and decreased population inadequacy of several key micronutrients without affecting intake of calories, fat or sodium and thus have beneficial effects on the diets of children (9–18 years) and adults (19+ years).Inadequate intake of micronutrients has been consistently associated with adverse health effects such as neural tube defects, poor bone health (osteoporosis), impaired immune function, and impaired cognitive function, as well as chronic diseases, such as certain cancers, age‐related eye diseases, hypertension, and possibly coronary heart disease and stroke Ames, . Accordi2 contents  Ethics Review Board. All subjects or proxies provided written informed consent to participate in the study.


Intracellular mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial oxygen consumption were measured. SD male rats were randomly divided into three groups: control, DM, and DM+WXKL groups. Rats in the DM+WXKL group were treated with daily gavage of WXKL at 3 g/kg. After eight weeks, echocardiography, hemodynamic examination, histology, electrophysiology study, mitochondrial respiratory function, and western blots were assessed. H2O2 treatment led to increased ROS and decreased intracellular MMP and mitochondrial oxygen consumption in primary atrial fibroblasts. WXKL improved the above changes. DM rats showed increased atrial fibrosis, greater left atrial diameter, lower atrial conduction velocity, higher conduction heterogeneity, higher AF inducibility, and lower mitochondrial protein expression, and all these abnormal changes except for left atrial diameter were improved in the DM+WXKL group. WXKL improves atrial remodeling by regulating mitochondrial function and homeostasis and reducing mitochondrial ROS in diabetic rats.Mitochondrial dysfunction and oxidative stress play an important role in the pathogenesis of both atrial fibrillation (AF) and diabetes mellitus (DM). Wenxin Keli (WXKL), an antiarrhythmic traditional Chinese medicine, has been shown to prevent cardiac arrhythmias through modulation of cardiac ion channels. This study tested the hypothesis that WXKL can improve atrial remodeling in diabetic rats by restoring mitochondrial function. Primary atrial fibroblasts of neonatal SD rats were divided into four groups: control, hydrogen peroxide (H Atrial fibrillation (AF) is the most common arrhythmia observed in the clinic. In recent years, its prevalence has increased with the aging of the population. AF is a risk factor for thromboembolism, which increases the risk of stroke and all-cause mortality , 2. The Diabetes mellitus (DM) is an independent risk factor for AF , 5. Our INa) and thus effectively suppresses AF in experimental models of AF [Wenxin Keli (WXKL) is a traditional Chinese medicine, which is composed of Codonopsis, Rhizoma Polygonati, Notoginseng, Amber, and Nardostachys . In Chinls of AF –14. Our ls of AF . WXKL wals of AF .2O2) as a potent oxidant induces mitochondrial dysfunction by increasing ROS production, which leads to reduced antioxidant activity and induced mitochondrial apoptotic signaling [2O2-induced oxidative stress in vitro and the atrial remodeling, atrial muscle mitochondrial function, and atrial electrophysiological properties in HFD/STZ-induced type 2 diabetic rats and the potential therapeutic effects of WXKL.Hydrogen peroxide  solution without Ca2+ and Mg2+. Atrial tissues were repeat digested by trypsin type II collagenase buffer  until the tissue completely dissipated. All the cell suspensions were centrifuged for 5 min at 1000 r/min. Isolated cells were resuspended in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum  and 100 IU/mL penicillin-streptomycin (Gibco), and plated on 10 cm culture dishes for 2 h until the fibroblasts adhered to the dishes in a humidified atmosphere at 37°C under 5% CO2. The preseeding medium containing cardiomyocytes was removed, and the culture medium was added to continue to culture the atrial fibroblasts. The culture medium was changed every 48 hours. Atrial fibroblasts were used in subsequent experiments and incubated in FBS free medium for 24 h before treatment. The cells were pretreated with vehicle or WXKL (1 g/L or 3 g/L) for 1 hour and then stimulated with 300 μM hydrogen peroxide (H2O2) for 24 hours. The cells were randomly divided into the following groups: control, H2O2, H2O2+WXKL 1 g/L, and H2O2+WXKL 3 g/L groups.Primary cultures of atrial fibroblasts were isolated from 3-day-old neonatal Sprague-Dawley (SD) rats. In brief, neonatal SD rats were sacrificed by cervical dislocation, then sterilized in 75% ethanol, and subjected to subsequent operations in a clean bench. The left atrium was separated from the exposed heart; then, it was cut to small pieces of 1 mmβ-cell toxin STZ, which results in a severe reduction in functional β-cell mass [SD male rats aged 8 weeks (average body weight of 200 ± 20 g) were purchased from Beijing HuaFuKang Bioscience Co., Ltd. . These rats were fed under controlled conditions with a constant temperature of 25 ± 2°C, a humidity of 60 ± 5%, and a 12 h day/night cycle. All rats accepted normal chow diet of 1 week for acclimation before initiation of the study. All rats were assigned randomly to the following 3 groups by a random number table: control, DM, and DM+WXKL groups. These rats of a diabetic group, including DM and DM+WXKL groups, were fed with HFD , while the control group was fed with normal chow diet throughout the whole study. After 4-week feeding, all animals were overnight fasted; then, the diabetic group was induced by a single tail vein injection of STZ  in 0.1 M citrate buffer (pH 4.5). The rats in the control group were injected with the citrate buffer vehicle. 72 hours after injection, fasting blood glucose levels (FBG) were measured, and rats in the STZ injection group with blood glucose levels > 11 mmol/L were considered diabetic models and would be used in future experiments. This model involves a combination of a diet high in fat to bring about hyperinsulinemia, insulin resistance, and/or glucose intolerance followed by treatment with the ell mass –21. WXKL2+ and Mg2+. The processes of measurement were used with Olympus FV1000 LSCM under excitation wavelength of 488 nm and 543 nm. The decreased relative fluorescence proportion of aggregate JC-1 (red)/monomeric JC-1 (green) indicates a decrease in the mitochondrial membrane potential (MMP). ROS were detected using the 2′,7′-dichlorofluorescin diacetate  fluorescent probe. The cells were incubated with 10 μM DCFH-DA in PBS without Ca2+ and Mg2+ in the dark for 20 min at 37°C. Fluorescence was determined at 488 nm for excitation and 525 nm for emission. Microscope images were saved as TIFF files and processed for densitometric quantification with ImageJ version 1.46 (NIH). Software settings were kept the same for every image analyzed.The atrial fibroblasts were incubated with JC-1 staining buffer in the dark for 20 min at 37°C and washed twice with PBS without Ca2 in XF calibration buffer at 37°C before assay. 10 μM oligomycin, 10 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 10 μM antimycin A (AA)+retenone were injected in turn during the process of assay.The mitochondrial oxygen consumption rate (OCR) of atrial fibroblasts was measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience), and this process was according to instructions of the manufacturer and Lay et al. . BrieflyAfter 8 weeks of treatment in each group, rats were allowed to inhale isoflurane (1.5 L/min) for anesthesia and placed on a 37°C thermal table in the supine position to measure transthoracic echocardiography. Echocardiographic parameters, including left atrial (LA) diameter (LAD), left ventricular posterior wall thickness (LVPWT), interventricular septal thickness (IVST), left ventricular end-diastolic dimension (LVEDD), and left ventricular end-systolic dimension (LVESD), were obtained in the parasternal long-axis view using a specified small animal ultrasound system  by a senior blinded operator. These parameters were measured by 2-dimensional imaging during 3 consecutive cardiac ejection cycles. Left ventricular ejection fraction (LVEF) was acquired in line with ventricular volumes calculated by the Teichholz formula:At the end of echocardiographic examination, each rat underwent right carotid artery cannulation allowing measurements of hemodynamic parameters using a Millar catheter during electrocardiographic (ECG) monitoring. Aortic systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) were recorded carefully after a stabilization period. Subsequently, the cannula was inserted through the aortic valve to the left ventricle to record the left ventricular end-diastolic pressure (LVEDP) and maximum increasing or decreasing rate of left ventricular pressure (±dp/dt max). 10 stable waveforms of electrocardiogram were selected to measure heart rate, P wave duration, PR interval, QRS duration, and QT interval. Finally, rats were instantly euthanized. Then, blood samples were obtained from the right ventricle and LA tissues were rapidly separated. Samples for histological assessment were soaked in neutral buffered 10% formalin solution, and samples for molecular biological experiments were rapidly frozen in liquid nitrogen and stored at -80°C.FBG levels were detected with the blood glucose meter and test strips each week. Serum biochemical parameters including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and creatinine (Cr) level were detected using a fully automatic biochemical analyzer. Rat ELISA kits were used to detect the serum levels of insulin and high-sensitivity C-reactive protein (hs-CRP) ; also, lipid peroxidation malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured according to instructions of the manufacturer .2 and 95% O2. Preperfusion was performed for 20 minutes to minimize endogenous release of catecholamine residual effects. Some hearts could not maintain normal rhythms after 20 minutes and were abandoned.The rats were intraperitoneally injected with 3% pentobarbital sodium (50 mg/kg) for anesthesia. The heart was quickly isolated and placed in 4°C Tyrode's solution to wash out residual blood and to remove residual lung tissue. Then, the aorta was cannulated and connected to a Langendorff perfusion system. The Langendorff perfusion system was filled with 37°C Tyrode's solution equilibrated with 5% COEpicardial activating electrical mapping was recorded by a 6 × 6 electrode microelectrode  on the epicardial surface of the left and right atrium. Data were recorded by multichannel systems . Activation waveforms acquired were amplified by a filter amplifier and thereby transmitted to the connected computer. All activation time was digitized and then used to draw activation maps. The activation times were calculated as the point of maximal negative slope of activation waveforms. Conduction velocity (CV), inhomogeneity index, and absolute inhomogeneity were calculated by EMapScope 4.0 software .Three silver bipolar electrodes were placed on the left atrium, right atrium, and right ventricular apex after recorded epical mapping, and the entire recorded process was connected to a custom-made computer software program . There were 3 basic cycle lengths (BCLs) of basic stimuli (S1), including 200 ms, 150 ms, and 100 ms. When the sinus cycle length (SCL) was shorter than 200 ms, we used the SCL subtracted by 10 ms to replace BCL of 200 ms. The interatrial conduction time (IACT) was measured during right atrium pacing. Then, atrial-ventricular Wenckebach cycle length conduction (AVWCL) was measured by right atrial (RA) incremental pacing. The S1S1 interval was decreased in 5 ms steps until ventricular rhythm was not 1 : 1 following atrial pacing. 8 S1 were followed by a premature extra stimulus (S2) to measure the atrial effective refractory period (ERP). The S1S2 interval was decreased in 2 ms steps until the longest S1S2 interval that failed to capture the depolarization. The stimulation order of burst pacing was from the right atrium to the left atrium. AF induction was verified by burst pacing  for 3 seconds, respectively, which was performed 5 times with 30-second intervals. AF was defined as rapid, irregular atrial response longer than 1 second. AF inducibility was defined as the percentage of burst pacing leading to AF episodes.μg of mitochondrial protein in 2 mL of respiration medium were incubated with JC-1 working solution at 37°C in the dark for 10 min.Rats were euthanized with 3% pentobarbital sodium (50 mg/kg), and median sternotomy was immediately performed. The LA mitochondria were isolated, and the mitochondrial protein content was assayed according to our previous studies. Atrial MMP was assessed by JC-1 staining . 300 μg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred by electrophoresis to a polyvinylidene difluoride (PVDF) membrane. Subsequently, PVDF membranes were blocked for 1 h with 5% nonfat dry milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST), then incubated at 4°C overnight with a primary antibody. Then, the membrane was washed with TBST and incubated with the horseradish peroxidase-conjugated secondary antibody for 1 hour. The primary antibodies used were as follows: β-actin , mitochondrial transcription factor A , dynamin-related protein , mitofusin 2 , transforming growth factor-β , nuclear factor kappa-b , Bcl-2 , Bax , collagen I , collagen III , and α-smooth muscle actin . β-Actin was evaluated as a loading control. The reactions were visualized using Tanon 5200 Multi Chemiluminescent Imaging System .Frozen LA tissues were homogenized in liquid nitrogen, and the total protein was extracted by RIPA lysis buffer. The lysates were centrifuged at 15,000g for 20 minutes, and the supernatants were collected. Protein concentrations were measured by the bicinchoninic acid (BCA) protein assay regent kit . All sample proteins loaded equally with 60 μm cross sections, then stained with hematoxylin and eosin (HE) to observe the morphological changes and Masson's trichrome stain to evaluate interstitial fibrosis. Digital images were scanned by Image-Pro Plus 6.0, and the extent of fibrosis was determined by dividing the area of collagen deposition by the entire cardiac tissue area. Five randomly selected sections of each group were utilized for quantification. The cardiac collagen volume fraction and cross-sectional area were analyzed using SPSS 17.0.The tissues were cut into 5 P values < 0.05 were considered significantly altered.Data were presented as mean ± standard deviation (SD). Differences between the groups were analyzed for statistical significance using the one-way analysis of variance (ANOVA) followed by the Least Significant Difference (LSD) test. All data were analyzed using SPSS 17.0, and 2O2 group compared with the control group , while ROS already had a decreased trend in the H2O2+WXKL 1 g/L group (P = 0.053) compared with the H2O2 group.DCFH-DA fluorescence was measured in atrial fibroblasts from each experimental group . ROS wasectively . The ROS2O2 group compared with the control group, and the above changes were improved in the H2O2+WXKL 3 g/L group . The MMP was similar between the H2O2 group and the H2O2+WXKL 1 g/L group  and DM+WXKL group  compared with the control group (7.12 ± 0.61 mmol/L), and there was no difference in FBG between the two diabetic groups (P = 0.28). Blood glucose was significantly higher in the DM  and DM+WXKL groups  compared with the control group (5.98 ± 0.93 mmol/L). Serum insulin level, quantified by ELISA, was significantly higher in the DM (32.65 ± 8.69 pmol/L) and DM+WXKL groups (31.32 ± 7.25 pmol/L) when compared to the control group (20.44 ± 5.71 pmol/L).We successfully established a rat model of type 2 diabetes by high-fat feeding+low STZ injection. FBG was stabilized in a hyperglycemic state within 8 weeks of the whole study process. The general characteristics of the 3 groups are presented in P < 0.01). The MMP of the DM+WXKL group was also lower than that of the control group . However, the MMP of the DM+WXKL group was improved compared with that of the DM group .After 8 weeks of WXKL administration, mitochondria of the left atrium were extracted for MMP detection . MMP wasP = 0.001, P = 0.691). There were no differences in LVEF, IVS, and LVPW between the three groups (Representative echocardiographic images of the three groups are shown in e groups . For ECGe groups .P < 0.01) and 5069.63 ± 355.39 vs. 7113.05 ± 632.80 (P < 0.01), We measured hemodynamics through a Miller pressure catheter. SBP, DBP, and MBP were not significantly different between the three groups . HoweverP = 0.01, P = 0.03, P < 0.01, index 3.07 ± 1.80 vs. 1.87 ± 0.59, P = 0.046; Figures Electrophysiological parameters were obtained from Langendorff-perfused hearts. We firstly recorded electrical conduction mapping of LA  and RA . MappingP < 0.01, P < 0.01, P = 0.93).There were no significant differences of SCL and AVWCL between the three groups . The IACP < 0.001, H&E and Masson's trichrome staining was used to detect morphological changes in the RA . CardiomP = 0.67). Lower SOD and higher MDA levels were detected in the DM group compared to the control group , reducing intracellular calcium overload [press AF , 16, 39.ntricles . Hu et antricles . Ranolazntricles . The prooverload , 42. We 2O2 treatment occurred with the outbreak of ROS, which was related to the change of mitochondria . H2O2 can diffuse across the membrane and generate other ROS. Superoxide released into the mitochondrial matrix can react with mitochondrial DNA (mtDNA), and ROS-induced mtDNA damage leads to respiratory complex enzyme dysfunction [2O2 delivered to the cytoplasm is involved in a variety of signaling pathways, including mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK1), and TNF-α/NF-κb [2O2 to prevent profibrotic cellular activities. Low concentrations of WXKL inhibit INa and suppress acetylcholine-induced AF, while higher concentrations inhibit fast sodium current and prevent ventricular reentrant arrhythmias. It exerts a tonic block on peak INa without significant use dependence and interacts with its inactivated state [INa and dissociates rapidly. WXKL is speculated to possess antisinus arrhythmia, antiheart failure, and antihypertensive properties [Our research was aimed at clarifying the signaling pathway of WXKL intervention. Hfunction . H2O2 de-α/NF-κb –46. Thro-α/NF-κb . We demoed state . WXKL bioperties . The mecin vitro, and we could not further clarify that ROS was a sole signaling pathway to improve mitochondrial function by WXKL. OCR of atrial fibroblasts was used as a marker of mitochondrial function. Secondly, we only assessed the effects of WXKL in atrial fibroblasts, and potential effects of WXKL on atrial cardiomyocytes were not investigated. Thirdly, ion channel currents of WXKL intervention were not studied in this study. Moreover, detailed electrophysiological mechanisms of the antiarrhythmic effects of WXKL, such as alterations in autonomic tone, calcium handling, and depolarization and repolarization dynamics, remain to be elucidated. In addition, action morphology will be measured in the presence and absence of WXKL in single atrial myocytes isolated from normal and diabetic hearts. WXKL may have non-DM-related biological effects in control rats. Further studies need to detail all the mechanisms of WXKL on the atrium by regulating the DM-induced mitochondrial oxidative stress.Several limitations of our study should be noted. Firstly, we did not block the pathway of mitochondrial oxidation 2O2 and in vivo induced by DM, alleviate atrial remodeling, and reduce AF incidence in DM rats.WXKL can inhibit oxidative stress and improve mitochondrial function in vitro induced by H


Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins . The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some  Enterococci are LAB, and they are found in the gastrointestinal tract of humans and animals as well as in feed, food, and healthcare units3. Most strains of Enterococcus faecalis are harmless commensals that may be beneficial to health (probiotics). In contrast, other strains are opportunistic pathogens, especially vancomycin-resistant enterococci (VRE) and multidrug-resistant enterococci (MDRE), causing nosocomial infections and acquiring antibiotic resistance determinants that become intrinsically resistant to commonly used antibiotics, leading to an increasingly serious problem in public health worldwide4. E. faecalis and E. faecium are recognized as the most common causes of nosocomial infections in their genus, causing urinary tract infection, postoperative wound infection, endocarditis, bacteremia, pneumonia, and meningitis5. The search for novel effective antimicrobial agents has become a global concern due to the increasing prevalence of MDRE and VRE. Over the past three decades, the rate of antibiotic discovery has slowed, and few antibiotics have been developed against VRE6. Furthermore, some enterococci have evolved resistance to the most recently-developed antimicrobial agents, such as quinupristin-dalfopristin, linezolid, daptomycin, and tigecycline7.Lactic acid bacteria (LAB) are defined as aerotolerant Gram-positive cocci or bacilli that ferment sugars to produce lactic acid10. There are three classes of bacteriocins. Briefly, class I consists of small, heat-stable, post-translationally modified peptides. Lantibiotics, due to the presence of the modified residues lanthionine and methyl-lanthionine, are a well-known example of that class. Some other bacteriocins are also considered members of class I, namely, those belonging to ribosomally-synthesized and post-translationally modified peptides that function as bacteriocins, such as circular bacteriocins, sactibiotics, and glycocins11. Class II consists of small, heat-stable peptides without modified residues. Class III consists of abundant, heat-labile proteins13.Antimicrobial peptides (AMPs) have drawn attention as alternatives to conventional antibiotics. Enterococci produce a variety of ribosomally-synthesized AMPs or bacteriocins16. The use of bacteriocins as a replacement for currently used antibiotics is promising15. Furthermore, resistance toward bacteriocins has rarely been observed17. Because enterococci are part of the healthy microbiota in humans, they are considered relatively safe but are not endowed with GRAS status18. However, some of them are used as probiotics19, and bacteriocin production adds an essential feature in their protection against pathogenic bacteria20.Bacteriocins have been used in the food industry for over 50 years and are Generally Recognized As Safe (GRAS)12. These killing mechanisms include membranous pore formation, disruption of the cell wall or protein synthesis, and degradation of nucleic acids21. Narrow-spectrum antibacterials, such as bacteriocins, are an attractive approach in the battle against multidrug-resistant bacterial pathogens due to their limited resistance acquisition and trigger no significant disruption to the host microbiota. It worth mentioning that the use of traditional broad-spectrum antibiotics can disturb the host microbiota, affecting the crucial role that it plays in various functions, including nutrient and certain amino acids supply, vitamin production, and protection against pathogens22. Exposure to broad-spectrum antibiotics during infancy and early childhood is mostly unfavorable, as the early microbiota lacks diversity and stability, making it distinctively sensitive to disruption, besides the importance of the gut microbiota in the early development and education of the immune system23. Thus, if the infection's causative pathogen is well-known, the use of narrow-spectrum antimicrobials, such as bacteriocins, could potentially avoid these issues of hurting gut microbiota.Bacteriocins differ from antibiotics in that they are ribosomally synthesized and target bacteria closely related to the producing strain by different mechanisms of actionE. faecalis isolates, obtained from food and clinical samples, for their ability to produce bacteriocins. Attempts were performed to purify and characterize BLIS with activity against nosocomial enterococci. One putative bacteriocinogenic isolate was studied in detail, and two BLIS were characterized from this isolate.In these perspectives, the main objective of this study was to screen different The bacterial strains used in this study and their culture conditions are presented in Table g for 10 min) and 1 g of each of the food and stool samples were inoculated into tubes containing 5 mL of GM17 broth  and incubated aerobically at 37 °C for 24 h.Different food samples  and clinical samples (urine and stool) were collected in sterile plastic cups from Beni-Suef City, Egypt, and refrigerated for 24 h prior to processing. Urine sample precipitates (collected after centrifugation at 5000×3+ to form a dark brown to black precipitate on the agar plate. The resultant black colonies were presumptively identified as Enterococcus sp. using the l-pyrrolidonyl-β-naphthylamide (PYR) test and then identified at species level using the API 50 CHL system  according to the manufacturer’s instructions.Enterococci were isolated by selective culture on bile esculin agar (Oxoid), where they grow in the presence of 4% (w/v) bile and hydrolyze esculin to esculetin that reacts with Fe24. Briefly, 1.5 ml of an overnight culture was centrifuged for 10 min at 3000×g, and the supernatant was discarded. The pellet was resuspended in 200 μL of spheroplast buffer , vortexed, and incubated at 37 ºC for 10 min until cell lysis occurred. Then, 50 μL of each of 5% (w/v) sodium dodecyl sulfate (SDS) (lysis buffer 1) and 5 M NaCl (lysis buffer 2) were added, mixed, and incubated at 65 ºC for 5 min. Next, 100 μL of neutralizing buffer  was added. The sample was vortexed, placed on ice for 5 min, and centrifuged at 18,000×g at 4 ºC for 15 min. The supernatant (approximately 400 μL) was transferred to a new tube, mixed with an equal volume of isopropanol, left to stand for 5 min at room temperature, and centrifuged at 18,000×g at room temperature for 15 min to precipitate the DNA. The resultant pellet was washed with 70% (v/v) ethanol by centrifugation at 18,000×g at room temperature for 5 min and finally air-dried, resuspended in 50 μL 1× TE buffer [pH 8], and stored in a refrigerator at 4 ºC. The DNA purity and concentration were determined by optical density measurement using a NanoDrop ND1000 spectrophotometer .It was performed with some modifications of a previously described protocol25 using 1% (w/v) agarose gels with reference to 1 kbp DNA ladder . Any impurities were removed from the PCR product using the QIAquick PCR purification kit . Quantification of the PCR products was determined by optical density measurement using a NanoDrop ND1000 spectrophotometer . The PCR product was sequenced and analyzed using an ABI Prism BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit on an ABI Prism 3100 Genetic Analyzer, according to the manufacturer’s instructions. The sequences were assembled by BioEdit v7.0.9.0 software, and a homology search of the National Center for Biotechnology Information (NCBI) database was performed using BLAST. Multiple sequence alignment was performed, and molecular phylogenies were evaluated using MEGA7 software using the neighbor-joining method, and the evolutionary distances were computed using the Kimura two-parameter method26.These were accomplished using primers 11F and 4R Table . The PCREnterococcus spp. were determined by PCR amplification using specific primers purchased from Invitrogen  (Table 25 using 1.5% (w/v) agarose gels with reference to a 100-bp DNA ladder (Fermentas).The presence of genes encoding 17 known bacteriocins in A) Table . DNA sam27. Briefly, the colonies were overlaid with 5 mL of soft agar containing 70 µl of the indicator strains which were grown to late exponential phase . One arbitrary activity unit (AU) was defined as the reciprocal of the highest dilution of bacteriocin, causing 50% growth inhibition of the treated indicator strain by measuring growth turbidity inhibition at 620 nm in the microtiter plate reader compared to the untreated control29. The bacteriocin activity was calculated according to the following equation; Bacteriocin activity (AU/mL) = (d × 1/m), where “d”; is the used dilution factor and “m” is the highest dilution causing 50% growth inhibition.Quantitative estimation of BLIS in the cell-free culture supernatant was performed using a microtiter plate assayE. faecalis isolates onto a fresh plate of Columbia blood agar (Oxoid) containing 5% (v/v) defibrinated horse blood and incubating at 37 °C for 24 h. The formation of a clear zone around the colonies indicated beta complete hemolytic activity. In contrast, the formation of greenish discoloration surrounding the colonies indicated alpha partial hemolytic activity, and the absence of any hemolysis was referred to as gamma or no hemolysis. Proteolytic gelatinase activity was detected by inoculating E. faecalis isolates into tubes containing gelatin medium composed of 0.5% (w/v) peptone, 2% (w/v) gelatin, 0.3% (w/v) beef extract, 0.5% (w/v) NaCl and final pH was adjusted to 7.2. All tubes, including the control (uninoculated), were incubated at 37 °C for 24 h. Gelatin liquefaction observed after keeping the tubes at 4 °C for 3 h, indicated positive gelatinase activity. Putative bacteriocinogenic E. faecalis isolates were cultured on both control MRS agar and MRS agar containing 0.5% (w/v) thioglycolate and 0.5% (w/v) sodium glychodeoxycholate  to investigate bile salt hydrolase activity. The formation of white precipitate around the colonies indicated bile salt hydrolase activity.Hemolytic activity, being alpha, beta, or gamma, was determined by streaking putative bacteriocinogenic E. faecalis isolates to check whether they are relevant to each other or from different clones, and to overview their biosafety for further applications, the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method in microtiter plates30 was used to determine the antibiotic resistance phenotype and MIC of these isolates against 13 antibiotics with different concentration ranges 31. The ST distribution among all putative bacteriocinogenic E. faecalis isolates, included in the MLST database, was evaluated using eBurst v332.An MLST scheme based on seven housekeeping genes was performed to investigate the epidemiology and population structure of the 11 putative bacteriocinogenic 33. Briefly, 2 L of E. faecalis OS13 cultivated in MRS broth at 30 °C for 18 h was used for further purification steps. After centrifugation at 14,500×g for 10 min at 4 °C  and filtration , the supernatant was subjected to ammonium sulfate precipitation 50% (w/v) and shaken vigorously at 4 °C for 30 min. The protein pellet was harvested by centrifugation at 14,500×g for 10 min at 4 °C and then redissolved in 40 mL of distilled water to obtain a 50× concentrated crude BLIS solution. Whenever necessary, the pH was adjusted to 3.5 using 0.1 M HCl.The BLIS was purified as previously describedThe crude BLIS solution underwent cation exchange chromatography using a 10-mL SP Sepharose Fast Flow column  pre-washed with 200 mL distilled water and equilibrated with 10 mL sodium acetate . After applying the crude BLIS, the column was washed with 50 mL sodium phosphate buffer  and finally eluted using 20 mL of 1 M NaCl. The BLIS was further purified by reverse-phase chromatography on an ÄKTA FPLC purifier system . First, the sample was applied to a Resource RPC 1 mL column (Amersham Pharmacia Biotech) pre-equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) in water (Buffer A) and eluted using a 30 column volume (CV) linear gradient from 0 to 100% (v/v) isopropanol containing 0.1% (v/v) TFA (Buffer B). Fractions of 2 mL each were collected. The most active fraction (determined by microtiter plate assay) was further diluted in 20 mL 0.1% (v/v) TFA in water and applied to a Sephasil Peptide C8 5-µm ST 4.6/250 column (Amersham Pharmacia Biotech). Elution was performed using 0.5 CV (10% (v/v) Buffer B), 1 CV (10–30% (v/v) Buffer B), 5 CV (30–45% (v/v) Buffer B), and finally 2 CV (45–100% (v/v) Buffer B) at a flow rate of 0.4 mL/min. The peaks were monitored at 214, 254, and 280 nm, and fractions (1 mL each) were collected.L. sakei LMG 2313) and incubated overnight at 30 °C. The molecular weight of the purified peptide after reverse-phase chromatography was further confirmed with matrix-assisted laser desorption ionization (MALDI)-TOF/MS , as described35. Briefly, 0.5 μL of the sample was mixed with 0.5 μL of the matrix mixed with 15 mg/mL alpha-cyano-4-hydroxycinnamic acid and deposited on a ground steel MALDI plate . The mass window was adjusted between 4000 and 10,000 Da in the linear positive ion mode, with an acceleration voltage of 25 kV. A peptide mass similarity search was performed using ExPASy with the TagIdent tool .After cation exchange chromatography, the partially purified BLIS was analyzed by one-dimensional SDS-PAGE  using a 4% (v/v) stacking gel and 15% (v/v) separating gel. A pre-stained molecular mass protein marker with a range of 7000–70,000 Da was used. The BLIS was separated at 180 V for 45 min, and the gel was divided into two identical vertical parts, one of which was stained and visualized using silver nitrate. The other part was used to identify antibacterial activity by first washing the gel three times with distilled water (40 min/wash) to remove excess SDS. Then, the gel was transferred to an MRS agar plate and overlaid with 10 mL of soft MRS agar containing the indicator microorganism  at a final concentration of 1 mg/mL was determined for both the crude and purified BLIS by incubating samples (pH 7.5) with or without enzyme at 37 °C for 60 min. Then, the activity of each sample was compared with the untreated sample using a microtiter plate assay with the indicator strain l-pyroglutamic acid beta-naphthylamide hydrolysis on PYR discs (results not shown). They were identified at the species level as E. faecalis using an API 50 CHL carbohydrate fermentation kit and were positive for glycerol, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetyl glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, saccharose, trehalose, melezitose, and tagatose. Furthermore, 16S rDNA sequence analysis confirmed their identity as E. faecalis. The obtained sequences of the bacteriocinogenic isolates were submitted to the NCBI GenBank database under specific accession numbers  that were collected from various food and healthcare units were positive for esculin hydrolysis on bile esculin agar and for rs Table . The parE. faecalis isolates were screened by PCR for genes corresponding to any of 17 known bacteriocin genes, none of these genes were found in 11 of the isolates . Of them, the food-isolated E. faecalis OS13 produced the most potent antimicrobial activity and was chosen for further study and purification of its biologically active antimicrobial substance. Some Gram-positive bacteria, such as L. sakei, L. plantarum, E. faecalis, and E. faecium were sensitive to the antimicrobial activity of the OS13 isolate. Still, other Gram-positive bacteria, such as Listeria, Bacillus, and Staphylococcus, and all tested Gram-negative bacteria were resistant. The most potent antimicrobial activity was shown against L. sakei LMG 2313, which was then used as the primary indicator microorganism during purification.Interestingly, when the putative bacteriocinogenic 31 revealed that these isolates belonged to ST 6 and ST116  6 or CC9, which are commonly spread in the hospital environment  and 40.5% (v/v) isopropanol, respectively . Also, the antimicrobial activity was decreased to 1280 AU/mL (12.5%) after proteinase K treatment. Repeating the same treatment with the concentrated purified enterocin OS13α with initial activity  and enterocin OS13β with initial activity  confirmed the sensitivity of both peptides to heat treatment, and their activities were entirely abolished. Proteinase K treatment reduced the activity of enterocin OS13α to 512,000 AU/mL (50%), but enterocin OS13β remained unaffected with an activity of 128,000 AU/mL.Enterococcus faecalis are widely spread in the environment and are commonly isolated from foodstuffs and clinical samples37. E. faecalis and other LAB have attracted more attention over the last few years due to the production of many useful AMPs or BLIS, with several safe applications in the food industry and medical treatments39. The identification of antimicrobial agents to treat antibiotic-resistant nosocomial enterococci is crucial because the treatment of such infections is becoming increasingly problematic. In addition, enterococci gained greater medical and public attention since the mid-1980s when vancomycin resistance was first present in clinical isolates (VRE)40. BLIS with the potential to inhibit the growth of such bacteria could remedy infections without affecting endogenous beneficial microbiota or risking dissemination of R-factors to non-enterococcal pathogens .E. faecalis isolates from Egypt of food and clinical origin. The 11 unknown putative bacteriocinogenic E. faecalis isolates were characterized epidemiologically by MLST and tested for antibiotic resistance, virulence traits, and antimicrobial activities. Our study showed that, E. faecalis OS13 produced highly active (> 106 AU/mL) BLIS designated enterocin OS13α and OS13β that had antimicrobial activity against some Gram-positive bacteria, such as L. sakei, L. plantarum, E. faecalis, and E. faecium, but not against Gram-negative bacteria. Generally, the bacteriocins produced from Gram-positive bacteria are active against closely related species and not directly active against Gram-negative bacteria. However, some other bacteriocins from LAB showed antagonistic activities against both Gram-positive and Gram-negative bacteria, like enterocin E76041, enterocin E50-5242, and salivaricin SMXD5143. Owing to the narrow-spectrum capabilities of enterocin OS13α and OS13β against target pathogens, they are favored when compared with conventional broad-spectrum antibiotics that can interfere with the host microbiota, affecting their crucial beneficial roles22. Especially during infancy and early childhood, exposure to broad-spectrum antibiotics is often unfavorable, as the early microbiota lacks diversity and stability, rendering it distinctly vulnerable to disturbance, in addition to the importance of intestinal microbiota in early development and education of the immune system23.Our study searched for suitable candidate BLIS in 65 Enterococcus faecalis V583, which was the first VRE Enterococcus isolated in the United States, and it was also resistant to high levels of aminoglycosides, macrolides, lincosamides, and streptogramin B44. Therefore, BLIS could represent a non-conventional alternative of interest to combat pathogens like enterococci, recognizing that the presence of natural resistance prevents a variety of modern drugs widely used, such as cephalosporins and quinolones, from working effectively against enterococcal infections45. Most VREs are also resistant to antibiotics as ampicillin, vancomycin, and aminoglycosides, widely used in the hospital environment. Therefore, only a few therapeutic options are commercially available to treat multidrug-resistant VRE infections, including linezolid, tedizolid, tigecycline, and daptomycin47. The increased use of these last-source antibiotics and the ability of enterococci to readily acquire new resistance determinants are currently raising the need to find alternative therapeutics and explore the effectiveness of natural antimicrobials such as bacteriocins.Noticeably, bacteriocins from isolate OS13 were active against multi-resistant nosocomial enterococci as 51. At each purification level, the antimicrobial activity decreased, especially after reverse-phase chromatography, which was observed in other studies52. SDS-PAGE did not accurately determine the molecular weight of both BLIS and was unable to differentiate between them54.In this study, purification of two BLIS, OS13α and OS13β, from strain OS13 and based on their chromatographic properties exposed that both BLIS were cationic and hydrophobic, which is in agreement with most bacteriocins purified from LAB56. The larger size of enterocin OS13α and OS13β being 8079.1 Da and 7858.6 Da, respectively, could interpret the reasons behind their heat sensitivity58. Although the crude BLIS with an initial activity of 10,240 AU/mL was decreased to be 1,280 AU/mL (12.5%) after proteinase K treatment but the purified enterocin OS13α was sensitive to proteinase K (50% reduction in its activity), which strongly suggests its proteinaceous nature59. Also, enterocin OS13β was resistant to proteinase K, and this is probably not due to the absence of digestion sites but more likely due to a tightly folded structure, making it inaccessible to enzymatic digestion61. The variation in sensitivity of crude BLIS compared to purified ones to proteinase K could indicate that the OS13 isolate might also produce other BLIS besides OS13α and OS13β that is/are more sensitive to proteinase K but was/were not isolated and purified.Although both enterocin OS13α and OS13β were heat-labile, and their activity was wholly abolished after heating at 100 ºC for 30 min, their activity together as a crude BLIS in the supernatant was decreased to 6.25% following heat treatment. There are previous reports of many BLIS becoming less heat-stable during subsequent purification stepsEnterococcus spp. found in food are presumed less virulent than clinical isolates63. Regarding the virulence traits of strain OS13, neither hemolytic activity nor bile salt hydrolase activity was demonstrated, while gelatinase activity was observed. Cytolysin  is a lantibiotic, but it differs from most lantibiotics because it is both antibacterial and hemolytic64. A higher incidence of cytolysin genes has been shown in clinical isolates (33%) compared with food isolates (6%)65. In our study, PCR analysis was used to confirm the absence of the cytolysin gene cluster (cyl). Although E. faecalis can be an opportunistic pathogen that possesses bile salt hydrolase activity66, such activity might be a desirable trait for a probiotic bacterium since it could maximize its ability to survive in the hostile environment of the gastrointestinal tract67. The gelatinase enzyme is involved in the virulence of Enterococcus sp. and hydrolyzes gelatin, casein, hemoglobin, and other bioactive peptides65. The primary role of gelatinase in enterococcal pathogenesis is to provide nutrients to the bacteria by degrading host tissue. However, it also appears to play a role in biofilm formation68.Generally, Enterococcus sp. behave as opportunistic pathogens in nosocomial infections and spread antibiotic resistance through the food chain with potential risks to humans69. In biosafety evaluations of enterococci, their resistance to glycopeptides, such as vancomycin, should be considered70. In our study, the pathogenic potential of the food isolated E. faecalis OS13, and the spectrum of resistance to various antibiotics was determined. OS13 was sensitive to ampicillin, penicillin G, vancomycin, erythromycin, kanamycin, and gentamicin. It was resistant to chloramphenicol, streptomycin, tetracycline, clindamycin, fusidic acid, and polymyxin B. These results are in agreement with previous publications72, where all tested E. faecalis strains were sensitive to ampicillin, penicillin, and vancomycin. Resistance to chloramphenicol, tetracycline, erythromycin, gentamicin, and kanamycin is becoming common among enterococci from different sources due to the abuse of antibiotics in clinics, animal and fish farming, and agriculture. Thus, enterococcal antibiotic resistance is not exclusive to the clinical field and is also prevalent in the food industry71.The food chain is considered the primary transmission route of antibiotic-resistant bacteria between populations. 73. MLST analysis of E. faecalis OS13 revealed that it belongs to ST116 and CC116. E. faecalis with ST116 has been reported as isolated twice from human clinical samples; vancomycin-resistant E. faecalis from a blood sample in Denmark and E. faecalis DQ287 from a catheter in Cuba75. To our knowledge, ours is the first report of the isolation of E. faecalis with ST116 from non-human samples.Although MLST is a tool for global epidemiological studies, the possible role of plasmids in transferring virulence between different strains should be considered76.The ability of CC116 to spread in hospitals and cause infection is unknown. However the most common clonal complexes that spread in the hospital environment are CC2, CC6, and CC9E. faecalis OS13 produced BLIS named enterocin OS13α (8079 Da) and OS13β (7859 Da), with activities against VRE and MDRE. MLST of E. faecalis OS13 revealed its belongings to ST116, being the first from non-human samples. E. faecalis OS13 was non-hemolytic, bile salt hydrolase-negative, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin, suggesting several safe applications in the food industry and medical treatments. Although our BLIS showed sensitivity to heat treatments, they could be used for preservation of raw or minimally processed foods stored at low temperatures besides their likely role in the treatment of clinical infections with VRE and MDRE. Future works are still required to figure out the amino acid sequences, structures, encoding genes, and mode of actions of such BLIS.The food-isolated Supplementary Information.