New strain Brevibacillus laterosporus TSA31-5 produces both brevicidine and brevibacillin, exhibiting distinct antibacterial modes of action against Gram-negative and Gram-positive bacteria.

The growing prevalence of antibiotic resistance has made it imperative to search for new antimicrobial compounds derived from natural products. In the present study, Brevibacillus laterosporus TSA31-5, isolated from red clay soil, was chosen as the subject for conducting additional antibacterial investigations. The fractions exhibiting the highest antibacterial activity (30% acetonitrile eluent from solid phase extraction) were purified through RP-HPLC. Notably, two compounds (A and B) displayed the most potent antibacterial activity against both Escherichia coli and Staphylococcus aureus. ESI-MS/MS spectroscopy and NMR analysis confirmed that compound A corresponds to brevicidine and compound B to brevibacillin. Particularly, brevicidine displayed notable antibacterial activity against Gram-negative bacteria, with a minimum inhibitory concentration (MIC) range of 1-8 µg/mL. On the other hand, brevibacillin exhibited robust antimicrobial effectiveness against both Gram-positive bacterial strains (MIC range of 2-4 µg/mL) and Gram-negative bacteria (MIC range of 4-64 µg/mL). Scanning electron microscopy analysis and fluorescence assays uncovered distinctive morphological alterations in bacterial cell membranes induced by brevicidine and brevibacillin. These observations imply distinct mechanisms of antibacterial activity exhibited by the peptides. Brevicidine exhibited no hemolysis or cytotoxicity up to 512 µg/mL, comparable to the negative control. This suggests its promising therapeutic potential in treating infectious diseases. Conversely, brevibacillin demonstrated elevated cytotoxicity in in vitro assays. Nonetheless, owing to its noteworthy antimicrobial activity against pathogenic bacteria, brevibacillin could still be explored as a promising antimicrobial agent.

Stereospecific NANOG PEST Stabilization by Pin1.

NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.

A qualitative exploration of exercise motivation among colorectal cancer survivors: an application of the theory of planned behavior.

PURPOSE: The purpose of this qualitative study was to use semi-structured interviews and thematic analysis to elicit key influencing factors (i.e., behavioral, normative, and control beliefs) related to physical activity and exercise in colorectal cancer survivors. METHODS: Colorectal cancer survivors (N = 17) were recruited from exercise programs designed for colorectal cancer survivors at the Yonsei Cancer Center, Seoul, South Korea. A purposive sampling method was used. Interview questions were informed by the theory of planned behavior (TPB). Semi-structured face-to-face interviews were conducted, and open-ended questions addressed the research question. Interviews were transcribed verbatim and analyzed using thematic analysis. RESULTS: Participants were on average 2.2 years post-treatment. The mean age of the sample was 55.9 years. Key behavioral, normative, and control beliefs emerged in the data. For behavioral beliefs, colorectal cancer survivors believed that exercise would result in physical and psychological improvements, and improve their bowel problems. For normative beliefs, most colorectal cancer survivors wanted their oncologists' approval for participation of exercise. Family members, more specifically the spouse, were also influencing factors for colorectal cancer survivors adopting physical activity. The most frequently mentioned control belief was that supervised exercise with an exercise specialist made exercise participation easier. CONCLUSIONS AND IMPLICATIONS: Beliefs identified in this study can inform TPB-based physical activity interventions tailored for colorectal cancer survivors. While information alone may not lead to behavior change, integrating these beliefs with other influential factors can potentially enhance intervention efficacy and promote physical activity in this population.

Multifunctional Properties of BMAP-18 and Its Aliphatic Analog against Drug-Resistant Bacteria.

BMAP-18, derived from the N-terminal region of bovine myeloid antimicrobial peptide BMAP-27, demonstrates potent antimicrobial activity without cytotoxicity. This study aimed to compare the antibacterial, antibiofilm, and anti-inflammatory properties of BMAP-18, rich in aromatic phenylalanine residues, with its aliphatic analog, BMAP-18-FL. Both aromatic BMAP-18 and aliphatic BMAP-18-FL exhibited equally potent antimicrobial activities against Gram-positive and Gram-negative bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Mechanistic investigations employing SYTOX green uptake, DNA binding, and FACScan analysis revealed that both peptides acted by inducing membrane permeabilization and subsequent intracellular targeting. Moreover, both BMAP-18 and BMAP-18-FL effectively prevented biofilm formation and eradicated existing biofilms of MRSA and MDRPA. Notably, BMAP-18-FL displayed a superior anti-inflammatory activity compared to BMAP-18, significantly reducing the expression levels of pro-inflammatory cytokines in lipopolysaccharide-stimulated macrophages. This study emphasizes the similarities and differences in the antimicrobial, antibiofilm, and anti-inflammatory properties between aromatic BMAP-18 and aliphatic BMAP-18-FL, providing valuable insights for the development of multifunctional antimicrobial peptides against drug-resistant bacteria.

Fusarium solani infection disrupts metabolism during the germination of roselle (Hibiscus sabdariffa L.) seeds.

Fungal infections adversely influence the production and quality of seeds. Previously, Fusarium solani was reported as the causal agent of roselle (Hibiscus sabdariffa L.) seed rot. This study was designed to evaluate the effect of F. solani infection on the germination, biochemical composition, energy reserves, and antioxidant activity of roselle seeds because there is currently a lack of information on the relationship between seed metabolism and infection with F. solani. The results showed that roselle seeds infected with F. solani exhibited a ca. 55% reduction in overall germination. Additionally, the fungal infection decreased antioxidant activity, total phenolic content, protein, sugar (sucrose, fructose, and glucose), and some amino acid (glutamine, serine, and arginine) contents. In contrast, some metabolites were more abundant in infected seeds, including alanine (2.1-fold) and some fatty acids (palmitic acid and heptadecanoic acid by 1.1- and 1.4-fold, respectively). The infection-associated changes in fatty acid profile resulted in the ratio of unsaturated/saturated fatty acids being 2.1-fold higher in infected seeds. Therefore, our results reveal that F. solani infection remarkably altered the biochemical composition of roselle seeds, which may have contributed to the loss of germination and quality of roselle seeds.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Cell selectivity and antibiofilm and anti-inflammatory activities and antibacterial mechanism of symmetric-end antimicrobial peptide centered on D-Pro-Pro.

This study aimed to develop a new symmetric-end antimicrobial peptide (AMP) with cell selectivity, antibiofilm, and anti-inflammatory activities. Two symmetric-end AMPs, Lf6-pP and Lf6-GG, were designed based on the sequence RRWQWRzzRWQWRR, which contains two symmetric repeat sequences connected by a ß-turn-promoting sequence (zz) that can be a rigid turn by D-Pro-Pro (pP) or a flexible turn by Gly-Gly (GG). Both Lf6-pP and Lf6-GG exhibited potent antibacterial activity without causing hemolysis, but Lf6-pP exhibited better cell selectivity, likely due to the more significant impact of the rigid pP turn. Compared to Lf6-GG, Lf6-pP demonstrated approximately three times higher antimicrobial activity against drug-resistant bacteria, had a low incidence of drug resistance, and maintained its activity in the presence of physiological salts and human serum. Additionally, Lf6-pP was more effective than Lf6-GG in inhibiting biofilm formation and eradicating mature biofilms. The BODIPY-cadaverine assay indicated that the potent anti-inflammatory activity of Lf6-pP may be attributed to its direct interaction with LPS, resulting in decreased TNF-α and IL-6 levels in LPS-stimulated macrophages. Mechanistic studies, including membrane depolarization, outer/inner membrane permeation, and membrane integrity change, demonstrated that Lf6-pP exerts its antibacterial action through an intracellular-target mechanism. Overall, we propose that Lf6-pP has potential as a novel antibacterial, antibiofilm, and anti-inflammatory agent against drug-resistant bacterial infections.

Discovery of structural and functional transition sites for membrane-penetrating activity of sheep myeloid antimicrobial peptide-18.

Cathelicidin antimicrobial peptides have an extended and/or unstructured conformation in aqueous solutions but fold into ordered conformations, such as the α-helical structure, when interacting with cellular membranes. These structural transitions can be directly correlated to their antimicrobial activity and its underlying mechanisms. SMAP-18, the N-terminal segment (residues 1-18) of sheep cathelicidin (SMAP-29), is known to kill microorganisms by translocating across membranes and interacting with their nucleic acids. The amino acid sequence of SMAP-18 contains three Gly residues (at positions 2, 7, and 13) that significantly affect the flexibility of its peptide structure. This study investigated the role of Gly residues in the structure, membrane interaction, membrane translocation, and antimicrobial mechanisms of SMAP-18. Five analogs were designed and synthesized through Gly → Ala substitution (i.e., G2A, G7A, G13A, G7,13A, and G2,7,13A); these substitutions altered the helical content of SMAP-18 peptides. We found that G7,13A and G2,7,13A changed their mode of action, with circular dichroism and nuclear magnetic resonance studies revealing that these analogs changed the structure of SMAP-18 from a random coil to an α-helical structure. The results of this experiment suggest that the Gly residues at positions 7 and 13 in SMAP-18 are the structural and functional determinants that control its three-dimensional structure, strain-specific activity, and antimicrobial mechanism of action. These results provide valuable information for the design of novel peptide-based antibiotics.

The long multi-epitope peptide vaccine combined with adjuvants improved the therapeutic effects in a glioblastoma mouse model.

Emerging data have suggested that single short peptides have limited success as a cancer vaccine; however, extending the short peptides into longer multi-epitope peptides overcame the immune tolerance and induced an immune response. Moreover, the combination of adjuvants such as lenalidomide and anti-programmed cell death protein 1 (PD1) with a peptide vaccine showed potential vaccine effects in previous studies. Therefore, the effects of a long multi-epitope peptide vaccine in combination with lenalidomide and anti-PD1 were analyzed in this study. Long multi-epitope peptides from two MHCI peptides (BIRC597-104 and EphA2682-689) and the pan-human leukocyte antigen-DR isotype (HLA-DR) binding epitope (PADRE) were synthesized. The therapeutic effects of long multi-epitope peptides in combination with lenalidomide and anti-PD1 were confirmed in the murine GL261 intracranial glioma model. Immune cells' distribution and responses to the long multi-epitope peptides in combination with these adjuvants were also estimated in the spleens, lymph nodes, and tumor tissues. The difference between long multi-epitope peptides and a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 was also clarified. As a result, long multi-epitope peptides combined with lenalidomide and anti-PD1 prolonged the survival of mice according to the suppression of tumor growth in an intracranial mouse model. While long multi-epitope peptides combined with these adjuvants enhanced the percentages of activated and memory effector CD8+ T cells, the increase in percentages of regulatory T cells (Tregs) was observed in a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 group in the tumors. Long multi-epitope peptides combined with these adjuvants also enhanced the function of immune cells according to the enhanced pro-inflammatory cytokines and cytotoxicity against GL261 cells in ex vivo. In conclusion, long multi-epitope peptides composed of MHCI peptides, BIRC5 and EphA2, and the MHCII peptide, PADRE, in combination with lenalidomide and anti-PD1 has the potential to improve the therapeutic effects of a vaccine against GBM.

A novel hybrid peptide composed of LfcinB6 and KR-12-a4 with enhanced antimicrobial, anti-inflammatory and anti-biofilm activities.

Hybridizing two known antimicrobial peptides (AMPs) is a simple and effective strategy for designing antimicrobial agents with enhanced cell selectivity against bacterial cells. Here, we generated a hybrid peptide Lf-KR in which LfcinB6 and KR-12-a4 were linked with a Pro hinge to obtain a novel AMP with potent antimicrobial, anti-inflammatory, and anti-biofilm activities. Lf-KR exerted superior cell selectivity for bacterial cells over sheep red blood cells. Lf-KR showed broad-spectrum antimicrobial activities (MIC: 4-8 µM) against tested 12 bacterial strains and retained its antimicrobial activity in the presence of salts at physiological concentrations. Membrane depolarization and dye leakage assays showed that the enhanced antimicrobial activity of Lf-KR was due to increased permeabilization and depolarization of microbial membranes. Lf-KR significantly inhibited the expression and production of pro-inflammatory cytokines (nitric oxide and tumor necrosis factor-α) in LPS-stimulated mouse macrophage RAW264.7 cells. In addition, Lf-KR showed a powerful eradication effect on preformed multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms. We confirmed using confocal laser scanning microscopy that a large portion of the preformed MDRPA biofilm structure was perturbed by the addition of Lf-KR. Collectively, our results suggest that Lf-KR can be an antimicrobial, anti-inflammatory, and anti-biofilm candidate as a pharmaceutical agent.

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability.

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 µM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 - Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.

Natural killer cell therapy potentially enhances the antitumor effects of bevacizumab plus irinotecan in a glioblastoma mouse model.

Various combination treatments have been considered to attain the effective therapy threshold by combining independent antitumor mechanisms against the heterogeneous characteristics of tumor cells in malignant brain tumors. In this study, the natural killer (NK) cells associated with bevacizumab (Bev) plus irinotecan (Iri) against glioblastoma multiforme (GBM) were investigated. For the experimental design, NK cells were expanded and activated by K562 cells expressing the OX40 ligand and membrane-bound IL-18 and IL-21. The effects of Bev and Iri on the proliferation and NK ligand expression of GBM cells were evaluated through MTT assay and flow cytometry. The cytotoxic effects of NK cells against Bev plus Iri-treated GBM cells were also predicted via the LDH assay in vitro. The therapeutic effect of different injected NK cell routes and numbers combined with the different doses of Bev and Iri was confirmed according to tumor size and survival in the subcutaneous (s.c) and intracranial (i.c) U87 xenograft NOD/SCID IL-12Rγnull mouse model. The presence of injected-NK cells in tumors was detected using flow cytometry and immunohistochemistry ex vivo. As a result, Iri was found to affect the proliferation and NK ligand expression of GBM cells, while Bev did not cause differences in these cellular processes. However, the administration of Bev modulated Iri efficacy in the i.c U87 mouse model. NK cells significantly enhanced the cytotoxic effects against Bev plus Iri-treated GBM cells in vitro. Although the intravenous (IV) injection of NK cells in combination with Bev plus Iri significantly reduced the tumor volume in the s.c U87 mouse model, only the direct intratumorally (IT) injection of NK cells in combination with Bev plus Iri elicited delayed tumor growth in the i.c U87 mouse model. Tumor-infiltrating NK cells were detected after IV injection of NK cells in both s.c and i.c U87 mouse models. In conclusion, the potential therapeutic effect of NK cells combined with Bev plus Iri against GBM cells was limited in this study. Accordingly, further research is required to improve the accessibility and strength of NK cell function in this combination treatment.

Structure-function relationship of fermented skate skin gelatin-derived bioactive peptides: a peptidomics approach.

In this study, we investigated the multi-functionality of bioactive peptides derived from fermented skate (Raja kenojei) skin gelatin hydrolysates. The extracted gelatin was hydrolyzed using a combination of food grade subtilisin and actinidin. The hydrolysates were then fractionated via ultrafiltration, and the fractions with the highest dipeptidyl peptidase-IV (DPP-IV) inhibitory, angiotensin-converting enzyme (ACE) inhibitory, and antibacterial proprieties were further purified via ion exchange, solid phase extraction, and reverse phase high performance liquid chromatography. Analysis of the obtained extract revealed a direct relationship between hydrolysis time, degree of hydrolysis, and biological activities. The peptides GRPGNRGE (P1) and AKDYEVDAT (P2), with a molecular weight of 841.42 and 1010.46 Da, respectively, were identified through tandem mass spectrometry. P1 had a lower ACE and DPP-IV inhibitory activity, with a half maximal inhibitory concentration [IC50] of 0.74 and 0.69 mg.mL-1, respectively, than P2 (0.52 and 0.58 mg.mL-1, respectively). Antibacterial analysis showed similar results, with a minimum inhibitory concentration of 0.52 and 0.46 mg.mL-1 against Staphylococcus aureus (highest activity) and 1.75 and 1.44 mg.mL-1 against Klebsiella pneumonia (lowest activity) for P1 and P2, respectively. Overall, this study revealed two fish gelatin-derived multifunctional peptides, exhibiting ACE inhibitory, DPP-IV inhibitory, and antibacterial activities, as natural nutraceuticals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00998-6.

Solitary Nitric Oxide Signaling Mediates Mild Stress-Induced Anxiety and Norepinephrine Release in the Bed Nucleus of the Stria Terminalis during Protracted Ethanol Withdrawal.

Ethanol withdrawal (EtOHW) alters the pattern of neurohormonal and behavioral response toward internal and external stimuli, which mediates relapse to alcohol use even after a long period of abstinence. Increased noradrenergic signaling from the nucleus tractus solitarius (NTS) to the bed nucleus of the stria terminalis (BNST) during EtOHW underlies withdrawal-induced anxiety, while nitric oxide synthase (NOS) inhibitors injected into the periaqueductal area attenuate EtOHW-induced anxiety. Therefore, this study investigated the involvement of NOS within the NTS in anxiety and increased norepinephrine (NE) release in the BNST during protracted EtOHW in rats exposed to a mild stress. Rats were intraperitoneally administered 3 g/kg/day EtOH for 21 days followed by 28 days of withdrawal, and on the 28th day of withdrawal, the rats were subjected to restraint stress for 7 minutes. The elevated plus maze test was employed to evaluate anxiety-like behavior in rats, and in vivo microdialysis was used to measure the extracellular NE level in the BNST. In elevated plus maze tests, EtOHW rats but not EtOH-naive rats exhibited anxiety-like behavior when challenged with 7-minute mild restraint stress, which was, respectively, mitigated by prior intra-NTS infusion of the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), or selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI). Each of these agents also decreased the plasma corticosterone levels in EtOHW rats. In in vivo microdialysis, prior intra-NTS infusion of carboxy-PTIO, L-NAME, or 7-NI attenuated the mild stress-induced NE release in the BNST of EtOHW rats. Additionally, EtOHW rats showed increased solitary nNOS gene and protein expression. Moreover, the anxiolytic effect of intra-NTS administration of 7-NI was abolished by subsequent intra-NTS administration of sodium nitroprusside. These results suggest that elevation of solitary nitric oxide signaling derived from nNOS mediates stress-precipitated anxiety and norepinephrine release in the BNST during protracted EtOHW.

Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.).

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10-37°C (optimally 25-30°C), at pH 7.0-8.0, and in NaCl 0-1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA-DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)-visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms.

Plant bacterial and fungal diseases cause significant agricultural losses and need to be controlled. Beneficial bacteria are promising candidates for controlling these diseases. In this study, Streptomyces sp. JCK-6131 exhibited broad-spectrum antagonistic activity against various phytopathogenic bacteria and fungi. In vitro assays showed that the fermentation filtrate of JCK-6131 inhibited the growth of bacteria and fungi with minimum concentration inhibitory (MIC) values of 0.31-10% and 0.31-1.25%, respectively. In the in vivo experiments, treatment with JCK-6131 effectively suppressed the development of apple fire blight, tomato bacterial wilt, and cucumber Fusarium wilt in a dose-dependent manner. RP-HPLC and ESI-MS/MS analyses indicated that JCK-6131 can produce several antimicrobial compounds, three of which were identified as streptothricin E acid, streptothricin D, and 12-carbamoyl streptothricin D. In addition, the disease control efficacy of the foliar application of JCK-6131 against tomato bacterial wilt was similar to that of the soil drench application, indicating that JCK-6131 could enhance defense resistance in plants. Molecular studies on tomato plants showed that JCK-6131 treatment induced the expression of the pathogenesis-related (PR) genes PR1, PR3, PR5, and PR12, suggesting the simultaneous activation of the salicylate (SA) and jasmonate (JA) signaling pathways. The transcription levels of PR genes increased earlier and were higher in treated plants than in untreated plants following Ralstonia solanacearum infection. These results indicate that Streptomyces sp. JCK-6131 can effectively control various plant bacterial and fungal diseases via two distinct mechanisms of antibiosis and induced resistance.

A native conjugative plasmid confers potential selective advantages to plant growth-promoting Bacillus velezensis strain GH1-13.

The conjugative plasmid (pBV71) possibly confers a selective advantage to Bacillus velezensis strain GH1-13, although a selective marker gene is yet to be identified. Here we show that few non-mucoid wild-type GH1-13 cells are spontaneously converted to mucoid variants with or without the loss of pBV71. Mucoid phenotypes, which contain or lack the plasmid, become sensitive to bacitracin, gramicidin, selenite, and tellurite. Using the differences in antibiotic resistance and phenotype, we isolated a reverse complement (COM) and a transconjugant of strain FZB42 with the native pBV71. Transformed COM and FZB42p cells were similar to the wild-type strain GH1-13 with high antibiotic resistance and slow growth rates on lactose compared to those of mucoid phenotypes. RT-PCR analysis revealed that the expression of plasmid-encoded orphan aspartate phosphatase (pRapD) was coordinated with a new quorum-sensing (QS) cassette of RapF2-PhrF2 present in the chromosome of strain GH1-13, but not in strain FZB42. Multi-omics analysis on wild-type and plasmid-cured cells of strain GH1-13 suggested that the conjugative plasmid expression has a crucial role in induction of early envelope stress response that promotes cell morphogenesis, biofilm formation, catabolite repression, and biosynthesis of extracellular-matrix components and antibiotics for protection of host cell during exponential phase.

Sauchinone Blocks Ethanol Withdrawal-Induced Anxiety but Spares Locomotor Sensitization: Involvement of Nitric Oxide in the Bed Nucleus of the Stria Terminalis.

Both the positive (manifested by locomotor sensitization) and negative (withdrawal symptoms) reinforcing effects of ethanol (EtOH) involve central nitric oxide (NO) signaling. Sauchinone (a bioactive lignan in Saururus chinensis) has been shown to improve methamphetamine-induced behavioral and neurochemical changes via the NO signaling pathway. Thus, this study evaluated the effects of sauchinone on locomotor sensitization and anxiety during EtOH withdrawal (EtOHW). Male adult Sprague-Dawley rats were treated with 1.5 g/kg/day of EtOH (20%, vol/vol) via intraperitoneal injection for 28 days, followed by a 3-day withdrawal. During withdrawal, the rats were given intragastric sauchinone (2.5, 7.5, or 25 mg/kg/day) once a day. EtOH locomotor sensitization was determined by challenging EtOHW rats with 0.75 g/kg EtOH, while EtOHW-induced anxiety was assessed using the elevated plus maze (EPM). None of the three doses of sauchinone affected EtOH locomotor sensitization. However, in the EPM, treatment of EtOHW rats with sauchinone at 7.5 or 25 mg/kg/day increased both the number of entries into and the time spent in the open arms. Moreover, the two doses of sauchinone inhibited the oversecretion of plasma corticosterone during EtOHW. In the bed nucleus of the stria terminalis (BNST), EtOHW increased NO production, enhanced gene and protein expression of both inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS), and also elevated protein levels of corticotropin-releasing factor, which were all inhibited by 25 mg/kg/day sauchinone. In an in vitro experiment, sauchinone (3, 10, and 30 µM) inhibited H2O2-stimulated nNOS protein expression in neuronal PC12 cells. Finally, intra-BNST infusion of sodium nitroprusside, a NO donor, after sauchinone (25 mg/kg/day) administration, abolished its expected anxiolytic effect. Taken together, these results indicate that sauchinone attenuates anxiety-like behavior in rats during EtOHW but spares EtOH locomotor sensitization, and the anxiolytic effect is mediated via the NO signaling pathway in the BNST.

Structure-function engineering of novel fish gelatin-derived multifunctional peptides using high-resolution peptidomics and bioinformatics.

The multifunctional properties of fish gelatin hydrolysates have not been completely elucidated. Here, the biological characterization of these peptides was performed to engineer multifunctional peptides. Bioactive peptides were produced from mackerel byproducts via successive enzymatic hydrolysis reactions using subtilisin A and actinidin as microbial and herbal proteases. The antibacterial activity against both gram-negative and -positive food-borne pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae, as well as the inhibitory potential of angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), was accessed in vitro. The synthesized peptides demonstrated multifunctional properties, which were further confirmed by in silico protocols. The ACE and DPP-IV inhibitory (IC50) values of P1, P2, and P3 were 0.92 and 0.87, 0.51 and 0.93, 0.78 and 1.16 mg mL-1, respectively. Moreover, the binding energy was sufficient for all three peptides to inhibit both ACE and DPP-IV enzymes with excellent three-dimensional conformation (RMSD = 0.000) for all six docking mechanisms.

Inhibitory Effects of Porphyra tenera Extract on Oxidation and Inflammatory Responses.

Porphyra tenera (laver) has long been a popular and traditional seaweed food in Korea, Japan, and China. Historically, it was known as a marine medicinal herb to treat hemorrhoids and cholera morbus in Donguibogam. We investigated the effects of P. tenera extract (PTE) for its antioxidant and anti-inflammatory activities. These activities were measured using assays for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging and its superoxide dismutase- (SOD-) like activity, and through the inhibitory production of inflammatory mediators (prostaglandin E2 (PGE2), NO, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)) in lipopolysaccharide- (LPS-) stimulated Raw 264.7 cells. The antioxidant assay results showed that PTE displayed DPPH radical scavenging activity (46.44%), NO radical scavenging activity (67.14%), and SOD-like activity (80.29%) at a concentration of 5 mg/mL. In the anti-inflammatory assays, treatment with PTE (1 mg/mL) significantly inhibited expression levels of LPS-induced COX-2 and iNOS, as well as the production of PGE2, NO, TNF-α, and IL-6. These results show that PTE has antioxidant and anti-inflammatory properties and provide scientific evidence to explain the antioxidative and anti-inflammatory properties of PTE.