The structures of these new derivatives were established by HRESIMS and NMR techniques. All the compounds (1–71) were evaluated for their antialgal and antiplasmodial activities. Among them, 14 compounds displayed antifouling activities against adhesion of the fouling diatoms. In particular, 9 and 34 exhibited strong and selective inhibitory effects against the diatoms Navicula laevissima and Navicula exigua (EC50 = 6.67 and 8.55 μmol/L), respectively, which were similar in efficacy to those of the positive control SeaNine 211 (EC50 = 2.90 and 9.74 μmol/L). More importantly, 38, 39, and 69–71 showed potent antiplasmodial activities against Plasmodium falciparum with IC50 values ranging from 3.54 to 9.72 μmol/L. Very interestingly, the five antiplasmodial derivatives displayed non-toxicity in the cytotoxicity assays and the zebrafish embryos model, thus, representing potential promising antiplasmodial drug agents. The preliminary structure–activity relationships indicated that biphenyl substituent at C-2, acetonide at positions C-5′ and C-6′, and tri- or tetra-substituted of acyl groups increased the antiplasmodial activity. Therefore, combining evaluation of chemical ecology with pharmacological models will be implemented as a systematic strategy, not only for environmentally friendly antifoulants but also for structurally novel drugs.Marine natural products play critical roles in the chemical defense of many marine organisms and are essential, reputable sources of successful drug leads. Sixty-seven 14-membered resorcylic acid lactone derivatives The online version contains supplementary material available at 10.1007/s42995-021-00103-0. Marine natural products play critical roles in the chemical defense of many marine organisms and in some cases can influence the community structure of entire ecosystems , and NO/cGMP pathways, among which the NO/cGMP pathway was considered to play a key role in barnacle larval settlement . To further investigate antifouling activity and antiplasmodial activity of the OH substituent groups of RALs, synthesis of the etherification products of the zeaenol were encouraging. The structure–activity relationships analysis indicated that the acetoxy and acetonide groups at C5′–C6′ had positive effects on the antiplasmodial activity  are polyketide natural products with a 14-membered macrocyclic ring fused to a resorcylic acid residue (Jana and Nanda 3–27 and 30–71 of the marine-derived zeaenol (1) were semisynthesized. The antifouling activity against adhesion of the fouling diatoms Phaeodactylum tricornutum Bohlin, Chlorella vulgaris, Chaetoceros socialis, Navicula laevissima, and Navicula exigua, and the antiplasmodial activities against P. falciparum of all the compounds (1–71) were evaluated. The preliminary structure–activity relationships of antiplasmodial activity are also discussed.As part of our ongoing investigation of new antialgal and antiplasmodial agents, a series of new 14-membered RAL derivatives Cochliobolus lunatus (TA26-46) was cultured in a 500-mL flask containing 200 mL of liquid medium . The fungus was fermented at 28 °C for 9 days on a rotary shaker at 120 r/min, and the fermented liquid medium was extracted with an equal volume of EtOAc. The organic extract (23.2 g) was subjected to silica gel column chromatography (CC), Sephadex LH-20 CC, and then recrystallization to obtain 1 (7.0 g). The chemical structure of 1 was elucidated by analysis of NMR data and compared with literature  and benzyl bromide reagents, which were prepared as described in the literature  underwent etherification reaction, and then acetal formation using a modified method from the literature with acetone/deuterated acetone to yield derivatives 28, 29, 37–55, and 58–68 (Stoessl and Stothers O-Acylation of the hydroxyl group of 1 (or 2) with acid anhydride or acyl chloride afforded acyl derivatives 3 and 69–71. Acetal formation of 1 (or 2) with acetone or deuterated acetone were subjected to acylation to afford derivatives 30–36, 56, and 57.The 14-membered RAL derivatives 1–71) against the stained diatoms N. laevissima and N. exigua and planktonic microalgae P. tricornutum Bohlin, C. vulgaris, and C. socialis were evaluated. As shown in Table 3, 4, 12–15, 24, 26, 45, 58, and 62 displayed selective and moderate activities with EC50 values ranging from 11.04 to 32.55 μmol/L. The natural product zeaenol (1) showed moderate activity against the microalgae P. tricornutum Bohlin, C. socialis, and N. exigua and showed strong activity against the diatom N. laevissima, with an EC50 value of 8.79 μmol/L. Significantly, 9 exhibited selective and strong activity against the microalgae P. tricornutum Bohlin, C. socialis, and N. laevissima . These represent an effective inhibitory adhesion structural class of potential antifouling leads.Diatoms have been identified as biofoulers of artificial surfaces placed in marine environments, the attachment of which was thought to be the initial stage of the biofouling process  and its derivatives 2–71 were evaluated for their in vitro antiplasmodial activities against P. falciparum, and the results are displayed in Table 38, 39, 50, 52, 65, 67, and 69–71  and the selective inhibitory effect on P. falciparum (IC50) calculated here as CC50/IC50. We decided to evaluate in vitro cytotoxicity of the active derivatives 38, 39, 50, 52, 65, 67, 70, and 71 by determining their 50% cytotoxic concentrations. The results are presented in Table 50 values > 100 μmol/L, reaching selectivity indexes > 10. The cytotoxic concentration at 150 μmol/L was not be determined because of a lack of solubility of the tested molecule in the culture medium. Moreover, the toxicity of these derivatives was also evaluated against two other cell lines: hepatocellular liver carcinoma cell line (Hep G2) and cervical carcinoma (HeLa), finding CC50 values > 100 μmol/L.Toxicity is a major concern of drug discovery and development, and the selectivity index (SI) is used as the evaluation parameter of drug potential of the test samples by comparing the toxicity on a human cell line  value of 25 μmol/L.Furthermore, compounds 3–27 and 30–71 of the natural product zeaenol (1) isolated from the marine-derived fungus C. lunatus were semisynthesized. Two derivatives (9 and 34) displayed strong and selective antialgal activities against the diatoms N. laevissima and N. exigua, with effects similar to those of the positive control SeaNine 211. Importantly, 38, 39, and 69–71 showed potent non-toxic antiplasmodial activities against P. falciparum, and their structure–activity relationships were also discussed. Our results offer important insights for the development of a new and safer class of antialgal and antiplasmodial agents.In summary, a series of new14-membered RAL derivatives 2. Silica gel (100–200 and 200–300 mesh particle size) was used for column chromatography. Thin layer chromatography (TLC) was precoated with silica gel GF 254 plates . NMR spectra were obtained at 500 MHz for 1H and 125 MHz for 13C on a JEOL JEM-ECP NMR spectrometer. Chemical shifts δ are reported in parts per million (ppm) values, using TMS as the internal standard, and coupling constants J are in Hz. HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer.Reagents and solvents were purchased from commercial suppliers and used without further purification. The progress of the reactions was monitored by TLC on SiO1  in dry CH2Cl2 (10 ml) was added a solution of SO2Cl2  in CH2Cl2 (4.0 ml) at 0 °C. After 1 h, the reaction was quenched by the addition of 15.0 ml of 5% aqueous NH4Cl solution. The organic phases were separated and evaporated. The residue was purified by silica gel column chromatography  to give derivative 2.To a solution of compound 1 or 2 , benzyl bromide reagent  and K2CO3  in acetone (15 ml) was stirred at 60 ℃ for 12–24 h. The reaction mixture was washed with aqueous saturated NaHCO3 solution. The organic layer was evaporated to dryness to leave the crude product. This was purified by silica gel column chromatography  to give the etherification derivatives 4–27.A mixture comprising compound 1 or above etherification derivatives , and p-toluene sulfonic acid  in acetone (8 ml) was stirred at 30 ℃ for 1–3 h. Progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was purified by silica gel column chromatography  to give the acetonide derivatives 28, 29, 37–52. Similarly, the deuterated acetonide derivatives 54, 55, and 58–68 were obtained with deuterated acetone instead of acetone as a solvent.A mixture of compound 1  , acid anhydride or acyl chloride reagent , and K2CO3  in acetone (15 ml) was stirred at 50 ℃ for 12–48 h. Progress of the reaction was monitored by TLC. The reaction mixture was washed with aqueous saturated NaHCO3 solution. The organic layer was evaporated to dryness to leave the crude product. This was purified by silica gel column chromatography to give the O-acylation derivatives 3, 30–36, 56, 57, and 69–71.A mixture comprising compound P. tricornutum Bohlin, C. vulgaris, C. socialis, N. laevissima, and N. exigua was evaluated by the method of Ortlepp et al. (5 cells/ml), which were used for microalgae growth inhibition assay. The compounds 1–71 were first dissolved in a small amount of DMSO and then diluted with 0.22 μm of filtered seawater (FSW) to achieve a range of concentrations from 0.625 to 50 μg/ml. Ten microliters of the compound solution was added to multiwell Petri dishes containing a 1-mL suspension of the microalgae (approximately 12 × 104 cells/ml). FSW with DMSO was used as a negative control, and solutions of SeaNine 211 in seawater were used as positive controls. After the plates were incubated for 48 h (16 ± 1 ℃) under continuous light, the FSW with non-attached microalgae was removed. The amount of chlorophyll a in attached microalgae was determined with a spectrophotometer . Then the EC50 (inhibits 50% of settlement of cyprids in comparison with the control) was calculated using the Probit software program.The antialgal activity against the microalgae p et al. . BrieflyP. falciparum, as reported by Trager and Jensen  at a hematocrit of 2% erythrocytes at 37 °C with a gas mixture containing 90% N2, 5% CO2, and 5% O2. Parasites were synchronized with 5% d-sorbitol Below is the link to the electronic supplementary material.


Staphylococcus aureus (MRSA) infections are considered a major public health problem, as the treatment options are restricted. Biofilm formation and the quorum sensing (QS) system play a pivotal role in S. aureus pathogenicity. Hence, this study was performed to explore the antibacterial effect of pyocyanin (PCN) on MRSA as well as its effect on MRSA biofilm and QS.Methicillin-resistant n = 30) with a MIC value equal to 8 µg/ml. About 88% of MRSA biofilms were eradicated by PCN treatment using the crystal violet assay. The disruption of MRSA biofilm was confirmed using confocal laser scanning microscopy, which showed a reduction in bacterial viability  and biofilm thickness . Additionally, the disruption of the formation of microcolonies and the disturbance of the connection between bacterial cells in the MRSA biofilm after PCN treatment were examined by scanning electron microscopy. The 1/2 and 1/4 MICs of PCN exerted promising anti-QS activity without affecting bacterial viability; Agr QS-dependent virulence factors , and the expression of agrA gene, decreased after PCN treatment. The in silico analysis confirmed the binding of PCN to the AgrA protein active site, which blocked its action. The in vivo study using the rat wound infection model confirmed the ability of PCN to modulate the biofilm and QS of MRSA isolates.Data revealed that PCN exhibited strong antibacterial activity against all test MRSA isolates (The extracted PCN seems to be a good candidate for treating MRSA infection through biofilm eradication and Agr QS inhibition.The online version contains supplementary material available at 10.1186/s12866-023-02861-6. Staphylococcus aureus is one of the most prevalent pathogens in the community and hospital-acquired infections . The biofilm eradication activity of PCN was further confirmed using a light microscope (LM), as described by [8 CFU/ml was transferred to a 6-well microtiter plate containing a 1 × 1 cm size of coverslips. After 48 h of incubation at 37 °C, planktonic cells were removed, and the wells were gently washed with normal saline three times. Thereafter, 500 µl of PCN  was added. Later, the entire plate was incubated at 37 °C for 24 h under static conditions. After incubation, glass coverslips were removed, washed with PBS, and stained with 0.1% crystal violet. After the removal of excessive stains by washing with deionized water and drying, the coverslips were examined at 100 × magnification using LM .The effect of PCN on the developed biofilms of MRSA was estimated using the method described by . About 2ribed by , 30. In To determine the effect of PCN on bacterial viability within pre-established MRSA biofilms, we determined the count of viable bacterial cells in the presence and absence of PCN, as previously described by . BrieflyThe effect of PCN on EPS production was assessed using an approach previously described by . In brieThe antibiofilm potential of PCN on the preformed MRSA biofilm was visualized under SEM, as prescribed by . In brieThe antibiofilm potential of PCN on the preformed MRSA biofilm was visualized under a CLSM, as prescribed by . The 8-wn = 10) were cultured in LB broth with an OD600 of 0.3 with 3/4, 1/2, and 1/4 MICs of PCN and without PCN at 37 °C. Samples were collected and the absorbance was read at 600 nm at 30-min intervals; 3 ml samples of each culture were collected immediately after the addition of PCN (zero time) and after 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, and 420 min [The most virulent MRSA strains ( 420 min . The subOvernight cultures of each tested MRSA strain in LB broth with and without 2 and 4 µg/ml PCN were centrifuged and then filtered using a 0.45 μm filter. Sterile supernatants (100 μL) were added to the wells made in the skim milk agar plates. Clear zones that formed around the wells after 24 h of incubation at 37 °C were measured .Qualitative and quantitative approaches were utilized for the detection of the effect of PCN on hemolysin production. In the qualitative approach, the overnight culture of a bacterial strain (20 μL) was inoculated into LB broth (180 μl) with or without PCN (4 μg/ml). After incubating at 37 °C for 18 h, the samples were streaked on human blood agar. The zone of red cell clearance around inoculated bacteria was observed after 24 h .The motility assay was conducted using motility plates alone and motility plates containing 2 and 4 µg/ml PCN. The plates were inoculated with a sterile toothpick and incubated at 37 °C for 24 h. Motility was assessed by measuring the zone formed by the colonies migrating away from the point of inoculation .n = 10) were cultured in LB broth with and without PCN (4 µg/ml). The kit manufacturer’s instructions  were followed for extracting total RNA from bacterial pellets. The RNA yield and purity were evaluated by measuring the absorbance, and samples with a ratio of 260/280 nm in the 1.8–2 range were used. Synthesis of cDNA was conducted according to the kit manufacturer’s instructions . The qRT-PCR was performed using a RT-PCR device, Rotor-Gene Q , to determine transcript levels of genes using oligonucleotides listed in Table 16S rRNA gene was used, and the cycling conditions were 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, and a final extension cycle of 5 min at 72 °C [The most virulent MRSA strains n = 0 were cuS. aureus (PDB ID: 3BS1) was downloaded from the Protein Data Bank (http://www.rcsb.org/). Ligands and water molecules not implicated in the binding were detached. Then, the ArgA protein was ready for docking analysis using the protonate 3D protocol in MOE with default options. The PCN structure was drawn in ChemBioDraw (version 14) software [The binding interaction between PCN and the AgrA protein was performed by the Molecular Operating Environment  software. The crystal protein structure of the AgrA protein of software .n = 12) were obtained from 6 to 8 weeks of age from the College of Veterinary Medicine of Cairo University  animal house. After wound creation, rats were individually housed to prevent cross-contamination and fighting. Rats were randomly divided into two groups (n = 6); group 1 was injected with vehicle (PBS) only, while group 2 received PCN dissolved in the vehicle at a concentration of 40 μg/ml. The procedures were established as previously performed by [6 CFU) of the most virulent MRSA isolate. Following inoculation, 30 min later, 20 μL of the vehicle (PBS) was injected subcutaneously in the control group (group 1), and PCN was injected subcutaneously in the treated group (group 2) on days 0, 3, and 6. Images of wounds were taken by the camera. Wound size was quantified by analysis using Image J software version 146. Wound contractions were presented as a percentage of the wounded area according to the following formula: percentage of wound closure = /initial wound size) × 100. At the end of our experiment, six animals from each group were euthanized by CO2 inhalation on day 6. The skin lesions were excised with 2–5 mm of the area and collected for histopathological examination using hematoxylin and eosin (H & E) at the Pathology Department, Faculty of Medicine, Tanta University, and for the counting of bacterial load at the wound area. After the homogenization of tissues by repeated application of a 1 ml syringe plunger in a microfuge tube that contained the tissue and distilled PBS, the homogenized suspensions were serially diluted and plated on mannitol salt agar for CFU count determination [The following procedures were permitted by the Faculty of Pharmacy-Tanta University Research Ethics Committee (TP/RE/12/22p-0068), and the study was conducted in accordance with ARRIVE guidelines. Male BALB/c rats of 120–150 g in weight  using GraphPad Prism version 5 software, and the threshold for significance was set at a S. aureus (n = 100) was isolated from different specimens (blood (10), wound (60), sputum (12), and abscess (18)). The percentage of MRSA isolates (resistant to both oxacillin and cefoxitin) was 30%. The percentage of antimicrobial resistance of MRSA strains involved in the study (n = 30) was the following: penicillin (80%), aztreonam (43.3%), chloramphenicol (40%), gentamycin (73.3%), rifampin (0%), vancomycin (0%), trimethoprim-sulfamethoxazole (43.3%), erythromycin (83.3%), linezolid (0%), levofloxacin (56.6%), tetracycline (36.6%), clindamycin (33.3%), oxacillin (100%) and cefoxitin (100%) as shown in Table Based on culture on mannitol salt agar plates, Gram-staining technique, and the catalase test,  sputum 1, and absBiofilm formation and virulence factors of MRSA isolates, such as protease, hemolysin, and motility, were estimated for detecting the strongest biofilm-forming and most virulent MRSA isolates. These MRSA isolates were used for evaluating the effect of PCN on the eradication of their pre-established biofilm and the production of their virulence factors. From Table P < 0.05) dose-dependent antibacterial activity against MRSA with inhibition zones ranging from (15-36 mm), (13-30 mm), and (10-19 mm) at concentrations of 40, 20, and 10 µg/ml, respectively, as shown in Fig. n = 30) were also determined by the microdilution technique, and all strains had the same MIC value of 8 μg/ml.The antimicrobial activity of PCN was examined using the well-diffusion method against MRSA strains. The results showed that PCN exhibited a significant  of pre-established biofilm by (83–88%), (69–79.4%) and (29.7–56.8%) for 40, 20, and 10 µg/ml of PCN, respectively, as shown in Figs. Pre-established (48 h) MRSA biofilms were treated with PCN at different concentrations  using a microtiter plate assay. PCN treatment led to a significant eradication (P < 0.05) after PCN treatment, as shown in Fig. P < 0.05) after PCN treatment, as shown in Figs. To understand how PCN could eradicate the pre-established MRSA biofilm, we assessed the effect of PCN treatment on bacterial viability within the biofilm by determining the viable bacterial count as well as the effect of PCN on EPS, the main matrix component, using the phenol–sulfuric acid method. We found that the bacterial viability significantly decreased (P < 0.05) the viability of bacterial cells by approximately 65 and 82% for 20 and 40 µg/ml of PCN, respectively, as shown in Fig. P < 0.05) by 50 and 60% for 20 and 40 µg/ml of PCN, respectively, as shown in Fig. The CLSM was used for further investigation of the antibiofilm activity of PCN against MRSA isolates. After PCN treatment of a 48-h pre-established MRSA biofilm, the viability and the biofilm thickness were visualized using acridine orange and propidium iodide for staining dead cells (red) and viable cells (green). PCN penetrated the biofilm matrix and significantly reduced  inhibited by (25–100%) and (18.8–63.6%) for 4 and 2 µg/ml of PCN, respectively, as shown in Fig. P < 0.05) inhibited, using a spectrophotometric assay, by (64.3–90.6%) and (35.3–80.1%) for 4 and 2 µg/ml of PCN, respectively, as shown in Fig. P < 0.05) inhibited by (25–57.6%) and (4.4–45.5%) for 4 and 2 µg/ml of PCN, respectively, as shown in Fig. The effect of PCN on virulence factors of MRSA isolates, such as proteolytic activity, hemolytic activity, and motility, was assessed. The proteolytic activity of MRSA isolates was estimated in the absence and presence of PCN using skimmed milk plates. Untreated MRSA showed a zone of clearance, while treated MRSA showed no zone of clearance (no proteolytic activity), as shown in Fig. d by 25–5.6% and  in the most virulent MRSA isolates (n = 10). The expression of the agrA gene significantly (P < 0.05) decreased after PCN treatment in all tested MRSA strains, as shown in Fig. agrA gene expression levels were 53.3, 63.3, 60, 73.3, 76.67, 60, 43.3, 53.3, 40, and 66.67% for MRSA strains Nos. 1, 2, 3, 14, 16, 22, 24, 25, 27, and 30, respectively.The Agr QS is considered a universal regulator of agrA gene decreased at the molecular level. To gain a better understanding of the plausible mechanism by which PCN can induce its anti-QS activity, we proceeded to examine the interaction of PCN with the main regulator protein of Agr QS (AgrA protein) (PBD ID: 3BS1). Molecular docking was accomplished in standard precision mode using the glide ligand-docking module. Results showed that PCN was appropriately oriented in the active site of the AgrA protein with a significant docking score ; PCN made a strong complex with the AgrA protein through Met 160, Phe 222, Phe 161, Cys 199, and Phe 203 amino acids, as shown in Fig. S. aureus virulence factors and genes.Based on our results, PCN phenotypically decreased Agr QS-dependent virulence factors, such as protease, hemolysin, and motility. Additionally, the expression level of the P < 0.05) higher in treated rats compared to untreated rats on different days of the experiment (Table P < 0.05) lower bacterial burden  than the untreated wounds  after 6 days of inoculation, as shown in Fig. The effect of PCN on MRSA infection was investigated using the rat wound infection model by measuring the wound closure in MRSA male rats with and without PCN treatment. The percentage of wound closure was significantly , the histopathological examination of tissues showed wide-ranging destruction, skin ulceration that was covered by heavy inflammation (acute and chronic inflammatory cells), and necrotic debris with no epithelization, as shown in Fig. The WHO has urged the scientific community to search for new approaches to combat antibiotic resistance, a dangerous public health problem as more and more bacterial pathogens have developed resistance to antibiotics and the present treatment options have become few and ineffective –45. NumeP. aeruginosa-derived PCN against MRSA were assessed in the present study.The great advantages of natural pigments, such as biodegradability and safety, compared to synthetic pigments, have captured the attention of the industry in human applications . DespiteS. aureus, Escherichia coli, Klebsiella species, Salmonella typhi, and Shigella species. Aziz et al. found that PCN inhibited the growth of S. aureus, K. pneumoniae, Enterococcus faecalis, Burkholderia cepacia, and E. coli [Bacillus cereus, S. aureus, Staphylococcus sciuri, E. coli, S. typhi, Salmonella enterica, K. pneumoniae, and Lactococcus lactis. Researchers consider that the key mechanism by which PCN exerts its antibacterial activity is that PCN simply permeates cell membranes and accepts electrons from NADH or NADPH. Under aerobic conditions, PCN passes those electrons to O2, and reactive oxygen species (ROS) are created, which leads to oxidative stress and the inhibition of the ion's interaction with the membrane, respiration, and the active transport of solutes; consequently, the bacterial growth is inhibited [According to our study, PCN showed a high killing power against MRSA isolates with a MIC value equal to 8 µg/ml. In agreement with our study, El-Fouly et al.  observed E. coli . Additio E. coli , PCN exenhibited –19.P. aeruginosa BTRY1 exhibited a remarkable reduction in foodborne pathogens’ biofilm formation, belonging to the genera Bacillus, Staphylococcus, Brevibacterium, and Micrococcus [B. cereus, S. aureus, and K. pneumoniae [Besides affecting the MRSA isolates in their planktonic mode, we found that PCN inhibited the growth of MRSA isolates in their biofilm mode. The crystal violet assay showed that PCN significantly eradicated biofilms of tested MRSA isolates, and the mechanism by which PCN eradicated pre-established MRSA biofilms was associated with decreasing bacterial viability and reduction of the EPS matrix. The reduction of the viability of bacterial cells and biofilm thickness by PCN treatment was further confirmed using CLSM. Moreover, SEM images proved that PCN disrupted MRSA biofilms, and the biomass of the MRSA biofilm was decreased after PCN treatment. In accordance with our study, PCN extracted from rococcus . Additioeumoniae .S. aureus largely depends on the production of plentiful extracellular virulence factors . Subsequently, an alternate strategy in the treatment of S. aureus is targeting bacterial virulence factors [S. aureus, the majority of virulence factors are regulated by QS, which is under the control of the agr (accessory gene regulatory) operon [agrBDCA, which encode all the main components of QS [The pathogenicity of  factors . In S. a) operon . The exp) operon . Upon Ag) operon . The RNAts of QS . Blockints of QS , 11, 62.agrA gene expression by 53.3, 63.3, 60, 73.3, 76.67, 60, 43.3, 53.3, 40, and 66.67% for MRSA strains Nos. 1, 2, 3, 14, 16, 22, 24, 25, 27, and 30, respectively. Additionally, the anti-QS activity of PCN was proven through diminished hemolytic activity, proteolytic activity, and motility of MRSA isolates after PCN treatment with 1/2 and 1/4 MICs of PCN without affecting bacterial viability. In addition, the in silico analysis revealed that PCN binds to the AgrA protein at a critical location that is required for proper folding, DNA binding, and exerting its QS regulation activity [agrA gene and the reduction of the hemolytic and proteolytic activities of S. aureus [hla, psmα, hysA, agrA, cap1A, and cap1C genes and Agr QS-dependent virulence such as hemolysin [In our study, PCN inhibited the . aureus . Blockinemolysin . Furtheremolysin , 62.P. aeruginosa by PCN [For further confirmation of the ability of PCN to compete with MRSA infection, we assessed the effect of PCN on the wound infection caused by virulent MRSA isolates, which is one of the most common bacteria isolated from chronic wounds , 65. We a by PCN . Furthera by PCN . Marreira by PCN .P. aeruginosa-derived PCN showed remarkable antibacterial and antibiofilm activities against MRSA as well as an anti-QS effect through binding to the AgrA protein. Hence, PCN is considered a promising antibacterial candidate for the treatment of biofilm-associated MRSA infections as well as a QS attenuation agent through blocking AgrA protein activity. Further studies are required to assess the effect of PCN in combination with antibiotics in the treatment of bacterial infections.In conclusion, Additional file 1: Supplementary data Table 1. The percentage of wound closure with and without pyocyanin treatment after 3 and 6 days of infection. The green column represents the size of the first wound, and the red column represents the size of the second wound in the same rat .


Erwinia amylovora is a plant pathogen that causes fire blight disease in Rosaceous plants, such as pear and apple. To develop an effective biocontrol method to suppress E. amylovora, a total of 16 bacteria were isolated from pear orchard soil in China and screened for antagonistic activity in vitro. Among them, 9 isolates that exhibited antagonistic activity against E. amylovora were identified, including Bacillus atrophaeus, Priestia megaterium (previously known as Bacillus megaterium) and Serratia marcescens based on the partial 16S rDNA sequence analysis and similarity search. The plate confrontation experiments showed that strain 8 (P. megaterium strain KD7) had strong antagonistic activity against E. amylovora. The methanolic extract from cell-free supernatant of strain KD7 displayed high antibacterial activities against E. amylovora. Furthermore, the active compounds of strain KD7 were separated by thin layer chromatography (TLC) and the amino acids were detected by the presence of a spot with retention factor (Rf) of 0.71. Next, three lipopeptides were identified with high-resolution mass spectrometry (HRMS), including C13-surfactin [M+H]+ at m/z 1008.14, C15-surfactin [M+H]+ at m/z 1036.50, and C14-iturin A [M+H]+ at m/z 1043.17. Strain KD7 showed multiple antibiotic resistance, such as ampicillin, erythromycin, penicillin and tetracycline. The detached pear leaves, twigs and fruits assay showed that both protective and curative action with strain KD7 had the ability to decrease the development of fire blight. Taken together, P. megaterium strain KD7 is a potential effective biocontrol agent against fire blight. Erwinia amylovora, is one of the most serious plant diseases that mainly affects Rosaceae, such as apple, pear, medlar and quince. The disease spreads easily and rapidly during warm and moist, especially during the bloom stages. All parts of the plant can be affected by fire blight, including leaves, shoots, flowers and fruits. The characteristic symptoms include wilt and death of flower clusters, wilting and dieback of shoots, and the affected shoots often bent into a ‘shepherd’s crook’ shape    showed hE. amylovora strain 0017 which was originally isolated from pear in Kyrgyzstan was used as a reference strain. This strain was identity by polymerase chain reaction (PCR) using the primers AMS3/AMS4c, based on chromosomal ams gene (E. amylovora cultures were maintained and grown on nutrient agar (NA)  (ams gene . E. amyl7.2–7.4) .Pyrus betulifolia Bunge) and Korla fragrant pear, were used to evaluate the biocontrol activity of antagonistic bacteria against E. amylovora. The detached leaves and twigs were selected from ‘duli’ pear, a wildtype pear widely used rootstock for grafting in China due to its resistance to biotic and abiotic stress (Pyrus sinkiangensis Yü), a popular cultivated pear variety in China.Two pear varieties, ‘duli’ pear  in SDW, and 200 μl of the dilutions (1.0 × 10−5 and 1.0 × 10−6) were plated onto NA medium at 28°C for 24–48 h. The colonies were picked up and streaked on nutrient broth (NB)   in plastic bags and stored at 4°C. Ten grams of soil sample was suspended in 90 ml of sterile distilled water (SDW), under shaken (200 rpm) for 30 min, and the 1.0 × 107.2–7.4)  separatePhenotypic and biochemical traits of antagonistic bacterial isolates were tested by conventional bacteriological methods. Morphological traits such as colony color and cell motility as well as physiological fingerprints were performed on NA medium. Gram staining was performed as previously described .Genomic DNA was extracted using CTAB/NaCl method . The bacE. amylovora 0017 and antagonistic bacteria were inoculated on NB medium, respectively, and cultured at 28°C with shaking at 200 rpm. After 24 h, the bacterial suspensions were diluted to an OD620 of 1.0 (about 1.0 × 108 to 1.0 × 109 /ml)  .E. amylovora suspension was poured into an NA plate (12 cm in diameter), spread and dried at room temperature for 5 min. A single colony of potential antagonistic bacteria was picked up and spotted on the NA plate containing E. amylovora. After incubation , the diameter of the inhibition zone was measured, and the antagonistic activity was determined by measuring the diameter of the inhibition zone (mm) around the isolated bacteria.The plate confrontation method was used to verify the functional activity of these isolated bacteria. 200 μl of diluted P. megaterium strain KD7, was investigated by the disk-diffusion method as described previously , and the supernatant was sterilized through a 0.22 μm filter. The bacterial cells were disrupted by an ultrasonic cell breaker and filter sterilized to obtain sterile liquid inclusion.E. amylovora was measured by disk-diffusion method , the diameter of the inhibition zone around the disk was measured. All experiments were repeated three times independently.The antibacterial activity of these two filtrates against n method . 10 μl oP. megaterium strain KD7 was cultured in 100 ml of NB medium for 24 h . Bacterial culture was centrifuged for 10 min , then the supernatant was collected and filtered through a 0.22 μm sterile filter.P. megaterium strain KD7. This method is a process of transferring compounds from one liquid phase into another based on their relative solubility in two different immiscible liquids , then placed on NA plate containing The antibacterial components of the extracted supernatant were detected by thin layer chromatography (TLC) . A chlor18 column  using H2O (A)/acetonitrile (B) both containing 0.1% HCOOH as a mobile phase. The injection volume was 2 μl and the flow rate was 0.3 ml min−1.To identify the substances produced by strain KD7, the crude methanol extracts were analyzed by High-resolution mass spectral (HRMS) on an Orbitrap Exploris 480 mass spectrometer and EASY-nLC liquid chromatography system (Thermo Fisher Scientific) equipped with an electrospray ionization (ESI) in positive mode. The ion source was set up as follows: capillary voltage: 2300 V, ion transfer tube temperature: 320°C. Detection was performed with resolution of 120,000, scan range from 200 to 1,200 m/z, 1.6 m/z isolation window. Separation of compounds was performed on ChromXP CP. megaterium strain KD7 against E. amylovora was investigated on detached pear tissues: leaves and twigs of ‘duli’ pear, and fruits of Korla fragrant pear. The streptomycin was used to evaluate the efficiency of the treatment.Under laboratory conditions, the potential protective and curative effect of E. amylovora 0017 (1.0 × 108 CFU/ml) instead of SDW. After 48 h of incubation, leaves were sprayed with suspensions of antagonistic bacteria strain KD7 (1.0 × 108 CFU/ml) and streptomycin solution, separately. For protective tests, leaves were sprayed with strain KD7 or streptomycin, separately. After 48 h, leaves were injected with 100 μl pathogenic bacterial suspensions. For curative tests, leaves were treated inversely to the protective treatments. To observe the effect of strain KD7 in the leaves, a treatment control was designed as follows: leaves were sprayed with suspensions of strain KD7. After 48 h, the suspensions of strain KD7 were sprayed on the leaves again. The treated leaves were incubated in a climate chamber  and symptoms were assessed from 3 to 15 days of post-inoculation (dpi). Three leaves were used for each treatment and all experiments were repeated three times. Disease severity was assessed by evaluating the percentage of leaf area affected  into the intercellular spaces on the hollow petiole of the mature leaves using a syringe and then sprayed with SDW to keep all the leaves in the same conditions . The samaffected . The sca8 CFU/ml). For protective tests, twigs were sprayed with suspensions of antagonistic bacteria strain KD7 (1.0 × 108 CFU/ml) and streptomycin, separately. After 48 h, twigs were sprayed with suspensions of pathogenic bacterial (1.0 × 108 CFU/ml). For curative tests, twigs treated reversely to the protective tests. Three twigs were used for each treatment with three independent experiments. All twigs were incubated at 28°C in darkness for a period of 7 to 15 days. After 15 dpi, disease severity was assessed by measuring the necrosis at the inoculation site based on the vascular browning index of twigs : 0 = absence of browning, 1 = 1–10% browning, 2 = 11–25% browning, 3 = 26–75% browning, and 4 = 76–100% browning  and surface sterilized as described above. Each twig was artificial injured before treatment. For negative control, twigs were sprayed with SDW. For positive control, twigs were sprayed with suspensions of pathogenic bacterial (1.0 × 10browning .8 CFU/ml) was injected instead of SDW. After 48 h, wounded fruits were sprayed with strain KD7 (1.0 × 108 CFU/ml) and streptomycin solution, separately. For protective tests, fruits were sprayed with antagonistic bacteria strain KD7 (1.0 × 108 CFU/ml) and streptomycin, separately. After 48 h, wounded fruits were sprayed with suspensions of pathogenic bacterial (1.0 × 108 CFU/ml). For curative tests, fruits treated reversely to the protective treatments. Symptoms were recorded from 6 dpi to 15 dpi. Disease severity was assessed based on a fruit infection index : 0 = no necrotic spots, 1 = necrotic spots of 1–5 mm in diameter, 2 = necrotic spots of 6–10 mm in diameter, 3 = necrotic spots of 11–20 mm in diameter, and 4 = necrotic spots over 21–30 mm in diameter  were made on each fruit and 100 μl of SDW was injected on each hole. For positive control, 100 μl pathogenic bacterial suspension (1.0 × 10diameter .p ≤ 0.05).SPSS 17.0 and GraphPad Prism 7 were used for statistical analysis and mapping of experimental data, and variance analysis was performed by LSD multiple comparison method , Priestia  and Serratia (1 and 6).Bacillus atrophaeus , which was identified as B. atrophaeus. Strain 5, 7 and 8 shared the highest sequence similarity (>99%) with B. megaterium (KJ155812.1 and MT626661.1), which was identified as P. megaterium. Strain 1 and 6 shared the highest sequence similarity (>99%) with Serratia marcescens (AJ233431.1 and AB594756.1), which was identified as S. marcescens. The 16S rDNA sequences of the 9 isolated strains were submitted to the NCBI for GenBank accession numbers as follows: strain 1 , strain 2 , strain 3 , strain 4 , strain 5 , strain 6 , strain 7 , strain 8 , and strain 9 .A phylogenetic tree based on partial 16S rDNA sequence of isolated strains and other bacterial species from the GenBank database was constructed. As shown in P. megaterium KD7) against E. amylovora was checked by disk-diffusion method. The cell-free supernatant (CS) of strain KD7 exhibited antimicrobial activity compared to the bacterial cell disruption (BD) , n-hexane (EH), ethyl acetate (EEA) and n-butanol (EB). After incubation, only the bacteria culture broth (BC) and methanol extracts (EM) exhibited an antibacterial activity  , indicatactivity ,B. The mD , indicactivity , suggest+ and C15-surfactin [M + H]+, respectively, and the major product of m/z 1043.17 assigned to C14-iturin A [M + H]+ (Using the HRMS(ESI) method, two types of lipopeptides were identified from the crude methanol extracts of strain KD7, including surfactin and iturin A . From th[M + H]+ . The resP. megaterium strain KD7 was tested using disk-diffusion method. As shown in Antibiotic resistance of E. amylovora. In detached ‘duli’ pear leaves assay , and reached the disease incidence by 100% at 15 dpi. Compared with positive control (index = 4.00), vascular browning index was reduced significantly in strain KD7-treated twigs in protective and curative treatments (index = 1.62 ± 0.27 and 2.32 ± 0.28 respectively), which was similar as streptomycin-treated twigs in protective and curative treatments (index = 1.71 ± 0.40 and 2.69 ± 0.27 respectively).In the second experiment on ‘duli’ pear twigs , 6B, no E. amylovora appeared necrosis after 6 dpi, and reached the disease incidence of 100% (index = 4.00) at 15 dpi. However, the infection index was significantly reduced both in both protective and curative treatments with strain KD7 , indicating that strain KD7 decreased the pathogen development compared with positive control.The third experiment on the fruits of Korla fragrant pear , 6C, posin vivo assays, disease symptoms and pathogen development in pear tissues treated with strain KD7 were significantly lower than positive control (p < 0.05). Furthermore, the effectiveness of protective and curative treatments of strain KD7 against E. amylovora were similar to those of streptomycin, indicating its potential biological control agent for controlling fire blight.In all E. amylovora in vitro and in vivo. A total of 9 bacteria with stable antagonistic against E. amylovora were isolated from pear orchard soil. These isolates were identified as B. atrophaeus, P. megaterium and S. marcescens. Among the isolated bacteria, strain 8 (P. megaterium strain KD7) showed the highest inhibition effect against E. amylovora in vitro experiments. P. megaterium is a key microorganism for the biological control of plant diseases which can enhance host plants defenses against diverse pathogens, such as Septoria tritici blotch in wheat  had high protective ability on apple branches, blossoms and shoots, and even more effective than the copper product  was evaluated in vitro and in vivo study on detached pear tissues, which is a potential source of a new biocontrol agent to control fire blight.From the soil environment of pear orchard in China 16 microbial isolates were obtained. By plate confrontation method, some The original contributions presented in the study are included in the article/ZC, LS, and LH designed the work. ZC and LH performed the experiments. WM, LZ, and DG analyzed the data. LS and ZC wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by College of Life Science, Shihezi University, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: A study was undertaken to determine the acute effects of a beverage made with Avela™ (R)-1,3-butanediol, on blood beta-hydroxybutyrate (BHB) levels (using the Keto-Mojo monitor), gastrointestinal (GI) tolerability , and sleepiness .Methods: Following a 12-h overnight fast, 26 healthy adults consumed one beverage containing 11.5 g of (R)-1,3-butanediol at each of 0, 30, and 60 min, culminating in a total intake of 34.5 g of (R)-1,3-butanediol. Blood BHB levels, GI tolerability, and sleepiness were assessed at baseline (0 min), and at 30, 60, 90, 120, 180, 240, and 300 min. At 240 min, a protein bar was consumed.Results: The mean (±SD) BHB fasting baseline level, maximal concentration, time at maximal concentration, and incremental area under the curve over 300 min were 0.23 ± 0.21 mmol/L, 2.10 ± 0.97 mmol/L, 133.85 ± 57.07 min, and 376.73 ± 156.76 mmol/L*min, respectively. BHB levels at each time point were significantly increased relative to baseline. In females, BHB Tmax was significantly greater (p = 0.046), and BHB iAUC0–300 min nearly significantly greater (p = 0.06) than in males.Discussion: The beverage formulated with Avela™ had no impact on sleepiness and was generally well-tolerated, with no or mild GI symptoms reported in most participants. Mild headaches were reported as an adverse event by five participants and judged possibly related to the study product in two of the participants. Ketogenesis is a process whereby the liver utilizes fatty acids to synthesize ketones: beta-hydroxybutyrate (BHB), acetoacetate, and, to a lesser degree, acetone. Ketones are produced in low amounts when dietary carbohydrates are limited  or during prolonged exercise, but they can also be produced in response to diets that are ketogenic. Under normal physiological conditions, blood BHB levels are <0.5 mmol/L  without the dietary restrictions imposed by a ketogenic diet . ExampleR form and the S form; the R stereoisomer is naturally occurring and appears to be the configuration that can be converted to physiological ketone bodies most proficiently -1,3-butanediol were assessed have been conducted -1,3-butanediol. In the study by R,S)-1,3-butanediol resulted in nausea, euphoria, and dizziness; however, these adverse outcomes were mitigated with the administration of divided servings (as opposed to a single intake). R form of 1,3-butanediol was administered to 12 healthy male and female adults. In this study, the administration of a single serving of 10 g of (R)-1,3-butanediol resulted in a peak blood BHB level of 1.2 mmol/L at 39 min, and an incremental area under the curve (iAUC) of 118 mmol/L x 240 min; furthermore, the serving was well-tolerated.1,3-butanediol, an organic compound that can be converted to BHB, has also been studied for its potential to increase ketone levels. 1,3-butanediol is converted to BHB, primarily in the liver, by alcohol and aldehyde dehydrogenases . There aiciently . To dateonducted . There wR)-1,3-butanediol in a beverage. The secondary objectives were to assess GI symptomology, as well as any effects on sleepiness.The primary objective of our study was to determine the level of ketones (measured as blood BHB) following the single-day consumption of 34.5 g of .This study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of PearlIRB . The study was registered at 2; and weighed at least 110 lbs. Subjects were not eligible for inclusion if they had previous disorders of the GI tract; gastroenteritis in the 2 weeks prior to the study; diabetes; a history of drug or alcohol abuse; a previous diagnosis of neurological disorders, depression, or mental illness with psychosis; unexplained alarm features ; used an antibiotic or any medication impacting gut transit during the 2 weeks prior to study; constipation or diarrhea; an allergy to tree nuts; a current pregnancy or were currently breastfeeding; or medical conditions affecting the pancreas, liver, thyroid, or gallbladder. Participants were enrolled in the study in groups of ten at a time.A total of 30 healthy male and female adults were intended to be enrolled in the study. Male and female subjects were eligible for inclusion if they were 18–65 years of age; had a body mass index (BMI) of 18 to <35.0 kg/mR)-1,3-butanediol purified from microbial fermentation and is liquid at room temperature. To formulate one beverage serving, 11.5 g of Avela™ were mixed with distilled water to a total volume of 118 mL. Avela™ (R)-1,3-butanediol was consumed in the form of 3 servings of the beverage, with consumption of each serving separated by 30 min and each serving providing 11.5 g of (R)-1,3-butanediol . (R)-1,3-butanediol has Generally Recognized as Safe (GRAS) status for intakes of up to 34.5 g/day. This dosing regimen for Avela™ is based on 3 servings per day (up to 11.5 g per serving), which is similar to the proposed uses of another ketogenic substance, D-beta-hydroxybutyrate ester, the parent substance for (R)-1,3-butanediol. D-beta-hydroxybutyrate ester has received a no questions letter from the Food and Drug Administration on its GRAS status for use in bars, gels, and beverages at use levels up to 75 g/day over 2 to 3 servings per day.Avela™, manufactured by Genomatica Inc.,  is ≥ 99.7% chirally pure . Invitations to participate in the study were extended to employees of Genomatica Inc.; accordingly, several steps were taken to safeguard the rights and confidentiality of the participants, in line with recommendations provided by R)-1,3-butanediol], an ALOHA Organic Plant Based Protein Bar , and study instructions.All subjects included in the study were provided with study-related materials, including lancets, sharps container, alcohol swabs, Keto Mojo BHB monitor and ketone test strips , bandages, pregnancy test [if applicable], measuring tape, three 4-oz. (118-mL) bottles of sealed water premixed with Avela™ . At the 30-min and 60-min time points, participants repeated the procedures followed at the baseline (0 min) time point; that is, they first completed the mVAS GI Symptoms Tool, followed by the SSS, and next, they measured and recorded their blood BHB levels, after which they consumed a second (at 30 min) and third (at 60 min) beverage prepared using Avela™. By 60 min, a total of 34.5 g of (R)-1,3-butanediol had been consumed. At each of the subsequent time points , the mVAS GI Symptoms Tool and the SSS were completed, and blood BHB levels were measured and recorded. Of note, at 240 min, following the completion of the questionnaires and the blood BHB assessment, participants consumed one, 56-g ALOHA Organic Plant Based Protein Bar , to provide sustenance to the study participants who, by 240 min, would have consumed nothing for 16 h , other than the investigational product.Following a 12-h (overnight) fast and prior to ingesting any investigational product, participants completed, using the online portal, baseline assessments of GI symptomology  followed by sleepiness . Each participant then measured their own baseline blood BHB level using a portable blood BHB monitoring device, the Keto-Mojo monitor , and recorded it using the online portal. Participants were then instructed to consume the first of three beverages prepared using Avela™ [each beverage contained 11.5 g of . The secondary outcomes of the study were the statistical differences in GI symptoms and sleepiness ratings at each time point relative to baseline, defined as time 0 min. GI symptoms were assessed using the mVAS GI Symptoms Tool and sleepiness was assessed using the SSS. Each of these tools is described further below.The mVAS GI Symptoms Tool is a self-administered questionnaire that is modelled after the validated “gold standard” measurement tool predominantly used to test clinical gastroenterology scenarios . GaskellThe SSS  is a subAt each time point in the study, study participants were queried about AEs and were asked to describe and rate the severity of each using a five-point analog scale as mild (score of 1 or 2), moderate (score of 3 or 4), or severe (score of 5). Each AE was rated by the Principal Investigator as unrelated or possibly, probably, or definitely related to the investigational product.2), overweight (25 to <30 kg/m2), or obese (≥30 kg/m2). Sex, age category , and BMI category  were presented as percentages.All subjects who participated in the study were included in the analysis. Summary statistics  were calculated for subject demographics, including BMI, height, weight, and age. Of note, BMI and age also were examined as categorical variables; specifically, the median age was used to classify participants as “younger” and “older,” and BMI was categorized as normal weight (18.5 to <25 kg/mmax), the time at which the maximal BHB level was reached (Tmax), and the BHB iAUC over the 300-min study are provided.The BHB mean, SD, median, minimum, and maximum at each time point are provided; in addition, the mean, SD, median, minimum, and maximum for the maximal BHB level  the baseline pre-intake GI symptom severity rating from the post-intake GI symptom severity rating, is presented. As well, the number of unique GI symptoms experienced by each individual, after adjusting for baseline GI symptom severity ratings, is presented, as is the average percentage of time points at which each GI symptom was reported among those reporting the symptom.For the SSS scores, the minimum, maximum, median, mean, and SD at each time point are provided, both before and after adjusting for baseline scores.p < 0.05.All statistical analyses (described below) were conducted using SAS software , and significance was defined as 2 (continuous variable) as fixed effects and participants as random effects, were intended to be used to determine relationships with BHB levels, each of the 4 GI symptom category ratings, and the SSS scores; however, for each model, the residuals were not normally distributed, even after log transformation. Mixed models were then used to evaluate the effects of time  on the mean differences in BHB levels, the GI symptom category ratings, and the SSS scores, using the respective baseline values as references; however, the residuals were not normally distributed. For each model, the data were log transformed and the mixed models were refitted; however, the residuals still were not normally distributed. Of note, for the GI symptom category scores, there was an abundance of zeros , and so the mixed model was determined to be inappropriate for the data. For comparisons of each post-baseline BHB level, GI symptom category score, and SSS score with the respective baseline (pre-intake) values, paired t-tests or Wilcoxon signed rank tests were conducted, as appropriate . For the primary outcome only , sensitivity analyses were conducted if there were any outliers .Mixed models with time (continuous variable) and timeA correction for multiple comparisons  is normally applied to the secondary outcomes. However, as this was a pilot study, no correction for multiple comparisons was applied. Furthermore, the application of a Bonferroni correction to the statistical analysis of the secondary outcomes would have increased the difficulty in identifying possible GI intolerance or changes in sleepiness.max, and Tmax were assessed using unpaired t-tests  or the Wilcoxon Mann-Whitney test . Correlations between age (continuous variable) and each of BHB fasting level, iAUC, Cmax, and Tmax, as well as BMI (continuous) and each of BHB fasting level, iAUC, Cmax, and Tmax, were assessed using Pearson’s correlation coefficient . As well, at each time point, correlations between blood BHB levels and scores for each GI symptom group as well as between blood BHB levels and SSS scores, were assessed using Pearson’s correlation coefficient .The effects of sex, age , and BMI  on BHB fasting level, iAUC, C2. Of the 26 participants, 12 (46.2%) were normal weight (BMI was 18.5 to <25 kg/m2), 12 (46.2%) were overweight (BMI was 25 to <30 kg/m2), and two (7.6%) were obese (BMI ≥30 kg/m2). Since there were only two obese participants, results from these individuals were included with those categorized as overweight in all analyses in which BMI was assessed as a categorical variable, resulting in two categories . The median age of 42.50 years was used to separate study participants as “relatively younger” (<42.50 years) and “relatively older” (≥42.50 years).The goal was for 30 participants to be enrolled in the study; however, only 26 participants were enrolled, given that recruitment was slower than had been anticipated. Participant demographics are summarized in max, which was 2.10 ± 0.97 mmol/L, was reached at an average of 133.85 ± 57.07 min, approximately 1 h following the consumption of the final beverage containing Avela™ (R)-1,3-butanediol. One individual reached a Cmax of 5.5 mmol/L at 180 min; this was not the same individual who had a high fasting BHB level of 1.10 mmol/L at baseline. The next two highest Cmax levels of 3.9 and 3.1 mmol/L were reached at 240 and 180 min, respectively. The average blood BHB iAUC0–300 min was 376.73 ± 156.76 mmol/L*min. At 300 min , the average blood BHB level was 1.11 ± 0.85 mmol/L.Blood BHB levels following the 12-h overnight fast  and at the subsequent time points are presented in Each post-baseline blood BHB measurement  was significantly greater than that at baseline . Seven it-test. Although the slope of the line between 240 and 300 min (−0.39 ± 0.31 mmol/L over 60 min) was slightly steeper than that between 180 and 240 min (−0.32 ± 0.60 mmol/L over 60 min), there was no significant difference in the slopes of the lines (p = 0.50).Although the design of the study does not allow for an assessment of whether or not the consumption of the plant-based protein bar at 240 min had any impact on ketone levels thereafter, in an unplanned analysis, the slopes of the lines between 180 and 240 min (the hour before the bar was consumed) and 240 and 300 min (the hour after the bar was consumed) were compared using a Student’s paired The maximum severity reported by each individual for each GI symptom over the 300-min study, after adjusting for the baseline symptom severity rating, is shown in In p = 0.08). No participants reported stitch; thus, the increase in post-baseline measures was due to the reporting of nausea and dizziness, each by 5 and 10 participants, respectively.Global scores (including the means and ranges) for upper GI symptoms, lower GI symptoms, defecation, and other GI symptoms at each time point are presented in .The ratings by individual subjects for these symptoms over the course of the 300-min study, after adjusting for baseline ratings, is shown in Unadjusted and baseline-adjusted SSS scores at each time point are summarized in max, Tmax, and iAUC0–300 min are shown in p = 0.02); likewise, fasting BHB levels were significantly negatively associated with BMI, with a Spearman’s correlation coefficient of −0.56 (p = 0.003)  . In unplanned analyses, the associations between body weight and each of fasting BHB, BHB iAUC0–300 min, BHB Cmax, and BHB Tmax also were evaluated. Body weight was inversely and significantly or nearly significantly associated with each of these variables  . Interestingly, when we looked at the difference in BMI between males and females, there was no significant difference (p = 0.2184). Significant relationships between blood BHB levels and each of the GI symptom category scores, as well as between blood BHB levels and the SSS scores, were not identified at any of the time points examined. Adjusting for body weight had no impact on these results (data not shown).The associations of age, BMI, and sex with fasting BHB levels, C= 0.003) . BHB Tma= 0.046) . Also inctively) . An unpaSpontaneous AEs were not reported at baseline, 30 or 60 min. The most frequently reported AE was headache, reported by five subjects and judged by the Principal Investigator to be possibly related to the study product for two persons. One of the individuals reported a persistent mild (score of 1) headache at 90, 120, 180, 240, and 300 min, with no other AEs reported. The second individual reported a mild headache (score of 1) at 180, 240 and 300 min. Of the three remaining persons, the headache was attributed to a positive COVID-19 diagnosis the following day (n = 1), fasting (n = 1), or resolved after one rating (n = 1). A feeling of warm and sweating was reported by one individual at 90 and 120 min; these symptoms, which disappeared within 60 min of a drink of water, were judged by the Principal Investigator to be possibly related to the study product.R)-1,3-butanediol were consumed in close succession at 0, 30, and 60 min by participants in a fasted state, and effects on BHB levels, GI symptomology, and sleepiness were assessed over a total period of 300 min.In the study reported herein, three servings of a beverage containing Avela™ and each providing 11.5 g of (R)-1,3-butanediol  resulted in a mean blood BHB Cmax of 2.10 ± 0.97 (SD) mmol/L, which was achieved roughly 1 h post consumption of the final beverage containing Avela™. In a recent publication, 12 healthy individuals who consumed a single serving of 10 g of (R)-1,3-butanediol following an 8-h fast achieved a mean peak blood BHB level of 1.2 ± 0.3 mmol/L (SD) approximately 40 min post consumption (0–300 min was 376.73 ± 156.76 (SD) mmol/L*min, a value approximately triple that which was measured (iAUC0–240 min of 118 ± 64 mmol/L*min) following the consumption of 10 g (R)-1,3-butanediol in the study by Consumption of three servings of .Statistically significant inverse associations between BMI and fasting BHB and between body weight and each of fasting BHB, BHB iAUCogenesis . Indeed,max (p = 0.046) and nearly significantly greater BHB iAUC0–300 min (p = 0.06) determined for females versus males also are interesting, given that sex differences have been reported in metabolic changes following fasting. Increases in plasma fatty acid and ketone body concentrations . Of course, the sample sizes are very small  and so these results should be interpreted with caution. Additional studies are needed to better understand the effects of gender and body weight on ketone levels.To further evaluate the effects of body weight on fasting BHB, BHB CR)-1,3-butanediol was well-tolerated with no or mild GI symptoms reported by most participants. Post-baseline adjusted symptoms were rated as moderate by only one person, for each of urge to regurgitate, nausea and dizziness; this person tested positive for COVID-19 the following morning. The GI symptoms that were most frequently reported were mild belching, mild nausea, and mild dizziness, each of which was reported by eight, four, and nine persons, respectively, along with mild headache reported by 5 subjects. Past studies have reported diarrhea, decreased appetite, and headache with KS (R)-1,3-butanediol. While symptoms of nausea and dizziness/light headedness have been reported following consumption of other ketone beverages ((R)-1,3-butanediol has a bitter taste which may have contributed to the nausea. Of note, neither “other GI Symptoms” (which included nausea and dizziness) nor any of the other GI symptom categories was associated with BHB levels, even after adjusting for body weight (data not shown).GI symptomology was assessed at multiple time points throughout the study to capture the presence or absence, as well as the severity, of 19 symptoms. Overall,  on an empty stomach, without food, was considered to represent a best-case scenario in terms of increasing BHB levels, but a worst-case scenario regarding GI tolerability.R)-1,3-butanediol beverage, when a total of 34.5 g (R)-1,3-butanediol would have been consumed. (R)-1,3-butanediol was well tolerated. Unlike reports following consumption of KME and KS, no subjects reported experiencing diarrhea following consumption of the study product. The most frequently reported symptoms of belching, nausea, headache and dizziness, were mild and transient in nature. No effect on subject sleepiness was reported.In this study, peak blood BHB levels of 2.10 ± 0.97 mmol/L were achieved roughly 1-h post consumption of the last (R)-1,3-butanediol to achieve blood BHB levels typical of physiologic ketosis. There is a high likelihood of creating regimens for chronic administration which could potentially sustain a desired level throughout the day. Next steps require testing of such regimens in patients with measurable disease or performance endpoints.This study demonstrates the feasibility of administering oral (


Chromosome 16p13.11 duplication is a well‐known genetic risk factor for schizophrenia (SCZ) (odds ratio = 1.84). However, no case reports focusing on patients with SCZ and 16p13.11 duplication have been published. Therefore, here, we report the detailed clinical cases of four patients with SCZ and 16p13.11 duplication who were identified in our previous whole‐genome copy number variant (CNV) study.In the four patients with SCZ and 16p13.11 duplication detected by array comparative genomic hybridization, one patient was found to have treatment‐resistant SCZ and an additional pathogenic rare CNV. Two of the four patients in this study had environmental risk factors that may have been involved in the development of SCZ.The results of this case series suggest that a genetic cohort study would be useful for evaluating which genetic and environmental risk factors could better explain the variable expressivity of 16p13.11 duplication. Furthermore, this work could be useful for elucidating the pathophysiology of SCZ. Chromosome 16p13.11 duplication is a well‐known genetic risk factor for schizophrenia (SCZ) (odds ratio = 1.84). Therefore, here, we report the detailed clinical cases of four patients with SCZ and 16p13.11 duplication who were identified in our previous whole‐genome copy number variant (CNV) study. However, the detailed clinical course of carriers with these high‐risk variants remains unclear. Therefore, it is important to accumulate knowledge about carriers with high‐risk variants for psychiatric disorders to improve future genetic counseling.NDE1).Chromosome 16p13.11 duplication is one of the most well‐known risk factors for schizophrenia (SCZ)22.1All patients were of Japanese ancestry and had been diagnosed with SCZ according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition.2.2Genomic DNA was extracted from blood samples. CNVs in the 16p13.11 region were identified in four patients with SCZ using two types of array comparative genomic hybridization: NimbleGen 720 k Whole‐Genome Tiling Arrays (Roche NimbleGen) and Agilent SurePrint G3 Human CGH 400 K (Agilent Technologies).2.3NDE1 mRNA transcription, we compared the relative expression of a patient (Patient 2) and 16p13.11 duplication with 55 healthy controls (40.5 ± 11.8) using lymphoblastoid cell lines (LCLs). LCLs were established by Epstein–Barr virus transformation of lymphocytes and cultured in RPMI‐1460 medium containing 20% fetal bovine serum, penicillin, and streptomycin. Total RNA was extracted from LCLs using the RNAqueous Kit (Invitrogen) and treated with DNase using the TURBO DNA‐free™ Kit (Invitrogen), then reverse‐transcribed to cDNA using the high‐capacity RNA‐to‐cDNA Kit (Invitrogen). Two housekeeping genes, hypoxanthine‐guanine phosphoribosyltransferase (HPRT) and glucuronidase‐beta (GUSB), were selected as internal control genes to normalize the polymerase chain reaction (PCR) tests. Real‐time quantitative PCR was performed with the probes of the predesigned TaqMan Gene Expression Assay  using a real‐time PCR instrument . Measurement of the cycle threshold was implemented in duplicate. The data, including the amplifying efficiency and relative expression on quantification, were analyzed using the 2–ΔΔCT method.To investigate the effect of 16p13.11 duplication on 2.4Figure Patient 1 was a 79‐year‐old female with hypertension and hyperlipidemia. At age 7 years, her father passed away from pneumonia. Her grades at junior high school were above average. After graduating from junior high school, she was employed as an office worker. At age 22 years, she developed delusions and hallucinations with soliloquy and excitation. Subsequently, she was diagnosed with SCZ and hospitalized repeatedly. At the time of this study, she had been hospitalized for 30 years from age 49 years. Due to her negative symptoms, such as abulia and social withdrawal, she did not engage with other patients in the hospital. At study recruitment, she was prescribed risperidone (2 mg/day) and estazolam (1 mg/day). However, her response to these psychotropic drugs was poor.VPS13B) deletion  and levomepromazine (40 mg/day) at study recruitment, her response to antipsychotics was poor.Patient 4 was a 19‐year‐old female. She was born by normal delivery and showed no abnormalities in growth or development. She had many friends and her junior high school grades were average. In the third grade of junior high school (age 15 years), she experienced auditory hallucinations, persecutory delusions, increased irritability, unrestrained deviant behavior, verbosity and hyperactivity, and insomnia. She was subsequently hospitalized and diagnosed with SCZ. Her psychiatric symptoms improved with treatment with antipsychotics. Although she was enrolled in high school, she refused to attend. After dropping out at age 18 years, she experienced hyperthymia and unrestrained behavior, followed by a manic state with verbosity, hyperactivity, extravagant behavior, and violence, leading to hospitalization. At study recruitment, she was prescribed levomepromazine (50 mg/day), chlorpromazine (200 mg/day), and carbamazepine (400 mg/day); this pharmacotherapy was effective for her psychiatric symptoms.3To our knowledge, this is the first case series focusing on patients with SCZ and 16p13.11 duplication.Although 16p13.11 duplication is a well‐known risk factor for SCZ (OR = 1.84),VPS13B deletion. Patients with SCZ with two pathogenic CNVs are more likely to show severe clinical manifestations.Patient 2 with TRS had 16p13.11 duplication and In patients 1 and 3, environmental risk factors may have been involved in the development of SCZ. These patients experienced childhood adversities such as parental loss or separation. Such adversities are associated with a more than threefold increased risk of SCZ.Although 16p13.11 duplication has been reported to be associated with neurodevelopmental disorders  and cardiovascular disease,H.K., I.K., and N.O. designed the study. I.K. and A.Y. performed the genetic analysis. H.K., I.K., A.B., M.B., T.I., and N.O. recruited the participants and/or collected DNA samples or phenotype data. H.K. and I.K. wrote the first draft of the manuscript, and the other authors commented on and refined the manuscript. All authors carefully read the manuscript and approved the final version for submission.This research was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Ministry of Health, Labour and Welfare of Japan; the Japan Agency for Medical Research and Development (AMED) under Grant Nos. JP21dm0207075, JP21ak0101113, JP21dk0307103, JP21ek0109488, JP19km0405216, JP22tm0424222, and JP21wm0425007; the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. 15 K19720, 18H04040, 20 K20602, 21 K07543, 21H00194, 17H05090, 18 K19511, 21H02848, and 21H04815; the Uehara Memorial Foundation; and the SENSHIN Medical Research Foundation.H.K., I.K., M.B., A.B., and T.I. declare no conflict of interest. N.O. has received research support or speakers' honoraria from, or has served as a consultant to, Sumitomo Dainippon, Eisai, Otsuka, KAITEKI, Mitsubishi Tanabe, Shionogi, Eli Lilly, Mochida, DAIICHI SANKYO, Nihon Medi‐Physics, Takeda, Meiji Seika Pharma, EA Pharma, Pfizer, MSD, Lundbeck Japan, Tsumura, Novartis, Boehringer Ingelheim, Viatris, Kyowa, Janssen, Yoshitomi Yakuhin, Kyowa Kirin, Ono, Astellas, UCB, Taisho Toyama, Medical Review, and Woolsey outside the submitted work.This study was approved by the ethics committee of Nagoya University Graduate School of Medicine and other participating institutes. Written informed consent was obtained from all participants. This study complied with all the provisions of the Declaration of Helsinki.N/A


Acute physical fatigue was induced through the T-SAFT90 test. Results showed that under acute physical fatigue players with high decision-making skills maintained tactical behavior efficiency and had their paces reduced, in addition to displaying reduced total distance covered (p < 0.001), number of accelerations (p < 0.001), and decelerations (p < 0.001), and average movement speed (p < 0.001). On the other hand, players with low decision-making skills displayed reduced tactical behavior efficiency (p = 0.002) and maintained their movement pace under physical fatigue. It is concluded that decision-making skills contribute to players’ tactical behavior efficiency under acute physical fatigue, besides promoting reduced physical strain in movement actions throughout the field.The purpose of this study was to verify whether decision-making skills influence the tactical behavior and physical performance of soccer players under acute physical fatigue, assessed in an actual game-play. The sample was comprised of 24 trained soccer players (18.25 ± 1.48 years old) from two Brazilian clubs grouped into two categories with 12 players each (with high and low decision-making skills). The assessment of decision making, tactical behavior, and physical performance were carried out using TacticUP Over the last decade, decision-making has drawn attention and received growing research interest with the purpose of understanding the processes and mechanisms that sustain this skill in different game contexts . In geneResearch has indicated that players who are efficient in performing tactical actions display higher decision-making skills . BesidesAcute physical fatigue is described as a reduction in maximal voluntary muscle strength and inability to maintain a certain intensity or power level during exercises . ConsequConsidering the above, although decision-making has not been explored as a characteristic for group categorization, this skill may mediate an aspect of players’ performances under the state of physical fatigue. Studies on this topic indicate that players with higher levels of decision-making skills display better performance indexes . Also, iThe sample was comprised of 24 trained male soccer players (mean age: 18.3 ± 1.5 years) from two Brazilian clubs . As inclThe participants were previously informed about the objective of the study. The present study was approved by the Ethics Committee in Research with Human Beings (no. 3.208.190) and conformed to the norms of the National Health Council (CNS 466/2012) and the Declaration of Helsinki (2013). To take part in the study, the participants aged 18 years and older signed an informed consent form; the participants who were minors signed an assent form, while the participants’ legal guardians signed an informed consent form.Participants were assessed on tree different days .®). In the second day, the “control” condition was applied, in which participants performed the “GK+3 vs. 3+ GK” field test, monitored by GPS units. Teams were composed by one defender, one midfielder and one forward, and were assembled according to players’ technical levels following the head coach’s criteria in order to balance the level of the games , so as to meet the estimated required 18% of energy to each participant . Water intake during the experiment was This study employed the CR10 Scale, proposed by ® video test for soccer, an online assessment platform (www.tacticup.com.br) based on the core tactical principles of soccer , in actions with and without the ball, that take place near and far from the ball. Participants performed 36 trials in random order with three scenes for each tactical principle, besides having three scenes for familiarizing with the test. In each scene, four response options are displayed and participants are asked to select the most appropriate course of action for every scene. At the end, the test score is given. In the present study, the TacticUP® measure that described players’ decision-making skills was the overall score, i.e., the mean of the performance indexes of the offensive and defensive phases . The test procedure was carried out in a closed environment (indoors) and without external interference. The entire test procedure lasted around 15 min for each participant.All procedures were clarified to the participants prior to the start of the tasks, and the researcher was always available to answer potential questions. The TacticUPThe System of Tactical Assessment in Soccer (FUT-SAT) was used to assess participants’ tactical behaviour . TacticaThe FUT-SAT protocol comprises three procedures: the first consists of analyzing the players’ actions during a game, in which ball possession is considered a unit to distinguish between defensive and offensive phases; the second procedure refers to the assessment, classification, and recording of tactical actions based on spatial references of the field; and the third and last procedure refers to the calculation of variables within the “Tactical Actions” and “Percentage of Correct Actions” categories. In the present study, the percentage of accuracy and the number of tactical actions were the FUT-SAT variables used to describe the efficiency and frequency of the tactical actions, respectively. Following data collection, data analysis was performed according to the procedures proposed by Costa and colleagues (2011).®, Canberra, Australia) were used to obtain data on players’ physical performance, with a sampling rate of 5 Hz for tracking players’ positions in the field. The categorization of the intensity zones of distance covered was based on -1), walking (0.8–7.1 km/h-1), jogging (7.2–14.3 km/h-1), running (14.4–19.7 km/h-1), high-speed running (19.8–25.1 km/h-1), and sprinting (>25.1 km/h-1). In addition, the following parameters were also considered: total distance covered (m), average speed (km/h), number of sprints (AU), number of accelerations (AU) and number of decelerations (AU). The GPSports® built-in software was used to analyze physical performance.During the field and the T-SAFT90 tests, GPS units ; 5.58 km jogging (∼10.5 km h-1); 1.68 km striding (∼15 km h-1) and 0.36 km sprinting . In addition to these, they performed ball dribbling 0.36 km, 24 short passes and 12 shoots on target. In relation to the acceleration and deceleration, the players performed 1,269 changes in speed (every 4.3 s), 888 changes in direction (180°), 444 cutting maneuvers and 12 jumps ; 3.48 km walking  was named “High Skills Group” (n = 12), whereas the group with lower value (<50th percentile) was named “Low Skills Group” (n = 12). The values obtained for the groups were compared using the t-test for independent samples.Descriptive analysis (means and standard deviation) was used to describe the sample characteristics. Distribution normality for all variables was verified using the Shapiro-Wilk test. The sample was categorized in two groups according to the score in the “general” item (average performance index in the offensive and defensive phases) of the TacticUPt-test and Wilcoxon test were used to compare tactical behavior and physical performance between the “control” and “physical fatigue” conditions among groups. The test-retest method was used to calculate reliability, and the values of Cohen’s Kappa were used. Intra-observer reliability was carried out following a 3-week interval, so as to avoid task familiarity issues  for Windows®, version 24.0.The significance level was set at p < 0.001; large effect size), which indicates that, on average, players considered the physical fatigue condition “very hard”.Participants displayed higher perceived effort under the physical fatigue condition (7.9 ± 1.1 AU) when compared to the control condition (1.1 ± 1.1 AU) (z = −6.06 (11) = −7.19; p < 0.001; d = 2.93)  .t(11) = 6.40; p < 0.001; d = 1.73), running (t(11) = 3.89; p = 0.003; d = 1.71), high-speed running (t(11) = 4.32; p = 0.001; d = 1.33), average speed (t(11) = 8.20; p < 0.001; d = 2.82), number of sprints , accelerations (t(11) = 5.13; p < 0.001; d = 1.27), and decelerations (t(11) = 5.46; p < 0.001; d = 1.40) under the “physical fatigue” condition  = 3.98; p = 0.002; d = 1.31) and overall tactical behavior efficiency (t(11) = 4.18; p = 0.002; d = 1.07) were found lower under the physical fatigue condition (Table 3). On the other hand, with respect to physical performance, participants displayed a decrease only for the distances covered in running (t(11) = 2.44; p = 0.033; d = 1.12) under the state of physical fatigue  Players with higher decision-making skills under acute physical fatigue are able to maintain the quality of their actions and display reduced physical strain when tired.2) Players with lower decision-making skills under acute physical fatigue committing more mistakes when moving and positioning themselves on the field and displayed similar physical effort compared to when they were rested.3) Players with lower decision-making skills compensate the decrease of tactical behavior efficiency maintenance of physical performance.4) Decision making ability well establish enables players to find solutions for the problems the game systematically poses, despite their motor limitations.5) Developing players’ decision-making is an aspect that helps maintaining performance and even on the reduction of injury risk during matches and seasons


Mucus derived from many land snails has been extensively utilised in medicine and cosmetics, but some biological activities of the mucus need to be well documented. Nevertheless, most mucus is obtained from land snails, while mucus from freshwater snails has yet to be attended.Lissachatina fulica and the freshwater snail Pomacea canaliculata. ABTS, DPPH, reducing power and total antioxidant activity assays were used to evaluate the antioxidant capacity. Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 cells was performed to determine the anti-inflammatory activity. Additionally, the histochemical analysis of mucous cells in each snail foot was conducted to compare the distribution of mucous cells and types of mucins using periodic acid-Schiff and Alcian blue staining.This study aims to determine and compare mucus’s antioxidant and anti-inflammatory activities from the land snail L. fulica and P. canaliculata exhibited antioxidant and anti-inflammatory activities in different parameters. L. fulica mucus has higher total antioxidant (44.71 ± 2.11 mg AAE/g) and nitric oxide inhibitory activities (IC50 = 9.67 ± 0.31 µg/ml), whereas P. canaliculata mucus has better-reducing power activity (43.63 ± 2.47 mg AAE/g) and protein denaturation inhibition (IC50 = 0.60 ± 0.03 mg/ml). Histochemically, both species’ dorsal and ventral foot regions contained neutral and acid mucins in different quantities. In the dorsal region, the neutral mucins level in L. fulica (16.64 ± 3.46%) was significantly higher than that in P. canaliculata (11.19 ± 1.50%), while the acid mucins level showed no significant difference between species. Levels of both mucins in the ventral foot region of L. fulica  were significantly higher than those of P. canaliculata . This study revealed scientific evidence of the biological capacity of mucus from L. fulica and P. canaliculata as well as provided helpful information on the region of the foot which produces effective mucus.Mucus from  Skin acts as a protective barrier against external threats such as UV radiation, pollutants, and pathogens. However, the skin is constantly exposed to these harmful factors, which can induce oxidative stress. An excess of reactive oxygen species (ROS), including free radicals, in the body can lead to cellular damage and dysfunction. Oxidative stress has been implicated in various health issues and skin problems such as wrinkling, fine lines, allergies and cancer . MoreoveLissachatina fulica, the giant African snail, is a terrestrial snail whose mucus has been used since ancient times. Aqueous extracts of mucus from L. fulica showed in vitro anti-inflammatory activities, including anti-proteinase, anti-lipoxygenase and anti-protein denaturation  using histological and histochemical techniques  and golden apple snails  were collected from Chiang Mai province and identified based on shell morphology and compared with original descriptions , 2,2-azino-bis-(3-ethylbenzothiozoline-6-sulphonic acid) (ABTS), hematoxylin, eosin, alcian blue 8GX (AB), sulfanilamide, N-naphthylethylenediamine dihydrochloride, Dulbecco’s Modified Eagle Medium (DMEM) and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich . Methanol, trichloroacetic acid, sulfuric acid, deionised water, dimethyl sulfoxide (DMSO), formaldehyde, hydrochloric acid (HCl), sodium sulphate, potassium persulfate and potassium ferricyanide were purchased from RCL Labscan . Ascorbic acid, ferric chloride and ammonium molybdate were purchased from Ajax Finechem Pty. Ltd. .The antioxidant activity assays were performed to evaluate the capacity of snail mucus to inhibit different free radicals DPPH, and ABTS radical scavenging assays were conducted based on hydrogen atom transfer and electron transfer reactions, respectively. Reducing power ability was evaluated by measuring the transformation of Fe (III) to Fe (II), and total antioxidant activity was evaluated by measuring the reduction of Mo (VI) to Mo (V).0 is the absorbance of the DPPH solution and A s is the absorbance of the sample at 30 min .The DPPH radical scavenging activity of mucus and gallic acid (used as a standard) was determined in terms of hydrogen-donating ability following the method of 0 is the absorbance of the ABTS solution and As is the absorbance of the sample .The ABTS assay was conducted according to the method of The reducing ability of samples was estimated using a Fe (III) to Fe (II) reduction assay . 100 µl The total antioxidant activity of samples was determined using the phosphomolybdate assay  based onThe anti-inflammatory activity was evaluated by measuring the inhibition of nitric oxide (NO) production using a macrophage inflammatory assay and the inhibition of protein denaturation.2 humidified incubator. The cell viability was performed using a modified MTT assay (6 cells/well), the medium was discarded and replaced with 100 µl of the sample (0.4 to 200 µg protein/ml) then the cells were re-incubated for 24 h at 37 °C in 5% CO2. After incubating, the sample in each well was removed, and 30 µl of MTT solution  was added to each well and incubated at 37 °C for 4 h. Subsequently, 200 µl of DMSO was added to each well to dissolve the formazan crystals, and the absorbance was measured at 540 nm. The concentration of mucus that could reduce the absorbance to 50% compared to untreated cells was used as the median lethal concentration (LC50) value.RAW 264.7, a mouse macrophage cell line, was obtained from the Division of Microbiology, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. The cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a 5% COTT assay . After o6 cells/ml) were incubated with lipopolysaccharide (LPS) (1 µg/ml) and treated with 100 µl of mucus for 24 h. The amount of NO was indirectly measured in the form of nitrite in the culture medium. Briefly, the culture medium was incubated with an equal volume of Griess reagent for 15 min in the dark at room temperature, and the absorbance was measured at 550 nm. The inhibition of NO production was calculated as a percentage using the following formula, and each sample’s half-maximal inhibitory concentration (IC50) was determined. c is the absorbance of the control LPS and As is the absorbance of the sample.The level of NO, an acute inflammatory mediator released from macrophage cells, was indirectly determined based on the Griess reaction. RAW 264.7 cells (1 × 1050) of each sample was determined. c is the absorbance of control and As is the absorbance of the sample.The method was conducted according to Ten adults of each snail species were used to prepare tissue sections. Small pieces of the foot were dissected and fixed in 10% neutral buffered formalin for 24 h. The fixed tissues were decalcified by soaking in 10% HCl for 2 h and neutralised by sodium sulphate for 12 h. The tissues were washed in running tap water before processing using a standard paraffin technique and cut into 6 µm-thick sections for histological and histochemical study.The sections from every tissue sample were stained with Harris’s haematoxylin and eosin (H&E) for standard histological study . For hisThe PAS- and AB-stained sections were used to identify the different types of mucin-secreting mucous cells in the dorsal and ventral foot surfaces. Images of each surface were taken . Image analysis software, ImagesJ , was used to measure the area of stained mucin following the colour deconvolution and quantification method . The rest-test was used to compare the percentage area of mucin between dorsal-ventral areas and between different species at a 5% level of significance.The results were analysed using SPSS version 17 for Windows. For antioxidant and anti-inflammatory activities, independent sample t-tests and one-way ANOVA, followed by Tukey’s test for multiple comparisons, were used to compare the ability between mucus of different species. For the study of mucin quantification, an independent sample L. fulica mucus and the P. canaliculata mucus were 1.49  ±  0.37 and 1.13  ±  0.19 mg/ml, respectively. Therefore, the mucus concentration was adjusted to 1 mg/ml, which was used as the maximum concentration for this study.Protein concentration in the filtered fresh mucus was estimated before use. The results showed that the protein contents of the L. fulica and P. canaliculata at 1 mg/ml showed no free radical scavenging activity of DPPH and weakly scavenged ABTS radical cation. The L. fulica mucus exhibited the reducing power of 36.02 mg AAE/g protein, while P. canaliculata mucus showed a higher reducing power of 43.63 mg AAE/g protein at the same concentration. Furthermore, the result of total antioxidant activity revealed that the L. fulica mucus showed a higher total antioxidant activity of 44.71 mg AAE/g.Antioxidant activities of the snail mucus are shown in 50 value for cytotoxicity and the IC50 value for inhibition of NO production. The concentrations of the mucus that yielded cell viability higher than 90% were used for NO inhibitory analysis. It was observed that 90% of the RAW 264.7 macrophage cells were viable at the concentration of 0.39 to 12.5 µg/ml for L. fulica mucus, and the LC50 value is 96.89 µg/ml. The percent viability of the RAW 264.7 cells treated with P. canaliculata mucus was lower than that treated with L. fulica mucus at the same concentrations with a non-significant difference  exhibited only 16.79% NO inhibition; therefore, the IC50 of P. canaliculata mucus could not be determined. L. fulica mucus exhibited a significant dose-dependent decrease in NO production. The highest concentration of L. fulica mucus (12.50 µg/ml) showed 61.90% NO inhibition, and its IC50 value was 9.67 ± 0.31 µg/ml. Strong inhibitory activity was found in L. fulica mucus at 3.13, 6.25, and 12.5 µg/ml concentrations, presenting a significant difference from the untreated group (control LPS) at p < 0.01.The inhibitory potentials of the mucus on proinflammatory molecule NO are presented in L. fulica and P. canaliculate exhibited the inhibitory capacity of 66.28% and 71.56%, respectively  to Fe (II) by donating a hydrogen ion. This reducing capacity reduces metal ions, especially ferrous ion which is pro-oxidant. In addition, our findings presented a different consequence from reducing power, where L. fulica mucus exhibited better total antioxidant activity than P. canaliculata mucus. These results indicated that antioxidant activity of the snail mucus was attributed to reducing capacity. This finding agrees with previous research which indicated that the extract from L. fulica mucus was particularly effective in reducing capacity, but it showed no DPPH free radical scavenging activity  in cells like macrophages and reacts with superoxide anions, generating peroxynitrite, which leads to the oxidation of low-density lipoprotein and causes cell apoptosis and subsequent inflammation  may have potential applications in medical and cosmetic products, akin to those of land snails (L. fulica).Our research has shown that the mucus from 10.7717/peerj.15827/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15827/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15827/supp-3Supplemental Information 3Click here for additional data file.