Caenorhabditis elegans is a suitable model to study the toxic effects of nanoparticles as it is amenable to comprehensive phenotyping, such as locomotion, growth, neurotoxicity and reproduction. In this study, we systematically evaluated the effects of silver (Ag) and five metal oxide nanoparticles: SiO2, CeO2, CuO, Al2O3 and TiO2. The results showed that Ag and SiO2 exposures had the most toxic effects on locomotion velocity, growth and reproduction, whereas CeO2, Al2O3 and CuO exposures were mostly neurotoxic. We further performed RNAseq to compare the gene expression profiles underlying Ag and SiO2toxicities. Gene set enrichment analyses revealed that exposures to Ag and SiO2consistently downregulated several biological processes  and pathways , with opposite effects on genes involved in innate immunity. Our results contribute to mechanistic insights into toxicity of Ag and SiO2 nanoparticles and demonstrated that C. elegans as a valuable model for nanotoxicity assessment.Engineered nanoparticles are used at an increasing rate in both industry and medicine without fully understanding their impact on health and environment. The nematode  The use of engineered nanoparticles has increased enormously over the last decade, and the nanotechnology industry has grown from a 10-billion-dollar enterprise in 2012 to being valued over one trillion dollars in 2015 . HoweverCaenorhabditis elegans is a free-living soil nematode reaching approximately one millimeter in length in adult stage and has a relatively simple life cycle that can be grown on solid  or liquid media   . The smaminthics . In termpathways .C. elegans as a model for nanotoxicity, as this bacterivore worm constantly interacts with microbes in nature, which are ingested through the pharynx, the main potential route of exposure to nanoparticles is consequently oral  to study underlying molecular mechanisms. For instance, using gene expression microarray, Rocheleau et al. found that C. elegans exposed to nano-TiO2 showed increased expression of the glutathione S-transferase gene gst-3 and the cytochrome P450 gene cyp33-11; while the oxidative stress response, as measured by the stress resistance regulator scl-1, showed increased expression after exposure to both nano- and bulk-sized TiO2  nanoparticles , and measured four endpoints  in C. elegans after exposure to these nanoparticles. Worms that exhibited the most significant effects were subjected to RNAseq to identify the affected biological processes and pathways. Hence, we offer a novel perspective to study nanoparticle toxicity using the soil nematode C. elegans.The fast phenotyping of zed TiO2 . In addiexposure . Based oarticles . To the C. elegans N2 strain was obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota. Escherichia coli OP50 was also obtained from the CGC and was grown for 18 h at 37 °C in Luria-Bertani (LB) broth  broth . The N2 eir eggs . Eggs weeir eggs .2, CuO, Al2O3, and CeO2 nanoparticles were purchased from Sigma-Aldrich . TiO2 nanoparticles were obtained from the Joint Research Center of (JRC) the European Commission. All nanoparticles were less than 100 nm in size as described by the manufacturer and commission. Product details are shown in 2O and sonicated using an Ultrasonic Processor VCX (GEX) 750 at an amplitude of 40% for a 3-minute pulse, followed by 1 min on ice. This step was repeated five times to ensure complete disaggregation of the nanoparticles. Nanoparticle solutions were then diluted to working concentrations of 200 µg/ml in S-medium , 10 or 50 µg/ml of each nanoparticle, supplemented with E. coli OP50 at a final optical density at 595 nm (OD595) of 1, for 72 h at 21 °C until they reached the day 1 adult stage. For the locomotion velocity endpoint assay, worms were then washed once in 1X M9 buffer and placed on unseeded NGM plates and allowed to explore their surroundings for 10 min. Worms (n = 14 to 111 per condition) were then recorded using a Nikon camera (SMZ1270) linked to a computer. The average locomotion velocity of each worm was calculated for 30 s at an interval of 0.500 ms using the software  accompanying the camera. The average locomotion velocity was calculated by averaging the locomotion velocity (in µm/s) over the 30 s of recording. For the growth (body length) endpoint assay, worms were grown in the same manner as in the locomotion velocity endpoint assay, washed once in 1X M9 buffer and killed with 10 mM sodium azide. Dead worms were transferred to an unseeded NGM plate to take pictures. The body length of worms (n = 20 to 43 per condition), measured in µm, was calculated using the camera’s software .200 L1 stage C. elegans N2, obtained by synchronization, were grown for 48 h at 21 °C to the L4-young adult stage on E. coli OP50-seeded NGM plates. Five L4-young adult hermaphrodites were transferred to an individual well in quadruplicate of a 12-well plate containing S-medium supplemented with either 0 (control), 10 or 50 µg/ml of each nanoparticle and E. coli OP50. The L4-young-adult hermaphrodites were then allowed to grow and lay eggs for 96 h, and resulting progeny were counted by dilution.200 L1 C. elegans N2 were grown to the adult day 1 stage (72 h at 21 °C) in individual wells of a 6-well plate containing S-medium containing either 0 (control), 10 or 50 µg/ml of each nanoparticle and E. coli OP50. A total of 1.0 ml of the well contents were centrifuged at 1,000 rpm for 2 min, the supernatant was decanted, leaving the worm pellet undisturbed. Worms were then washed in 1X M9 buffer and centrifuged at 1,000 rpm for 2 min. Worms were then transferred to an unseeded NGM plate containing 60 µl of K-medium (2.36 g of KCl and 3.0 g NaCl per liter of media dissolved in ddH2O). Individual adult day 1 stage C. elegans were transferred into the drop of K-medium. Worms were allowed to swim freely for 1 min. Afterwards, the number of head thrashes of each individual worm (n = 8 to 55 per condition) were counted for 1 min as described by 200 L1 C. elegans N2 were grown in individual wells of a 12-well plate containing either 0, 10 µg/ml Ag nanoparticles or 10 µg/ml SiO2 nanoparticles and supplemented with E. coli OP50, until the worms reached the adult day 1 stage (72 h at 21 °C). Each condition was repeated six times. The contents of the wells were centrifuged at 1,000 rpm for 2 min, the supernatant was decanted and the worm pellet was washed twice with 1X M9 buffer. 200 µl of Trizol  was then added to the worm pellet. The worm pellet then was flash-frozen in liquid nitrogen, followed by a quick thaw. These two steps were repeated once. RNA from the resulting worm pellet-Trizol solution was extracted using the Direct-zol RNA miniprep kit  according to the manufacturer’s instructions. Quantity and purity of total RNA were checked using a spectrophotometer . The RNA samples were then sent to the McGill University and Génome Québec Innovation Centre (http://gqinnovationcenter.com) for quality analysis with Bioanalyser and for single-end read (100 base) RNA sequencing under HiSeq 2500 (Illumina).400 L1 https://www.bioinformatics.babraham.ac.uk/projects/). The genome sequence of C. elegans and GTF file (Caenorhabditis_elegans.WBcel235.91.gtf) were downloaded from ENSEMBL (https://www.ensembl.org/). Reads were aligned to the C. elegans genome with HISAT2 (version 2.1.0) (C. elegans was downloaded from GO2MSIG (C. elegans was extracted from a current Bioconductor package (version 3.7)  and adapter related sequences were removed using Trim Galore (version 0.4.5) (n 2.1.0)  and sortn 2.1.0) . Raw rean 2.1.0) . Entrez n 2.1.0)  and assin 2.1.0) . Differen 2.1.0) . For genn 2.1.0) . The val GO2MSIG  and KEGGion 3.7)  and convion 3.7) . Furtherion 3.7)  and Wormion 3.7) , respection 3.7) .P value less than 0.05 was accepted as statistically significant.Statistical analysis was performed using GraphPad Prism (version 8.0.0). The statistical difference between the groups in the toxicity assays was evaluated with One-way ANOVA followed by Dunnett’s multiple comparison test and C. elegans grown under control conditions was 150.1 µm/s across all treatments, indicating that worms are active after growth in S-medium for 72 h at 21 °C. In comparison, the average locomotion velocity of worms decreased to 91.5 µm/s in the presence of 10 µg/ml Ag nanoparticles and further reduced to 44.2 µm/s in the presence of 50 µg/ml Ag nanoparticles, which was statistically significant for each concentration  . Surprisingly, it appeared that TiO2 nanoparticles increased C. elegans average locomotion velocity at 10 µg/ml (P = 0.0257), although this could be a statistical artifact due to sampling effect based on the data distribution , displaying locomotion velocities of 104.4, 114.8 and 112.2 µm/s, respectively .The pooled average locomotion velocity for ctively) . Additioribution . Al2O3-,ectively –1E. CeO2P < 0.0001 for both concentrations) (2 nanoparticles induced significant decrease at 10 µg/ml (P < 0.0001) (P < 0.0001 for both concentrations)   assay, Ag nanoparticles had the greatest effect. The pooled average body length of worms grown in the control S-medium after a three-day incubation at 21 °C was 1199.6 µm, indicating that the worms grew efficiently in this medium. As with the locomotion velocity endpoint assay, a concentration-dependent decrease in body length was observed when worms were exposed to different concentrations of Ag nanoparticles, with average body lengths of 1017.8 µm under 10 µg/ml and 859.3 µm under 50 µg/ml, respectively (rations) . Exposur 0.0001) , but no rations) . For CeO= 0.001) . In summC. elegans reproduction, we incubated five L4-young adults in S-medium supplemented with E. coli OP50 and the respective nanoparticles for four days (96 h) at 21 °C. It was expected that each worm would lay approximately 300 eggs in that time span, so that the total number of progeny per 5 worms would be near 1,500 under control conditions  (C. elegans brood size (P < 0.0001)   (P > 0.05)  (To measure the effects of the various nanoparticles on nditions . The ave 0.0001) . The eff 0.0001) . On the  0.0001) , indicaton range . CeO2 na 0.0002) . Interes > 0.05) . This ob > 0.05) , whereas > 0.05) .2 nanoparticles showed no significant impact of the number of head thrashes based on a population of worms in 60 s at 10 µg/ml versus control worms (2 nanoparticles showed a slight effect on the neurotoxicity assay at 50 µg/ml (P = 0.0033), whereas Ag nanoparticles had no such effect on neurotoxicity. Similar results were observed for TiO2 and nanoparticles at both concentrations tested , although the effect on neurotoxicity of CuO nanoparticles at 50 µg/ml was not significant (P > 0.05) (Both Ag and SiOol worms  and 4B. s tested . In cont > 0.05)  and 4F.C. elegans exposed to Ag and SiO2(10 µg/ml) as these two nanoparticles demonstrated the most outstanding effect on majority of the parameters . For each sample , around 20 million reads were obtained. Approximately 97% of reads were mapped to the worm’s genome and a total of 18,861 gene sequences were identified, using a minimal total read count of 3 across samples. Sample distribution by principal component analysis (PCA) is shown in Based on the phenotyping results, we further performed RNAseq analysis on Apoptotic process showed gene enrichment with positive NES in both Ag and SiO2 groups under significant levels (FDR < 0.0001). Phenotype reflecting BPs including regulation of locomotion, reproductive process and cell growth were enriched with negative NES significantly   based on Gene Ontology biological processes (BPs) showed various statistically enriched positively or negatively based on the fold changes and running enrichment scores . The top2 group) . A numbe2 group) .2 nanoparticles were foreign substances for the worm, we further examined the expression profiles of genes associated with innate immunity. Compared to Ag and Control, SiO2 exposure led to significant downregulations of genes related to the innate immune response (FDR < 0.0001)  .The enriched BPs by GOATOOLS were mainly related to the physiological events, of which, some were identical to those observed by the GSEA analysis . HoweverC. elegans , which were tachykinin receptor family (tkr-2), serotonin/octopamine receptor family (ser-1) in neuroactive ligand–receptor interaction pathway, skp1 related  in wnt signaling pathway, protein ver-1 (ver-1) and heat shock protein (hsp 70) in MAPK signaling pathway , but not on neurotoxicity. In this model, the transcriptome profile is concordant with the phenotype characteristic for both exposures  and MiRP K channel accessory subunit (mps-1) (regulation of locomotion), caveolin (cav-1) (regulation of reproductive process), and cyclic nucleotide-gated cation channel (tax-4) and protein let-756 (let-756) (regulation of cell growth), have been previously shown to hinder the worm’s biological events . Inactiv2 nanoparticles induced a remarkable downregulation pattern in innate immune response, compared to Ag and Control. In particular, several C-type lectins, which are known to be important components in innate immunity , wnt and MAPK signaling pathways  and mediate protein folding, influencing various regulatory proteins, respectively , serotonin/octopamine receptor family (ser-1) (neuroactive ligand–receptor interaction pathway), skp1 related  , and protein ver-1 (ver-1) and heat shock protein (hsp 70) . The proteins encoded by tachykinin receptor and ser-1 genes regulate locomotion via mediate neurotransmission and indirect modulation of neuromuscular circuits, respectively . The pro elegans . Putativectively . Overall2 also showed opposite effects on some of the top 20 enriched biological pathways. Spliceosome, mRNA surveillance and endocytosis pathways were enriched with positive NES in Ag, but with negative NES in SiO2, despite no obvious phenotypic differences were observed in our studies.Apart from the many similarities observed between the two nanoparticle effects, Ag and SiO2 exposures with the findings in previous studies on metal toxicities indicated that metallothionein-2 (mtl-2), a commonly observed responsive gene to the metal toxicities   , was up-2nanoparticles have been shown to affect locomotion velocity in C. elegans N2, as previously described  are relatively unknown based on the literature. In terms of neurotoxicity experiments, as determined by the number of head thrashes per minute in our study, our results differ from published results  were unconfirmed to adopt any shape at all. Further study is required to elucidate the answer to this question. It was shown that co-feeding nanoparticles with E. coli OP50 in S-medium leads to the uptake of these nanoparticles through the pharynx and absorption through the gut (2) nanoparticles were shown to inhibit the peptide transporter OPT-2/PEP-2, present on the apical layer of the intestinal membrane in C. elegans , demonstrated only slight growth defects, as determined by measuring bacterial density . With all these observations, we provide a novel angle to study the toxicity of nanoparticles on organisms, by exploring the mode of action of Ag and five metal oxide nanoparticles on different life history endpoints in C. elegans. To the best of our knowledge, this is the first study that integrates phenotype screening with RNAseq to investigate nanotoxicity in intact animals using C. elegans. Our RNAseq data not only confirmed positive enrichment of apoptotic process as reported in the literature  is shown.Sample distributionClick here for additional data file.10.7717/peerj.8684/supp-2Figure S2E. coli OP50 was grown in 50 µg/ml of CeO2, Ag, SiO2, TiO2, Al2O3 and CuO nanoparticles in S-medium in a 12-well plate at 21 °C. Bacterial density was measured by taking the OD595 nm values of three independent wells. Statistical significance, after five days incubation, is indicated with an asterisk. * P < 0.05, *** P < 0.01, **** P < 0.0001, compared to E. coli OP50 alone.Click here for additional data file.10.7717/peerj.8684/supp-3Data S1A total of 2648 genes were differentially expressed in Group Ag and a total of 1087 genes were differentially expressed in Group SiO2, in comparison with Control (FDR < 0.05).Click here for additional data file.10.7717/peerj.8684/supp-4Data S2P < 0.05). Genes with description for the top 20 enriched biological processes are provided.Enriched biological processes showing up- and down-regulation patterns are shown for both Ag and SiO2 groups . Genes with description for the top 20 enriched biological pathways are provided.Enriched biological pathways showing up- and down-regulation patterns are shown for the Ag and SiO2 groups (Click here for additional data file.10.7717/peerj.8684/supp-6Supplemental Information 1E. coli OP50 growth, measured at OD 595 nm, is shown with tested nanoparticles.Click here for additional data file.10.7717/peerj.8684/supp-7Supplemental Information 2Raw data/raw numbers for locomotion velocity, body length, reproduction capacity and neurotoxicity for Click here for additional data file.


Dorsal and median raphe nuclei  are members of the reticular activating system and play important role in the regulation of the sleep-wakefulness cycle, movement, and affective states. M-current is a voltage-gated potassium current under the control of neuromodulatory mechanisms setting neuronal excitability. Our goal was to determine the proportion of DR and MR serotonergic neurons possessing M-current and whether they are organized topographically. Electrophysiological parameters of raphe serotonergic neurons influenced by this current were also investigated. We performed slice electrophysiology on genetically identified serotonergic neurons. Neurons with M-current are located rostrally in the DR and dorsally in the MR. M-current determines firing rate, afterhyperpolarization amplitude, and adaptation index (AI) of these neurons, but does not affect input resistance, action potential width, and high threshold oscillations.These findings indicate that M-current has a strong impact on firing properties of certain serotonergic neuronal subpopulations and it might serve as an effective contributor to cholinergic and local serotonergic neuromodulatory actions. DR and MR are members of the reticular activating system and play important role in the regulation of sleep-wakefulness cycles, movement, and affective states. The activity of DR is increased during wakefulness, meanwhile, it is less active in paradoxical or REM-sleep Monti, .2C receptor  and sucrose (130 mm). All chemicals were purchased from Sigma–Aldrich , unless otherwise stated.Slice electrophysiological experiments were conducted in artificial cerebrospinal fluid (aCSF) with the following composition (in mM): NaCl, 120; NaHCOn = 26). Obtaining these mice, homozygous floxed-stop- tdTomato  and Sert-cre  strains  were crossed. Midbrain coronal slices (200 μm thickness) were prepared in ice-cold (0 to −2°C) low Na+ aCSF with a Microm HM 650V vibratome . Slices were incubated in normal aCSF for 1 h at 37°C before the experiment.Animal experiments were performed according to the appropriate national, international  and institutional guidelines and laws on the care of research animals . 12–22 days old mice expressing tdTomato fluorescent protein in a Serotonin transporter- (Sert) dependent way were used from both sexes  with an Axopatch 200A amplifier . Clampex 10.0 software was used for data acquisition, whereas data analysis was performed by Clampfit 10.0 software . Only stable recordings with minimal leak currents were considered, with series resistance below 30 MΩ and less than 10% change of the series resistance was tolerated. To eliminate action potential firing, 1 μM tetrodotoxin was used .Patch pipettes with 6–8 MΩ resistance were filled with the following internal solution (in mM): K-gluconate, 120; NaCl, 5; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10; NaDetails of voltage and current clamp protocols are shown in Patched neurons were labeled with biocytin and slices were fixed in 4% paraformaldehyde . For permeabilization, Tris-buffered saline  with 0.1% Triton X-100 and 10% bovine serum (60 min) was used. Slices were incubated with streptavidin-conjugated Alexa488  in TBS for 90 min.A 3-dimensional map of somata with or without M-current was drawn with NeuroLucida software  by using Paxinos atlas  was applied to assess correlations between parameters by plotting them against each other and by the linear fit of this dataset.Data are presented as mean ± SEM. Statistical significance was determined with a 2-sample Student’s n = 31 from the DR and n = 25 from the MR; The topographical organization of M-current possessing raphe serotonergic neurons was characterized and the impact of this current on other electrophysiological properties was described by patching 56 genetically identified serotonergic neurons (n = 24), holding current recorded at −20 mV was 153.94 ± 23.28 pA. Relaxation current was 25.9 ± 3.7 pA recorded at −60 mV repolarizing step; 39.16 ± 4.8 pA at −50 mV; 49.04 ± 6.64 pA at −40 mV and 44.26 ± 9.24 pA at −30 mV, respectively. When 20 μM XE991, M-current specific inhibitor was administered, the holding current was reduced to 120.78 ± 46.1 pA (p = 0.2), the relaxation current at −60 mV was 4.14 ± 2.64 pA (p = 0.0002), 11.72 ± 5.6 pA at −50 mV (p = 0.0033), 17.06 ± 9.27 pA at −40 mV (p = 0.013) and 19.14 ± 9 pA at −30 mV , holding current was 62.4 ± 9.12 pA at −20 mV; while relaxation current was 0.75 ± 0.32 pA at −60 mv, 4.1 ± 0.1 pA at −50 mv, 6.8 ± 1.18 pA at −40 mv and 7.6 ± 1.8 pA at −30 mv, respectively. Recorded currents were not altered significantly by XE991; since the holding current was 39.05 ± 9.38 pA at −20 mv (p = 0.12), 0.17 ± 1.42 pA at −60 mv (p = 0.45), 0.66 ± 1.12 pA at −50 mV (p = 0.24), 1.46 ± 0.74 pA at −40 mv (p = 0.22) and 2.17 ± 2.77 pA at −30 mV . 38.1% (8 from 21) of MR serotonergic neurons had M-current, whereas this proportion was somewhat lower in the DR  and 7.56 ± 0.84 Hz for those lacking M-current . Similarly, spike frequency adaptation, quantified by the adaptation index (AI), was also significantly different between the two groups. In neurons having M-current, the adaptation index was 0.77 ± 0.08 (n = 6), whereas this parameter was 0.44 ± 0.04 in those without M-current  while 15.37 ± 1.44 mV (n = 16) in neurons lacking M-current (p = 0.015). Medium AHP amplitude recorded 100 ms after the AP peak potential was 17.01 ± 2.95 mV in neurons possessing M-current, while in neurons lacking M-current this parameter was 11.65 ± 1.54 mV (p = 0.046). Slow AHP measured 300 ms after the AP peak was 10.87 ± 4.39 mV in neurons having M-current and 3.77 ± 1.25 mV in those without M-current , whereas the power maximum was 1.95 ± 1.31 mv2/Hz for cells with and 3.82 ± 3.39 mv2/Hz for ones without M-current  and rather located dorsally in the MR , whereas power amplitude was directly proportional to the input resistance  project to different brain regions, and these projections have topographical organization along the dorsoventral axis (Hale and Lowry, via the 5HT2C receptor (Roepke et al., via M-current might be involved in stress adaptation. These findings potentially provide further support to Zhao et al. (M-current is inhibited by muscarinic acetylcholine receptor activation (Brown and Passmore, o et al.  that M-cThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The animal study was reviewed and approved by Committee of Animal Research of the University of Debrecen.TB, AC and AK performed the experiments. AK, KP and BP analyzed the experiments and wrote the article. All authors contributed to the article and approved the submitted version.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.


Nα-deoxycholyl-l-lysyl-methylester was incorporated into ELNE, yielding ELNE#7. As expected, ELNE#7 showed 4.07- and 2.25-fold increases in artificial membrane and Caco-2/HT29-MTX-E12 permeability (appP), respectively, resulting in 224% greater oral bioavailability compared with the commercially available ETP emulsion. In contrast, inhibition of clathrin- and caveola-mediated endocytosis, macropinocytosis, and bile acid transporters by chlorpromazine, genistein, amiloride, and actinomycin D in Caco-2/HT-29-MTX-E12 monolayers reduced the appP by 45.0%, 20.5%, 28.8%, and 31.1%, respectively. These findings suggest that these routes play important roles in enhancing the oral absorption of ELNE#7. In addition, our mechanistic study suggested that P-glycoprotein did not have an inhibitory effect on the permeation of ELNE#7. Notably, ELNE#7 showed significantly enhanced toxicity in LLC and A549 cells compared with ETP-E. These observations support the improved oral absorption of ETP in ELNE#7, suggesting that it is a better alternative than ETP emulsion.In this study, a system for oral delivery of etoposide (ETP) was designed to avoid the problems associated with low and variable bioavailability of a commercially available ETP emulsion comprised of polyethylene glycol, glycerol, and citric acid anhydrous. ETP was complexed with low-molecular-weight methylcellulose (ETP/LMC) and loaded into a water-in-oil-in-water multiple nanoemulsion to formulate an ETP/LMC-nanoemulsion (ELNE). To further enhance the oral bioavailability, an ionic complex formed by anionic lipid 1,2-didecanoyl-sn-glycero-3-phosphate  and cationic  However, oral administration of antineoplastic agents is often compromised by their limited and variable bioavailability due to poor aqueous solubility and permeability of the drugs. To address these issues, they are generally solubilized in an excess of organic solvent/co-solvent and are administered as the maximum tolerated dose via the intravenous route  consisting of polyethylene glycol (PEG), glycerol, and citric acid anhydrous  and the anionic lipid 1,2-didecanoyl-sn-glycero-3-phosphate  (PA) (DCK–PA), as a functional ligand, into the oil phase of ELNE  and A549 cells and the oral bioavailability in rats were evaluated and compared with those of commercially available hydrophilic ETP emulsion.The main objective of our investigation was to design a drug delivery system capable of markedly increasing both the aqueous solubility and intestinal permeability of ETP. Multiple approaches were used. First, the aqueous solubility of ETP was increased by forming a physical inclusion complex with low-molecular-weight methylcellulose (LMC) (ETP/LMC). Second, the permeability was enhanced by loading the ETP/LMC complex into a water-in-oil-in-water (w/o/w) multiple nanoemulsion to formulate ETP/LMC-nanoemulsion (ELNE). Third, the oral absorption was further improved by actively targeting an apical sodium-dependent bile acid transporter (ASBT) present in the ileum by anchoring a complex of the cationic bile acid derivative 2.2.1.d-alpha-tocopherol polyethylene glycol succinate (TPGS), sodium carboxymethylcellulose (NaCMC), deoxycholic acid, chlorpromazine, methyl-β-cyclodextrin (MBCD), brefeldin A, genistein, actinomycin D, Cys A, ethylene glycol-bis-(2-aminoethyl ether)-N,N,N′,N′-Cys A, ethylene glycol-bis-(2-clofazimine) were obtained from Sigma-Aldrich Inc. . Cellulase (105 U/mg) was purchased from Worthington Biochemical Corporation . Propylene glycol monocaprylate (Capryol 90), caprylocaproyl macrogol-8 glycerides (Labrasol), diethylene glycol monoethyl ether (Transcutol HP), and Tween 80 were provided by Gattefossé . PA (10:0) was obtained from Avanti Polar Lipids . All other chemicals for high-performance liquid chromatography (HPLC) and liquid chromatography/tandem mass spectrometry (LC/MS) analyses were obtained from Thermo Fisher Scientific Inc. .ETP, teniposide , methylcellulose , hexafluro-2-propanol, polyoxyethylene sorbitan monooleate (Tween 80), 2.2.Sprague–Dawley rats  were provided by OrientBio . Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee of Mokpo National University  . All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the guidelines of the Institutional Animal Care and Use Committee.2.3.The physical complex ETP/LMC was prepared to enhance the solubility of ETP in water. First, LMC was prepared by enzymatic degradation of methylcellulose, as described previously . First, the primary w/o nanoemulsion was prepared. Briefly, ETP/LMC was dispersed in water followed by oil phase titration using Capryol 90, Labrasol, and Transcutol HP as the aqueous, oil, surfactant, and co-surfactant phases, respectively. The primary nanoemulsion was comprised of a 25.9% (w/w) aqueous solution of ETP/LMC, 68.3% surfactant/co-surfactant mixture , and 5.80% oil phase . Second, the w/o/w ETP/LMC was prepared by aqueous titration using the primary nanoemulsion, Tween 80, and deionized water as the secondary oil, surfactant, and aqueous phases, respectively. The final composition of w/o/w nanoemulsion was as follows: 70.0% (w/w) w/o nanoemulsion (oil phase), 9.69% (w/w) surfactant, and 19.3% (w/w) deionized water  , and zeta potential of ELNE were determined using a dynamic laser light scattering analyzer . Morphological evaluation of the selected ELNE was performed by transmission electron microscopy .2.4.2.4.1.eP) of each sample was calculated according to the formula:eP is the permeability (cm/s), A is the effective permeation area (0.288 cm2), VD is the volume of the donor well (0.2 mL), VA is the volume of the acceptor well (0.3 mL), t is the total incubation time in s, CA(t) is the concentration of the drug in the acceptor well at time t, and Cequilibrium represents CD(t) denotes the concentration of the drug in the donor well at time t.A parallel artificial membrane permeability assay was performed using pre-coated 96-well plates designed for this assay  according to the manufacturer’s instructions. Briefly, 300 µL PBS (pH 6.8) was added to the receiver plate, and 200 µL of each formulation diluted in PBS (pH 6.8) containing 100 µg/mL ETP was added to each well of the filter plate. The plates were then assembled, and the system was incubated at room temperature for five hours without shaking. After five hours, the plates were disassembled, and samples were withdrawn from both the donor and acceptor plates. The concentration of ETP that permeated through the artificial membrane was measured by HPLC at 254 nm. A C18 column  was used with a mobile phase consisting of acetonitrile and water  at a flow rate of 1 mL/min. The effective permeability  and HT29-MTX-E12  cells were seeded onto polycarbonate 24-well Transwell® filters at a density of 1 × 105 cells/well (8:2). The confluent monolayers obtained (18–21 days) were utilized to assess the in vitro permeability. Culture media in the apical and basolateral compartments were replaced at two-day intervals, and the integrity of the monolayer was monitored by measuring the transepithelial electrical resistance (TEER). Only cell monolayers with a TEER value of 450 ± 100 Ω·cm2 were used in the study. Before the experiments, culture medium in both chambers was replaced with pre-warmed Hank’s balanced salt solution (HBSS). Cultures were then stabilized at 37 °C for 20 minutes. For the permeation studies, 0.1 mL drug formulation diluted with HBSS  was added to the apical side, and the basolateral side was replaced with 0.6 mL fresh HBSS. The treated cells were incubated at 37 °C ± 0.5 °C. The amount of permeated drug was determined by collecting 0.2 mL samples from the basolateral compartment, followed by replacement with 0.2 mL fresh HBSS at 0, 0.5, 1, 2, 3, 4, and 5 hours. The collected samples were then filtered through 0.45-µm polyvinylidene difluoride filters, and the concentrations of ETP in the samples were determined by HPLC using a UV detector at 254 nm, as described above.Caco-2 (ATCCappP) of ETP in various formulations was calculated as follows:Q/dt is the slope of the cumulative drug permeated versus time (µg/h), A is the surface area of the monolayer (0.33 cm2), and C0 is the initial concentration of ETP on the apical side (µg/mL).The apparent permeability  and allowed to proliferate until they had formed a monolayer. Culture medium was replaced with an aqueous solution of ETP/LMC, ETP/LMC in 0.3% NaCMC, ENE, ELNE#1, and ELNE#7 formulations. All samples were co-loaded with coumarin-6 (10 µg/mL) as a model hydrophobic drug, then diluted with DMEM to a concentration of 10 µg/mL ETP. Notably, ETP and ETP/LMC cannot be conjugated directly with fluorescence dye. Subsequently, cells were incubated at 37 °C for three hours and treated with phalloidin-rhodamine (100 nM) to stain actin filaments; the nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride. The cells were rinsed three times with PBS (pH 7.4). Cellular uptake was then evaluated using a confocal laser scanning microscope .Enhanced cellular uptake of ELNE was assessed in Caco-2 cells. Briefly, Caco-2 cells were seeded at 5 × 104 cells/coverslip, then transfected with the human ASBT gene using Lipofectamine 2000® . After the transfected cells had formed a confluent monolayer, they were treated with coumarin-6-co-loaded ETP/LMC solution; ETP/LMC in 0.3% NaCMC; ENE; and ELNE#1, #6, and #7 diluted with DMEM . The cells were then incubated at 37 °C for three hours. Subsequently, the cells were fixed in 4% cold paraformaldehyde, blocked in blocking buffer , and incubated with an anti-human ASBT antibody. Further staining with 10 µg/mL Alexa Fluor 546-labeled secondary antibody was performed for one hour, and nuclei were counterstained with 4′,6-diamidino-2′-phenylindole dihydrochloride (1 µg/mL) for five minutes. Finally, fluorescence images were obtained using a confocal laser scanning microscope.After DCK–PA had been incorporated into the nanoemulsions, enhancement of the intestinal permeability of ELNE via interaction with the bile acid transporter was assessed using human ASBT gene-transfected MDCK cells. MDCK cells were seeded onto Cell-Tak-coated coverslips at a density of 1 × 102.6.2 had formed. To elucidate the transcellular pathway involved in ELNE absorption, 0.1 mL HBSS containing inhibitors of specific cellular uptake pathways and 0.6 mL HBSS were added to apical and basal chambers, respectively, and preincubated at 37 °C for 30 minutes . Thereafter, the solution in the apical compartment was replaced with 0.1 mL ELNE diluted in HBSS  along with the corresponding inhibitors, while the solution in the basolateral compartment was replaced with 0.6 mL fresh HBSS. The cells were then incubated at 37 °C. Aliquots (200 µL) of sample solution were withdrawn from the basolateral compartment of each well at 0, 0.5, 1, 2, 3, 4, and 5 hours and replaced with the same volume of fresh HBSS  were withdrawn from the basolateral chamber (receiver) at defined time points , followed by replacement with fresh HBSS to maintain the initial volume. For BA transport (efflux), 0.6 mL drug solution  without Cys A was added to the basolateral chamber (donor), and samples (0.05 mL) were withdrawn from the apical chamber (receiver) in a manner similar to that described for AB transport.Furthermore, to explore the role of P-gp-mediated efflux during the permeation of ELNE, a permeability bidirectional transport study of an aqueous solution of ETP/LMC and ELNE#7 was conducted, i.e. in both apical-to-basal  and basal-to-apical  directions, in the presence or absence of Cys A (specific inhibitor of P-gp efflux). For AB transport (uptake), 0.1 mL drug solution  with Cys A was added to the apical chamber (donor) of the Transwell2+ chelating agent (EGTA) was applied to reversibly open intracellular tight junctions in the Caco-2/HT29-MTX-E12 cell monolayers. Moreover, to investigate the effect of EGTA in combination with all inhibitors except Cys A (specific inhibitor of P-gp efflux) on ELNE transport, Caco-2/HT29-MTX-E12 cell monolayers with a TEER value >350 Ω·cm2 were treated with 0.1 mL HBSS (Ca2+-free medium) containing 2.5 mM EGTA with or without inhibitors except Cys A. The basolateral side was filled with 0.6 mL HBSS (Ca2+-free medium). The Transwell® was then preincubated at 37 °C for 45 minutes, and the integrity of the cell monolayer was monitored to ensure that the TEER value remained ≤70 Ω·cm2, thus confirming opening of the junctional complex. Medium in the apical compartment was replaced with 0.1 mL HBSS (with 1.8 mM Ca2+) containing ELNE#7  alone or with all inhibitors except Cys A. Next, 0.6 mL HBSS (with 1.8 mM Ca2+) was added to the basolateral side of each well, and the cells were incubated at 37 °C for an additional five hours. At predetermined time points, 0.2 mL medium on the basolateral side was withdrawn and replaced with the same volume of fresh HBSS. In addition, the integrity of the monolayer was checked by measuring the TEER value, which returned to same volume2 at 2 and 5 hours after drug loading, thus confirming restoration of transmembrane resistance , benzyl alcohol (3.06%), Tween 80 (8.16%), PEG 400 (66.33%), and ethanol (22.24%) (ETP-IV), ETP emulsion, or ELNE#7 diluted in DMEM or RPMI 1640 with 1% FBS. The cells were incubated for 24 hours. To determine cell viability, 10 μL 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt (WST-8) solution diluted in 100 µL DMEM or RPMI 1640 was added to each well. The cells were incubated for two hours, and the absorbance at 450 nm was measured using a microplate reader. Untreated cells were used as controls (100% viability). Wells without WST-8 were used as blanks to calibrate the spectrophotometer to zero absorbance. The percentage of viable cells was calculated by comparing the values of the treated cells with those of the untreated cells.LLC and A549 cells were seeded in 96-well plates at a density of 1 × 102.8.g, 15 minutes, 4 °C). Plasma samples were separated and kept frozen at −70 °C until the analysis. LC/MS was performed to determine the concentration of ETP in plasma. Briefly, 100 µL plasma was spiked with 100 µL internal standard (2.5 µg/mL teniposide). Next, 100 µL acetonitrile was added to the plasma spiked with internal standard. Extraction was performed using 3 mL tert-butyl methyl ether/n-hexane  by vortex mixing and centrifugation at 10,000 rpm for five minutes at 4 °C. After the mixture had been centrifuged, the organic phase was transferred to another vial and then evaporated to dryness using a rotatory evaporator at a reduced boiling point. Finally, residues were reconstituted in 100 µL acetonitrile and subjected to LC/MS using the Agilent 6120 Quadruple LC/MS system with a Luna C18 column  as the stationary phase. Aliquots (20 µL) from each sample were eluted in the mobile phase, consisting of acetonitrile (50%) with a 50% isocratic gradient of deionized water containing 0.1% (v/v) formic acid, for 15 minutes at a flow rate of 0.5 mL/min. ETP and internal standard were ionized using an electrospray ionization source in positive-ion mode under the following source conditions: capillary voltage, 4 kV; drying gas flow rate, 10.0 L/min; drying gas temperature, 300 °C. Quantitative analyses of protonated molecular ions were performed at ([M + Na]+=589.3) and ([M + Na]+=675.2) for ETP and teniposide, respectively , ETP in 5% DMSO (20 mg/kg ETP), ETP/LMC solution (20 mg/kg ETP), ETP emulsion (20 mg/kg ETP), and ELNE#7 (20 mg/kg ETP) were orally administered to rats. In addition, 200 µL ETP-IV (20 mg/kg ETP) was injected via the tail vein to evaluate oral bioavailability. Next, blood samples (150 µL) were collected into heparinized tubes from the retroorbital plexus at predetermined time points, then mixed with 50 µL 3.8% sodium citrate solution and immediately centrifuged . One-way analysis of variance followed by Tukey’s multiple-comparison test was used to compare more than two mean values. All data are expressed as means ± standard deviation. In all analyses, p<.05 was taken to indicate statistical significance.Pharmacokinetic parameters were assessed using a non-compartmental method in WinNonlin3.3.1.Supporting Information, Table S3). Next, ETP/LMC (1:5) was loaded into the inner aqueous phase of the optimum w/o/w nanoemulsion based on the pseudo-ternary phase diagram, thereby yielding ELNE . Moreover, successful incorporation of ETP/LMC (1:5) into nanoemulsion was confirmed by analysis of particle size, PDI, and zeta potential. ELNE#1 showed a particle size and PDI of 189 ± 10.3 nm and 0.377 ± 0.015, respectively, which confirmed the formation of an ETP/LMC-incorporated nanoemulsion . To further increase the permeability of ELNE via ASBT-mediated transport, DCK–PA was anchored into ELNE#1. The formation of an ionic complex between DCK and PA was confirmed by comparing the individual surface charges of PA, DCK, and DCK–PA. The zeta potential of PA was −83.4 ± 3.87 mV, and that of DCK was 29.4 ± 0.57 mV. However, after the formation of DCK–PA, the surface charge of the complex was −28.0 ± 0.21 mV. Therefore, the increase in surface charge of PA in DCK–PA micelles indicated mitigation of its negative charge by equimolar cationic DCK counterions, which supported formation of the DCK–PA complex. Next, to confirm the successful anchorage of the DCK–PA complex into the nanoemulsion, we incorporated PA into the ELNE#1. As expected, the surface charge of ELNE#1 decreased from −14.6 ± 1.68 mV to −47.4 ± 1.95 mV in PA-ELNE#7, whereas it was −11.1 ± 0.915 mV in DCK–PA-incorporated ELNE#1, i.e. ELNE#7, respectively. These findings indicate the successful anchorage of DCK–PA into the ELNE and confirmed that the hydrophobic tail component of PA was well adsorbed into the oil phase, whereas the anionic head remained exposed toward the o/w interphase.To enhance the aqueous solubility, loading ability, and stability of ETP in primary nanoemulsion, physical complexes between ETP and LMC (ETP/LMC) with different weight ratios  were formed, and their aqueous solubilities and artificial membrane permeabilities were evaluated. Based on the highest aqueous solubility and artificial membrane permeability, ETP/LMC (1:5) was selected for loading into the nanoemulsion  had a droplet size, PDI, and zeta potential of 172 ± 5.70 nm, 0.290 ± 0.005, and −11.1 ± 0.915 mV, respectively. The morphology and surface structure of ELNE#7, as determined by TEM, were also suggestive of nano-sized droplets based on the results of particle size analysis . These r3.2.Supporting Information, Table S3). However, the eP of ETP/LMC (1:10) was 201% lower than that of ETP/LMC (1:5). Thus, the enhancement of ETP/LMC (1:10) permeability did not appear to coincide with enhancement of solubility. The concomitant reduction in permeability of ETP/LMC with the proportional enhancement of solubility may have occurred because of the diminished membrane/aqueous partition, which resulted in a solubility–permeability tradeoff  and ETP emulsion were 165% and 326% lower than those values for ELNE#1, respectively , which enhanced eP via its well-known solubility- and permeability-improving properties   were performed using ELNE#7.Based on the results of 3.4.Supporting Information, Table S2) . Similarly, inhibitors of caveola-mediated endocytosis (MBCD and genistein) reduced the uptake of ELNE#7 by 35.7% and 21.0%, respectively, compared with the control (without inhibitors) (appP of ELNE#7 by 28.8% compared with the control (without inhibitors)  . Moreoveibitors) . The conappP of ELNE#7 by 33.8%, compared with the control (without inhibitors). This finding confirmed considerable lysosomal escape of ELNE#7, and demonstrated the roles of endocytosis and macropinocytosis, respectively  and Golgi complex are important regulators of the secretory ER/Golgi and endocytosis recycling pathways Burd, . To confectively .appP of ELNE#7 were compared with those on the appP of the control (without inhibitors)  (appP of ELNE#7 by 44.1% compared with that of the control (without inhibitors) (appP of ELNE#7 relative to the control (without inhibitors)  . After Aibitors) . The speibitors) . Similaribitors) . Taken tappP ratio  can be used to elucidate the role of P-gp in the absorptive direction. Furthermore, the net efflux ratio  was measured to evaluate the effects of P-gp during secretory (BA) transport. The P-gp-mediated efflux was considered substantial if the net efflux ratio  and net appP ratio  were >2 and >1, respectively. The ETP/LMC solution exhibited moderate efflux across the monolayer, with a net efflux ratio of 1.88. Similarly, the net appP ratio of ETP/LMC solution increased by 269% in the presence of Cys A  in the presence of the prominent P-gp inhibitor, Cys A . This finding indicated that 35.7% of ELNE#7 may have entered by passive diffusion or the paracellular route  was 1.89-fold greater than the appP of EGTA-untreated monolayers   and bile acids, which open tight junctions, in ELNE#7 may have favored its permeation via the paracellular route.To evaluate the roles of paracellular transport and passive diffusion, the effects of EGTA – a calcium-chelating agent that binds to free extracellular Caar route . Notablyt Cys A) . These rin vitro lipolysis model and in situ intestinal perfusion assay are necessary for more comprehensive understanding of the enhanced transport of ELNE#7.However, the transport mechanisms in this study implicate only transport pathways that can be followed by intact ETP/LMC-incorporated nanoemulsive vehicles; after oral administration, ETP in ELNE#7 can be absorbed via various other mechanisms. After the lipolysis of ELNE, the possible fates of the formulation can be summarized as follows. First, lipolysis or digestion of oil/lipid-based surfactants by gastric and pancreatic lipases can lead to the nanoprecipitation or partitioning of drug out of the oil phase into the surrounding aqueous environment. Second, byproducts of digested oil/lipid-based surfactants, combined with preexisting bile salts, can incorporate the precipitated drug in colloidal micelles and facilitate its uptake. In ELNE#7, we incorporated ETP/LMC into the nanoemulsive system; hence, despite lipolysis, the ETP/LMC can remain solubilized and reincorporated along with digested triglycerides, monoglycerides, fatty acids, lysophosphatidylcholine, and endogenous bile salts. It thus forms a series of colloidal structures, including micelles and uni-/multilamellar vesicles, in gastrointestinal fluid. These micelles will be absorbed by enterocytes, where they can be converted into chylomicron upon re-esterification and presumably reach the systemic circulation through the lymphatic pathway  and area under the plasma concentration–time curve (0–24 hours) (AUClast) were 2.55- and 2.23-fold greater, respectively, after oral administration of ETP emulsion compared with ETP in 5% DMSO. These changes led to enhancement of oral bioavailability, by 347%. Moreover, the Cmax and AUClast values of ETP emulsion showed 1.57- and 2.09-fold enhancements, respectively, compared with ETP/LMC solution. Notably, the Cmax and AUClast values of ELNE#7 were 3.36- and 2.23-fold higher than the respective values of ETP emulsion. Therefore, the oral bioavailability of ELNE#7 was 224% and 468% greater than the corresponding values of ETP emulsion and ETP/LMC solution. In addition, the times to reach the maximum plasma concentration of ETP dispersed in water, ETP/LMC solution, ETP emulsion, and ELNE#7 were 0.88 ± 0.25, 0.50 ± 0.00, 2.25 ± 1.26, and 1.00 ± 0.00 hours, respectively. The enhanced oral bioavailability of ELNE#7 was consistent with the results of the in vitro permeability and mechanistic studies. The increased eP and appP values of ELNE#7 across the artificial membrane and Caco-2 monolayer in vitro, compared with ETP in 5% DMSO, ETP dispersed in water, ETP/LMC solution, ETP emulsion, and ELNE#1, suggested the involvement of passive diffusion and various other transcellular transport mechanisms in the enhanced oral bioavailability of ELNE#7 . Furthermore, the significant reductions in appP after inhibition of cellular uptake pathways  confirmed that these routes played important roles in improving the oral bioavailability of ELNE#7  significantly enhanced the intestinal permeability and oral bioavailability of ETP. 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How tourist camp activities affect individual elephant welfare is an important and highly debated topic. Saliva and fecal samples were collected monthly for 1 year from 44 female Asian elephants that participated in three programs , and analyzed for glucocorticoids (GC) and immunoglobulin A (IgA). The hypothesis was that better welfare would be associated with low GC and high IgA concentrations. Both biomarkers showed significant variation with respect to camp size, riding activities, tourist-to-elephant ratios and seasonality, but not always consistently between feces and saliva, and not always in the predicted direction. However, there was no clear indication that riding per se negatively affected these two biomarkers. The lack of consistent responses highlights the difficulty in interpreting physiological data in relation to management factors, and suggests more work is needed to differentiate between potential chronic (feces) and acute  responses.Asian elephants have been an important part of wildlife ecotourism in Thailand for over two decades. Elephants in tourist camps are exposed to a variety of management styles and daily activities that can potentially affect health and welfare. This study investigated relationships between a novel welfare biomarker, immunoglobulin A (IgA), and daily camp activities, and compared results to glucocorticoid (GC) measures. Often no-riding camps are portrayed as providing better welfare than camps that offer riding. Therefore, we predicted that elephants at no-riding camps would have lower GC and higher IgA concentrations, and a low GC/IgA ratio. Forty-four female elephants from six elephant camps were divided into three groups based on riding activities: saddle-riding, bareback-riding, and no-riding. Fecal and salivary samples were collected monthly for 1 year along with evaluations of body condition, foot health, and wounding. Camp environment and management varied among camps, although the major difference was in riding activities. Concentrations of GCs and IgA varied among the working groups, but not always consistently between sample matrices. Overall fecal glucocorticoid metabolite concentrations were lowest in the saddle-riding group. Only in one bareback-riding camp did the elephants exhibit a potentially positive welfare response with a low GC/IgA ratio over time. Other results varied between the two biomarkers, with considerable variability across camps, suggesting there is more to good welfare than whether elephants participate in riding or not. Several other human-induced stressors, like chaining, ankus use, and limited social opportunities are likely to be impacting well-being and should be considered to ensure management practices meet physical and psychological welfare needs. Elephas maximus) is an iconic species in Thailand, and one that has played an essential role in wildlife-based ecotourism for decades [The Asian elephant  has been proposed as a novel biomarker for welfare assessments of wildlife species ,22,23, iThis study is a first step in trying to understand how captive elephants respond to different human-imposed stressors in the form of feeding, bathing, and riding. Because no-riding camps are often portrayed as providing better welfare than camps that do offer riding, the goal was to examine how GC and IgA varied among elephants at six tourist camps in Thailand, particularly in relation to riding activities. If riding is a significant stressor, we would expect to observe lower GC and higher IgA concentrations in elephants at no-riding camps. We also examined how these biomarkers related to other camp management factors, and to scores of body condition, foot health and wounding. Results could provide useful information and a new viewpoint to the use of IgA as a welfare measure, and identify how management practices affect the overall well-being of working elephants in Thailand.This study was approved by the Faculty of Veterinary Medicine, Chiang Mai University (CMU), Animal Care and Use Committee (Number S38/2561).Information on each of the six camps is summarized in Elephants were divided into three groups based on work activities: riding with and without a saddle, and no-riding. For saddle-riding (14 elephants), elephants carried one or two tourists in a wooden saddle (saddle weight ~30 kg) with a mahout on the neck. Elephants walked 20–45 min along a fixed trail in the jungle, with water  provided along the way. Both saddle camps organized elephant rides as rounds . Once each round was completed, elephants were given a 1 h break before beginning the next round. During the rest period, elephants were chained and offered browse. After the final round (~15:00 h), mahouts took elephants to bathe in the river before chaining them overnight with food. Supplemental feeding was not scheduled for riding elephants at the two saddle-riding camps.For bareback-riding (11 elephants), both camps allowed one or two tourists to sit on the neck with a mahout following on foot. Both camps had half- and full-day programs. Half-day programs provided ~3 h of activities  twice a day . The full-day program lasted ~4 h, with additional activities like making sticky rice balls to feed the elephants, and spending more time on the ground with them . In both programs, riding took place along a jungle trail for around 25–35 min. Tourists participated in activities such as feeding bananas and sugar cane (both camps), bathing in a river , and applying mud to the elephant’s skin (head and body) in a mud wallow (Camp C). Free foraging was allowed throughout the day along the jungle trail. In the half-day program, elephants were chained during the break and offered food , whereas elephants in the full-day programs were unchained and allowed to socialize and forage in the forest for 1 h (12:00–13:00 h). Elephants returned from the jungle after work  and were chained overnight with food provided until 7:00 h.Entada rheedii) vines (Camp F) or commercial shower cream . The additional activities of the full-day program included sticky rice ball making and feeding, paper-making from elephant dung, and extended time to spend with elephants. Elephants at these camps spent most of the time free-roaming with mahouts nearby in open dirt areas with access to a water source (pond or river). At Camp F, elephants were free-roaming during breaks away from tourist activities , where at Camp E, elephants were chained during break time ; both offered food during the breaks. Elephants were chained overnight after tourist activities  until 7:00 h.In the non-riding group (19 elephants), both camps provided half-day and full-day programs with 3 h (twice a day) and 4 h (once a day) of activities, respectively. Elephants were free to roam unchained with conspecifics in a grassy field area enclosed with (Camp F) or without (Camp E) a steel fence. For the first 30 min of each program, staff provided general information about elephants while tourists observed from afar. After that, tourists began their activities, which included feeding and touching with (Camp F) or without (Camp E) a protective barrier between elephants and tourists, walking, applying mud to the elephant’s body (Camp E), and bathing (both) in a river by actively scrubbed with herbal (n = 525) and salivary (n = 521) samples were collected monthly from January to December 2019 between 9:00 and 12:00 h, although some saliva data are missing due to sampling constraints. Fresh fecal samples were collected on the same day as saliva. The fecal ball was mixed by hand and two ~20 g subsamples were placed into two zip-lock plastic bags and frozen at −20 °C for separate GC and IgA extraction processes. Saliva was collected using a Salivette® kit  [g for 5 min at 15 °C. Two swabs were collected, and the saliva was pooled for an average volume of 500 µL (100–1500 µL) per sample. Saliva was stored frozen at −20 °C until analysis.Fecal  ,24 by wiBody condition (BCS), foot health (FS), and skin wound (WS) scores were assigned to each elephant at the time of sample collection. The 5-point BCS scale was based on Morfeld et al.  and adapg for 20 min and the supernatant was decanted into labeled tubes. Another 5 mL of 90% ethanol was added to the pellet, which was then vortexed briefly and centrifuged at 960× g for 20 min. The fecal extracts were combined and dried in a 90 °C water bath. Fecal extracts were re-suspended in 3 mL of 95% EtOH, dried down again, and finally re-suspended in 1 mL of 50% methanol. Samples were stored at −20 °C until analysis.Fecal samples were extracted following the procedure of Bansiddhi et al. . In brie®, pH 7.2) by vortexing overnight on a multi-tube pulse vortexer , with a motor speed of 50 and pulse ON. Samples were then vortexed briefly and centrifuged at 1800× g for 20 min at 4 °C. The supernatant was decanted into a clean tube and centrifuged again at 3500× g for 10 min at 4 °C to remove any remaining particulates. It was then decanted into a clean tube, dried in a lyophilizer at −40 °C overnight, and re-suspended with 0.5 mL ultra-purified water. Samples were stored at −20 °C until analysis.The fecal extraction protocol for IgA analysis was adapted from Edwards et al. . Frozen FGM concentrations were measured in fecal extracts diluted 1:3 in assay buffer using s double-antibody EIA with a polyclonal rabbit anti-corticosterone antibody  validated for Asian elephants in Thailand . SamplesTM, F96 Maxisorp immune plate, Roskilde, Denmark) were pre-coated with anti-rabbit IgG  per well as described by Plangsangmas et al. [Salivary cortisol was measured by a double-antibody enzyme immunoassay (EIA) validated for elephants  that uses et al. . Cortiso2SO4 was added to stop the reaction. Optical densities were determined at 450 nm by a microplate reader (TECAN). The EIA was validated for fecal IgA in this project by showing parallelism between serial dilutions of fecal extracts and the standards , and significant recovery of fecal IgA added to a low concentration sample before analysis . Assay sensitivity was 0.46 ng/mL. The intra- and inter-assay CVs were <10 and 12%, respectively.IgA in fecal and salivary samples was measured using commercially available components as described by Edwards et al.  with somR statistical software version 3.5.1 was used to conduct all statistical analyses in this study without transforming the data. Normality and variance of the data were examined by QQ plot . Mean dTourist camp activities are summarized in Summary statistics for fecal and salivary GC data are shown in p < 0.01), with no difference in FGM between bareback and no-riding elephants (p = 0.97). FGM concentrations differed (p < 0.01) in relation to high (25.8 ± 1.27 ng/g), moderate (32.5 ± 1.13 ng/g), and low (38.0 ± 1.33 ng/g) tourist-to-elephant ratios. There also was a difference in FGM concentrations for total chain hours, being lower for less  compared to more  chaining time (p < 0.01). More social time was associated with higher FGM concentrations (p < 0.01) across 0-h (28.4 ± 1.07 ng/g), 1-h (34.4 ± 1.31 ng/g), and 2-h (39.2 ± 1.72 ng/g) groups, as was tourist feeding (36.5 ± 1.02 ng/g) compared to no feeding (28.4 ± 1.50 ng/g) (p < 0.01).For FGM, elephants at Camp F had the highest, while those at Camp A had the lowest concentrations. Overall, those used for saddle-riding (28.4 ± 1.07 ng/g) had lower FGM concentrations than for bareback (36.2 ± 1.78 ng/g) or no-riding (36.7 ± 1.32 ng/g) (p < 0.01), the latter two of which were not different (p > 0.05). FGM concentrations also differed between high (43.9 ± 1.82 ng/g) and low (28.9 ± 0.67 ng/g) tourist seasons (p < 0.01).Across environmental factors, the highest concentration was observed in January (62.40 ± 3.51 ng/g), the lowest in June (25.10 ± 1.11 ng/g), and between March and July concentrations were generally low . Overallp = 0.31). Additionally, in contrast to feces, where there were no differences, salivary cortisol varied based on camp size, with large camps having the highest salivary measures (0.85 ± 0.04 ng/mL) compared to medium (0.59 ± 0.03 ng/mL) and small (0.44 ± 0.07 ng/mL) camps. In those with a high tourist-to-elephant ratio (0.89 ± 0.07 ng/mL), salivary cortisol was higher compared to low ratio camps . Salivary measures also differed between camps that provided 1-h of free time to socialize (0.57 ± 0.04 ng/mL) compared to 0-h (0.86 ± 0.04 ng/mL) and 2-h (0.83 ± 0.05 ng/mL), the latter two of which were not different. Camps with scheduled tourist feeding had elephants with lower salivary cortisol concentrations (0.68 ± 0.03 ng/mL) compared to camps that did not .For salivary cortisol, results often contrasted with FGM. For example, Camps A and F had the highest, while Camp D had the lowest measures. Elephants that participated in a bareback-riding program (0.49 ± 0.05 ng/mL) exhibited lower salivary cortisol concentrations than those in saddle- (0.86 ± 0.04 ng/mL) and no-riding (0.78 ± 0.03 ng/mL) groups, with the latter two being the same (p < 0.01).Salivary cortisol followed a seasonal pattern similar to feces, with the highest concentrations in January and February  and the lowest between August (0.36 ± 0.04 ng/mL) and November (0.39 ± 0.06 ng/mL), with more variable concentrations between March and July . ConcentBased on the univariable GLS model, significant factors that affected FGM included work activities, tourist-to-elephant ratio, work hours, chain hours, socialization time, tourist feeding and tourist and environmental seasons, while for salivary cortisol, type of work, camp size, tourist-to-elephant ratio, socialization time, tourist feeding, and seasons were significant . There wDescriptive statistics for fecal and salivary IgA data are displayed in p < 0.01), with no difference between the latter two (p = 0.86). Medium (1.65 ± 0.10 µg/g) camps had the highest fecal IgA concentrations, with large (0.85 ± 0.06 µg/g) (p < 0.01) and small (0.69 ± 14 µg/g) camps having similar values. Lower concentrations of fecal IgA were found in elephants at camps with a high (0.68 ± 0.09 µg/g) versus low (1.42 ± 0.08 µg/g) tourist-to-elephant ratio (p < 0.05). Fecal IgA concentrations also differed between camps that provided 1 h of socialization time (1.54 ± 0.09 µg/g) compared to 0 h (0.88 ± 0.08 µg/g) and 2-h (0.82 ± 0.08 µg/g), the latter two of which were similar.Camp C had the highest fecal IgA concentrations among camps. Overall, elephants in a bareback-riding program had higher fecal IgA concentrations (2.02 ± 0.18 µg/g) compared to saddle (0.88 ± 0.16 µg/g) and no-riding (0.77 ± 0.14 µg/g) programs (p < 0.01), but the patterns differed from those of GC (p < 0.01), and no clear seasonal pattern. Fecal IgA concentrations also differed between high and low tourist seasons , while the rainy season showed the lowest concentrations (0.79 ± 0.07 µg/g) compared to the summer (1.33 ± 0.11 µg/g) and winter (1.23 ± 0.09 µg/g) (p < 0.01).IgA varied throughout the year (se of GC . Monthlyp < 0.05).Similar to feces, camp C had high salivary IgA measures together with Camp B. In contrast to feces; however, salivary IgA was not affected by work type  or camp size . Lower concentrations of salivary IgA were found in elephants at camps with a high (5.65 ± 0.52 µg/mL) versus low (9.73 ± 0.58 µg/mL) tourist-to-elephant ratio (p < 0.01), similar to fecal measures. However, unlike feces, elephants exhibited the lowest concentrations in summer (6.14 ± 0.29 µg/mL) compared to the rainy (9.28 ± 0.64 µg/mL) and winter (10.8 ± 0.88 µg/mL) seasons (p < 0.01).The annual pattern of salivary IgA differed somewhat from feces, with low concentrations for the first 5 months of the year before increasing to a peak in December (18.3 ± 2.11 µg/mL). Salivary IgA concentrations differed between high (10.8 ± 0.88 µg/mL) and low (7.69 ± 0.36 µg/mL) tourist seasons . The majority of elephants (70.96%) had a FS of 1, mostly related to nail cracks. No elephants had a FS = 3. Both bareback-riding camps had better  FSs among the six camps (p < 0.05). No elephants had a WS = 2; the majority (83.53%) scored a 0. Overall, saddle-riding elephants had a higher average WS than those in the bareback-riding (p < 0.01) and no-riding (p < 0.01) programs. The majority with a WS = 1 were in a saddle program, and wounds were generally related to ankus use, with lesions apparent on the forehead; only one elephant had lesions related to saddle equipment (Camp A). However, one bareback-riding camp (Camp D) had a WS that was comparable to the saddle-riding elephants, which again was related to ankus use that caused cuts and abrasions. The other bareback-riding camp (Camp C) and both no-riding camps had the fewest skin wounds, with >90% scoring 0.Descriptive statistics for BCS, FS, and WS are shown in p > 0.05), except for salivary cortisol concentrations that were higher in elephants with a BCS = 3 (0.85 ± 0.13 ng/mL) compared to BCS = 5 , but not with a BCS = 4 . For FS, only salivary IgA differed between FS = 0 (12.49 ± 1.24 µg/mL) and FS = 1 (7.88 ± 0.87 µg/mL) or FS = 2 , with no differences between the latter two (p > 0.05). GC and IgA concentrations were not affected by WSs (p > 0.05).There were no differences in FGM, fecal or salivary IgA concentrations between the BCS categories (p < 0.01), but not with saliva. Camp size also affected only the fecal ratio, with elephants in small camps (but only camp D) having a higher ratio compared to medium and large camps. By contrast, the tourist-to-elephant ratio affected only the salivary GC/IgA ratio, with a high tourist-to-elephant ratio having higher GC/IgA compared to moderate and low ratios. The GC/IgA ratio did not differ across environmental and tourist seasons or the WS, but was affected by BCS = 3 having the highest ratio in both matrices, and FS = 2 having a higher ratio in feces.Ratios between GC and IgA within the same biological samples were calculated and averaged by elephant . Camp D p < 0.01); however, no other significant correlations between GCs and IgA were found.Repeated measures correlations between the two biomarkers in feces and saliva, and relationships between physical exam parameters are displayed in This is the first study to examine GCs and IgA in combination throughout the year in Thailand tourist camp elephants. While considerable work has been done using GCs as a welfare marker, including in Asian elephants ,7,14, feFGM results were comparable to our previous studies ,7, whereWhile saddle-riding elephants had the lowest FGM, the lowest salivary cortisol was observed in the bareback-riding elephants. One possible explanation for the difference may be the more invasive nature of saliva collection compared to fecal sampling . Even thIn examining how the tourist-to-elephant ratio affected GCs, we found an inverse relationship for feces and saliva, such that more tourists per elephant were associated with lower FGM. This was unexpected, given studies in other species that show higher visitor numbers are associated with increased GC activity in many zoo animals ,55,56, aFor IgA, there was considerable variability across individuals, comparable to earlier reports in elephants ,24 and oThere was a seasonal pattern of FGM similar to our previous findings ,7,74, whAnnual IgA patterns were not consistent between feces and saliva. Fecal IgA excretion was fairly consistent throughout the year, whereas for saliva, concentrations were clearly lower for the first 5 months of the study, corresponding to the mid- to late-summer season, before increasing in the months leading up to the high tourist season. In humans, Garde et al.  reportedIgA is the first line of defense against viral and bacterial infections ,27,28, aThis was the first study to use IgA in a welfare assessment of elephants participating in different tourist activities. Assuming low GC and high IgA are indicators of better welfare, our overall results showed that this was consistent for only one bareback-riding camp. This may suggest that other factors besides riding affect camp elephant welfare, such as those related to social management, ankus use, chaining, after-hours management, health care, and quality of the mahout–elephant relationship. For example, higher GC/IgA at camps with more tourists per elephant could indicate compromised welfare. The variability in results between saliva and feces for both biomarkers highlights potential differences between acute versus chronic stress responses, perhaps in relation to excretion lag times. It also is important to distinguish between physiological and pathological responses. Therefore, while low GCs often equate with better welfare, it also could be a sign of adrenal exhaustion resulting from chronic stress . Similar


One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria  Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A′ (compounds 1a/1b), as well as two new derivatives, BAC-D/D′ (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey’s method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola. Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host’s environment.The Hawaiian bobtail squid, IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro. One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds. Organisms that deposit eggs without subsequent parental care must have mechanisms to protect developing embryos from predation, infection, and fouling . To compEuprymna scolopes, these ANG bacteria are deposited into the JC surrounding developing embryos to protect eggs from fungal fouling during an approximately month-long embryonic period  –9. The A–c period .E. scolopes, the ANG and JC communities are composed of Alphaproteobacteria, Verrucomicrobia, Gammaproteobacteria, and Flavobacteriia  or nonribosomal peptide synthetase (NRPS) pathways, among others , inhibited Vibrio, Aeromonas, Staphylococcus, Streptomyces, and Escherichia coli strains in a disc diffusion assay  derivatives. Additional bioactivity profiling was conducted on purified BACs to understand how these bacterial secondary metabolites might contribute to the ANG symbiosis.In this study, we screened 19 ANG and JC bacterial isolates representing in vitro antibacterial activity of 19 ANG and JC isolates revealed that two strains, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, inhibited one or more of the marine bacteria tested (8 CFU/ml). The Alphaproteobacteria strain, Leisingera sp. ANG59, produced moderate zones of inhibition against two of the vibrio strains, Vibrio fischeri and Vibrio anguillarum. These results are comparable to those observed for an E. scolopes jelly coat isolate, Leisingera sp. JC1, which inhibited select vibrio strains in a similar bioassay  would be more representative of the concentrations of bacteria in natural seawater conditions where these isolates are exposed during the course of egg development. Significantly greater inhibition was observed at lawn densities between 104 and 105 CFU/ml and between 105 and 106 CFU/ml for all test strains except for Photobacterium leiognathi and Exiguobacterium aesteuri  cycle. Due to the absence of a phosphofructokinase ortholog, the Entner-Duodoroff or pentose-phosphate pathways are likely utilized instead of glycolysis. Nitrate and nitrite reductases formed a complete denitrification pathway. The essential structural genes were present for the type II, type IV, and type VI secretion systems. Flagellar biosynthesis and chemotaxis genes were present, suggesting that ANG59 may be capable of motility in response to chemoattractants. The antiSMASH analysis predicted seven secondary metabolite biosynthetic gene clusters, including two siderophore clusters, two “other” clusters, one bacteriocin cluster, one homoserine lactone cluster, and one ectoine cluster , Pseudoalteromonas sp. JC28 was placed in a well-supported clade containing mostly pigmented pseudoalteromonads isolated from seawater samples  was detected, the TonB-dependent receptor (iutA) and siderophore transport genes (fhuC and exB) were present, suggesting that JC28 may be able to utilize iron-chelating siderophores produced by neighboring bacteria under iron-limited conditions  clusters, four hybrid type 1 polyketide synthase-NRPS (T1PKS-NRPS) clusters, one hybrid ladderane-NRPS cluster, three bacteriocin clusters, and one thiopeptide cluster . Two of P. piscicida JCM20779  and a halogenase gene  which attaches the bromine to the alterochromide , produced by P. flavipulchra JG1 -producing cluster from JCM20779 . This opchromide , as has chromide  and the riformis , as wellsubtilis . Within subtilis . Of the chra JG1 . We did chra JG1 , tambjamchra JG1 , thiomarchra JG1 , or violchra JG1 , 28, whi+ peaks m/z 844.2899 and m/z 858.3034, respectively, which both agree with the calculated [M+H]+, 844.2881 and 858.3037, for C38H51BrN7O10 and C39H53BrN7O10. This also indicated that compounds 2a/2b have an additional CH2 unit compared to compounds 1a/1b. Extensive analysis by NMR spectroscopy revealed that compounds 1a/1b are an inseparable mixture of constitutional isomers of the known BACs  , with thown BACs . Furtherown BACs . Althougs KMM636 , their as KMM636  and e. T1.87 ppm , assigneds 2a/2b . This suds 2a/2b  between ds 2a/2b .During the course of structure determination, we noted several chemical shift assignments on the aromatic ring carbons  of BAC-A/A′ compounds 1a/1b that were incongruent with the previous assignments , necessil configurations and the Thr residue was given a threo assignment -MS analysis of the derivatized acid hydrolysate, all five amino acids in the peptide portion of BAC-A/A′ were tentatively assigned as having signment . These ssignment  and predtereomer , 20.3 Marfey’s method was performed on the acid hydrolysates of compounds 1a/1b and 2a/2b, as well as on all isomers of the standard amino acids (l-leucinamide (l-FDLA)-derived samples by LC-MS, compounds 1a/1b were found to bear d-Val, l- and d-Leu (1:6), d-Ile or d-allo-Ile, d-allo-Thr, and l- and d-Asp (1:2) -derived samples were prepared and analyzed using LC-MS, where d-allo-Ile, rather than d-Ile, was observed in the compound 1a/1b hydrolysate. Similar results were acquired for compounds 2a/2b, with the exception that a Val peak was not detected. Therefore, the complete structures were designated d-allo-Thr−d-Val−l-Asn1−d-Asn2−d-allo-Ile/d-Leu for compounds 1a/1b, and d-allo-Thr−d-allo-Ile−l-Asn1−d-Asn2−d-allo-Ile/d-Leu for compounds 2a/2b.To determine the absolute stereochemistry, the Cno acids . In addino acids , standarsp (1:2) . No peakBacillus algicola CNJ 803 and the Gram-negative strain V. fischeri ES114. BAC-A/A′ (1a/1b) exhibited an MIC of 7.4 μM against B. algicola and 59.3 μM against V. fischeri. BAC-D/D′ (2a/2b) had a similar MIC of 7.3 μM against B. algicola but without activity against V. fischeri (MIC of >58.3 μM). Interestingly, both BAC isolates were more active against B. algicola than V. fischeri.To investigate the potential functional role of these PKS-NRPS metabolites, BAC-A/A′ 1a/1b and BAC-D/D′ 2a/2b were tested against two marine bacteria, the Gram-positive strain Pseudoalteromonas sp. JC28 strongly reduced fungal hyphal growth of Fusarium keratoplasticum FSSC-2g  inhibition assay to understand potential interactions with the host immune response. Coincubation of lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells with 1a/1b resulted in inhibition of NO production with a 50% inhibitory concentration (IC50) of 16.9 μM.Since previous work showed that  FSSC-2g , BAC-A/AE. scolopes inhibit fungi  and green algae (Ulva lactuca) spores  spores , 45.Pseudoalteromonas species play important roles in interacting with marine invertebrates and are necessary for settlement or induction of morphogenesis in some invertebrate larvae, such as the polychaete Hydroides elegans  having the broadest antimicrobial activity , including new derivatives. These brominated derivatives of alterochromides were first identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) from the yellow-pigmented, sponge tissue-derived P. maricaloris KMM636  of the purified compounds 1a/1b compared to that of the Pseudoalteromonas sp. JC28 bacterial strain or its extract when tested in a similar assay . A simiinactive . These don assay . Our stu will also allow for the characterization of antimicrobial metabolite production under in vivo conditions.Here, we show that two strains associated with the squid ANG symbiosis demonstrated antimicrobial activity against other marine bacteria. One of these bioactive strains, ommunity , it remaE. scolopes were conducted in accordance with protocols approved by the Institutional Animal Care & Use Committee (A18-029), Office of the Vice President for Research at the University of Connecticut and in compliance with the Office of Animal Welfare, National Institutes of Health and the Association for Assessment and Accreditation of Laboratory Animal Care International.All experimental procedures involving E. scolopes  or jelly coats (JCs) of opes see , 13, 14.opes see  (13). Al10.1128/mSphere.00166-20.4TABLE S1Table S1, PDF file, 0.1 MB.Microbial strains used in this study. Download Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bacillus megaterium CNJ 778 and Exiguobacterium aesteurii CNJ 771) and Gram-negative bacteria  using an in vitro inhibition assay as previously described  was spotted (10 μl) onto the center of the plate. The plates were incubated at 28°C for 24 h. Due to the nonuniform morphology of the bacterial colonies, the zone of inhibition (ZOI) was defined here as the area (square centimeters), rather than diameter, around the ANG/JC isolate where no target bacteria grew  around Leisingera sp. ANG59 against a V. fischeri lawn was observed. (D to F) A decrease in V. anguillarum target strain growth was observed around Leisingera sp. ANG7, but the small ZOI (0.11 cm2) was not marked as positive for inhibition. (G to I) A large ZOI (4.1 cm2) around Pseudoalteromonas sp. JC28 against V. fischeri was observed. Images in the first column  depict the original plate image. Images in the second column  feature the yellow region of interest line drawn in FIJI with the freehand selection tool to measure the area of the entire ZOI. Images in the third column  feature the yellow region of interest line around the ANG/JC isolate to measure the colony area. All colony areas were subtracted from the entire ZOI areas to report the final ZOI. Images and measurements were taken as part of the initial ZOI screen summarized in FIG S1, PDF file, 0.8 MB.Representative images of zone of inhibition area measurements. Three examples of different interactions that were observed between ANG/JC isolates and target lawn strains. (A to C) A small ZOI area  1H NMR spectrum of compounds 1a/1b in DMSO-6d (600 MHz); (b) 1H-1H COSY NMR spectrum of compounds 1a/1b in DMSO-6d (600 MHz); (c) HSQC NMR spectrum of compounds 1a/1b in DMSO-6d (600 MHz); (d) HMBC NMR spectrum of compounds 1a/1b in DMSO-6d (600 MHz); (e) NOESY NMR spectrum of compounds 1a/1b in DMSO-6d (600 MHz). Download FIG S2, PDF file, 1.2 MB.NMR spectra of compounds 1a/1b in DMSO-Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00166-20.3FIG S36d. (a) 1H NMR spectrum of compounds 2a/2b in DMSO-6d (600 MHz); (b) 1H-1H COSY NMR spectrum of compounds 2a/2b in DMSO-6d (600 MHz); (c) HSQC NMR spectrum of compounds 2a/2b in DMSO-6d (600 MHz); (d) HMBC NMR spectrum of compounds 2a/2b in DMSO-6d (600 MHz); (e) NOESY NMR spectrum of compounds 2a/2b in DMSO-6d (600 MHz). Download FIG S3, PDF file, 0.9 MB.NMR spectra of compounds 2a/2b in DMSO-Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pseudoalteromonas sp. JC28, was chosen for further testing based on its ability to inhibit all target strains. To test for density-dependent effects of this inhibitory activity, inhibition trials were retested with target strains at lower lawn densities of 104, 105, and 106 CFU/ml as described above. Each test was performed in experimental and technical triplicates. For each target lawn strain, statistical significance between ZOIs at each lawn density was determined using one-way analyses of variance (ANOVAs). Multiple-comparison post hoc Tukey tests were performed to determine which lawn densities were significantly different.After the initial ZOI screen, the JC isolate, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28 were grown in 3.0 ml of SWT broth, 200 rpm, at 30°C to a density of ∼108 CFU/ml. Genomic DNA was extracted from 1.0 ml of culture using the MasterPure DNA Purification kit  following the manufacturer’s protocol. DNA quantity was measured with a high-sensitivity DNA Qubit fluorimeter , and quality was checked by absorbance measured on a Nanodrop spectrophotometer  and 1% agarose gel. DNA was prepped for sequencing using the Nextera XT DNA library prep kit . Library yield was checked with Qubit, and size and quality were measured on a high-sensitivity DNA bioanalyzer chip . Sequencing was performed on an Illumina MiSeq, reagent kit v.2 with 500 cycles at the Microbial Analysis, Resources, and Services (MARS) facility at the University of Connecticut.Leisingera sp. ANG59 and 3,976,074 raw, paired-end reads for Pseudoalteromonas sp. JC28, which were uploaded to the U.S. Department of Energy KBase server version 1.5.2 (https://kbase.us/) for analysis. Raw reads were trimmed and adaptors were removed using Trimmomatic  tree was assembled using 34 published Pseudoalteromonas genomes. Six single-copy housekeeping genes were selected for multilocus sequence analysis based on a previous study . Amino acid sequences for each gene were obtained from each genome by a tblastn search using the amino acid sequence from E. coli strain K-12 obtained from UniProt. Extracted sequences were aligned using MUSCLE (https://itol.embl.de/)  . Studiesmbl.de/) , are ref10.1128/mSphere.00166-20.5TABLE S2Pseudoalteromonas strains included in MLSA tree  are averages ± standard errors of the means from three trials. (b) Zone of inhibition areas of Pseudoalteromonas sp. JC28 assayed against the target bacteria. Download Table S3, PDF file, 0.1 MB.Zone of inhibition experiments. (a) Zone of inhibition areas of ANG/JC isolates tested against seven marine bacteria Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00166-20.7TABLE S4Leisingera sp. ANG59 genome. Download Table S4, PDF file, 0.1 MB.Indigoidine biosynthetic genes detected in the Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00166-20.8TABLE S5Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28 genomes. Download Table S5, PDF file, 0.1 MB.Secondary metabolite biosynthetic gene clusters predicted by antiSMASH in the Copyright © 2020 Suria et al.2020Suria et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00166-20.9TABLE S6Pseudoalteromonas genomes in MLSA tree  biosynthetic gene was obtained from the Minimum Information about a Biosynthetic Gene cluster (MIBiG) server . This clCM 20779  and contPseudoalteromonas sp. JC28 was prepared by inoculating the bacteria into 30 ml of marine broth  and incubating at 28°C for 24 h at 200 rpm. A 7.5-ml aliquot of the preculture was added into four 1-liter baffled flasks, each containing 500 ml of marine broth, before incubation at 28°C for 72 h at 200 rpm, protected from light. Solvents for extraction and semipreparative high-performance liquid chromatography (HPLC) purification were American Chemical Society (ACS) grade and HPLC grade, respectively, from Sigma-Aldrich . The extraction procedure was performed away from direct light exposure to prevent decomposition of the BACs (A preculture of the BACs .n-hexane (3 × 500 ml), followed by ethyl acetate (EtOAc) (12 × 300 ml) to afford 5.3 mg and 42.7 mg of extract, respectively. Meanwhile, the pellet was soaked in 100 ml of acetone overnight. The acetone extract was vacuum filtered and evaporated to dryness under reduced pressure before partitioning between n-hexane (200 ml) and 90% aqueous methanol (MeOH) (3 × 200 ml), to afford 7.8 mg and 69.7 mg of extract, respectively. Bromoalterochromides were detected by LC-MS analysis in the EtOAc supernatant extract, as well as in the MeOH pellet extract, using the following LC-MS conditions: column 150 × 4.6 mm Agilent Eclipse-XDB C18, 5 μm; column temperature of 24°C; flow rate of 0.7 ml/min; mobile phase A (0.1% HCOOH in water), mobile phase B ( 0.1% HCOOH in CH3CN), and a linear gradient of 40% mobile phase B to 100% mobile phase B over 30 min , monitoring at 395 nm. The EtOAc extract yielded 0.8 mg of compounds 1a/1b and 0.4 mg of compounds 2a/2b, while the MeOH extract yielded 3.3 mg of compounds 1a/1b and 0.7 mg of compounds 2a/2b. Isolated compounds were stored in amber-colored vials under an inert atmosphere at −20°C.Cultures were combined  and centrifuged at 6,300 rpm at 4°C for 10 min, and the supernatant was separated from the pellet via decantation. The supernatant was extracted with r 30 min . These t1H, 1H-1H correlation spectroscopy [COSY], 1H-13C heteronuclear single quantum coherence [HSQC], 1H-13C heteronuclear multiple-bond correlation [HMBC], and nuclear Overhauser effect spectroscopy [NOESY]; 6d . High-resolution mass data were obtained using a Waters Xevo G2-XS QToF mass spectrometer , equipped with a ultraperformance liquid chromatography (UPLC) column , and eluted using a gradient of mixtures of mobile phase A (0.1% aqueous HCOOH) and mobile phase B (0.1% HCOOH in CH3CN) beginning at 5% mobile phase B for 0.5 min, followed by a linear gradient from 5% mobile phase B to 60% mobile phase B for 3.5 min, then 60% mobile phase B to 98% mobile phase B for 4 min, and finally held at 98% mobile phase B for 1 min. The samples were dissolved in MeOH at 1 mg/ml, using 10-μl injections.The planar structures of the bromoalterochromides (BACs) were determined by extensive nuclear magnetic resonance (NMR) analyses , and d-allo-Thr and 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA) from TCI . The LC-MS data were collected on an Agilent ESI single quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system with a G1311 quaternary pump, G1322 degasser, and a G1315 diode array detector.All standards and reagents were obtained from Sigma-Aldrich, except for 3. Then, 40 μl of 1% l-FDLA or 1% 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA) in acetone was added prior to incubation at 35°C for 1 h. The reaction was quenched by the addition of 20 μl of 1 N HCl, then diluted with 370 μl of MeOH, from which 25 μl was injected for LC-MS analysis. The alkalinified solutions of the l and d forms of standard amino acids (100 μg), Val, Leu, Ile, Thr, Asn, and Asp, were individually derivatized in the same manner as the natural products. After quenching with 20 μl of 1 N HCl, they were diluted with 810 μl of MeOH, from which 25 μl was injected for LC-MS analysis. LC-MS conditions were as follows for FDLA-derived samples: column 150 × 4.6 mm Agilent Zorbax-SB C3, 5 μm; column temperature, 40°C; flow rate, 0.7 ml/min; mobile phase A is 5% CH3CN with 1% HCOOH in water and mobile phase B is 5% CH3CN with 1% HCOOH in MeOH; and linear gradient of 30% mobile phase B to 100% mobile phase B over 55 min. LC-MS conditions were as follows for FDAA-derived samples: column 150 × 4.6 mm Agilent Zorbax-SB C3, 5 μm; column temperature, 25°C; flow rate, 0.7 ml/min; mobile phase A is 7.5% CH3CN with 1% HCOOH in water and mobile phase B is 7.5% CH3CN with 1% HCOOH in MeOH; and linear gradient of 12.5% mobile phase B to 57.5% mobile phase B over 80 min.The absolute configuration of the BACs was determined by Marfey’s or advanced Marfey’s method as previously described , 74. TheBacillus algicola CNJ 803 and the Gram-negative bacterium V. fischeri ES114 in 96-well liquid assays, following previously described methodology  and V. fischeri on SWT agar, overnight at 28°C. Inocula of the target strains were prepared by adding colonies to nutrient broth or SWT broth and adjusting the optical density at 600 nm (OD600) to 0.08 to 0.10. A master mix was then prepared by mixing 7.84 ml of the appropriate broth, 6.40 ml water, and 1.60 ml of the inoculum. Into a 96-well round-bottomed plate , 199 μl of the master mix was added with 1 μl of sample or controls. The final testing concentrations were 250 μg/ml for extracts and 0.39 to 50 μg/ml for purified compounds, dissolved in dimethyl sulfoxide (DMSO) . The positive controls were 2.5 μg/ml of vancomycin  for B. algicola and 2.5 μg/ml of chloramphenicol  for V. fischeri, while the negative control was DMSO. The plate was incubated for 24 h at 28°C and with constant shaking at 200 rpm in the case of V. fischeri. The wells were observed visually for the presence or absence of bacterial growth to determine the MIC. The assays were done in technical triplicates with at least two experimental replicates.The BACs (compounds 1a/1b and compounds 2a/2b) were tested against the Gram-positive bacterium 4 cells/well, and incubated at 37°C in an atmosphere of 5% CO2. After 24 h, 2 μl of LPS  and 1 μl of compounds 1a/1b  or controls  was added to the well and incubated for an additional 24 h. Then, 100 μl of the supernatant was transferred into another flat-bottomed 96-well plate, to which 50 μl of 1% sulfanilamide (Sigma-Aldrich) in 5% phosphoric acid  was added. After 5 min, 50 μl of 0.1% aqueous N-(1-naphthyl)-ethylenediamine (NED) was mixed into the wells. The plates were read after another 5 min at 540 nm using a Synergy H1 hybrid reader . NO production was expressed as a percentage of the ratio between the sample absorbance and that of the negative control, DMSO. The assay was done in technical triplicates with two experimental replicates.Compounds 1a/1b were tested for inhibition of nitric oxide (NO) using previously described methods . BrieflyFusarium keratoplasticum FSSC-2g was spread onto an SWT agar plate and allowed to grow at 28°C for 24 to 48 h. An inoculum with an OD600 of 0.01 was prepared in SWT broth from F. keratoplasticum colonies on the plate. Sterile 6-mm paper discs (Difco Laboratories) were placed on top of SWT agar plates, to which 10 μl of sample or controls was added. Compounds 1a/1b, dissolved in DMSO, was tested at 10 μg to 100 μg/disc, with cycloheximide (Sigma-Aldrich) at 68 μg/disc as a positive control and 10 μl of DMSO as a negative control. Discs were allowed to dry for 5 min before adding the fungal inoculum onto the discs. The plates were then incubated at 28°C and observed for hyphal growth every 24 h for 3 days. Activity was measured by reduction in area of hyphal growth of the sample relative to the negative control.An antifungal assay was performed using previously described methodology with slight modification . FusariuPseudoalteromonas sp. JC28 draft genome assembly has been deposited in DDBJ/EMBL/GenBank under accession no. QOKX00000000 and is described in BioProject PRJNA480278. The version described in this paper is version QOKX01000000. The Leisingera sp. ANG59 draft genome assembly has been deposited at DDBJ/ENA/GenBank under the accession no. WLCM00000000 and is described in BioProject PRJNA589827. The version described in this paper is version WLCM01000000.The