AuNSps were subsequently dropped on a glass substrate before covered by thiophanate methyl, a broad-spectrum systemic fungicide. The AuNSps coated glass substrate was subsequently dried in the air for further surface-enhanced Raman spectroscopy (SERS) measurements. Optical properties, shape and size of AuNSps were confirmed by UV-vis spectroscopy, XRD, SEM-EDX and TEM. The results showed that AuNSps were successfully synthesized with the size of 53 nm, and their resonance peak was located at 560 nm. The Raman signal intensity of thiophanate methyl covered on AuNSps is higher than that without AuNSps, indicating SERS effects of AuNSps deposited glass substrate.It is essential to develop a simple and sensitive method to rapidly detect residual fungicides in agricultural products to protect human health. So far, little studies have been reported on potential application of gold nanospheres (AuNSps) as a surface plasmon resonance based sensor for in-situ detection of residual fungicides. Therefore, in this study, we investigated the potential application of AuNSps as a surface plasmon resonance based sensor for in-situ detection of fungicides. AuNSps were successfully synthesized via a seed-mediated method with some modifications. Firstly, gold nanoseeds were made during the reduction of chloroauric acid by trisodium citrate dihydrate (TSC). Then, AuNSps were grown from the seeds by using HAuCl The development of science and technologies has led to the introduction of modern analytical methods with high sensitivity and accuracy, such as high performance liquid chromatography (HPLC), fluorescence assay (FL), high performance liquid chromatography mass spectrometry/mass spectrometry (HPLC–MS/MS), electrochemical assay (EC) and gas chromatography/mass spectrometry (GC/MS). However, these methods have some limitations such as complicated sample preparation and analysis processes that require operators to be highly qualified, time-consuming operation, high consumption of organic solvents and restricted on-site measurement. Since the discovery of Raman spectroscopy took placed in early 1928, Raman spectroscopy has been thought to be a promising method to overcome these disadvantages . SERS isThiophanate methyl whose chemical structure is shown in In this work, AuNSps were synthesized by a facile method using TSC as a reducing agent and EDTA as a protective agent during the nucleation stage. The as-synthesized AuNSps were used as a platform to investigate the SERS signal in thiophanate methyl fungicide analysis.4·3H2O, ≥99.5%) and ethylenediaminetetraacetic acid  were purchased from Merck . Trisodium citrate dihydrate  were purchased from Prolabo . Deionized (DI) water  was used during the synthesis of AuNSps. A commercial product, called TOPSIN M 70WP (containing 70% of thiophanate methyl), was purchased from Nippon Soda Company, Ltd. All reagents were used without further purification.Tetrachloroauric (III) acid trihydrate (HAuClα radiation (λ = 1.54056 Å) operated at 40 kV and 30 mA with a scanning rate of 0.02° per step in the 2θ range of 10° ≤ 2θ ≤ 90° was carried out to characterize the crystal structure of AuNSps. UV-vis spectroscopy  was utilized to investigate the optical properties of AuNSps. The Raman spectra of AuNSps samples were recorded on a LabRam HR micro-Raman instrument with a 532 nm Ar+ ion laser at room temperature.The morphology of the as-synthesized samples was obtained by scanning electron microscopy  coupling with energy-dispersive X-ray (EDX) spectroscopy operated at 20 kV of the incident electron beam energy. Transmission electron microscope (TEM) images were obtained with a JEOL JEM-1400 (120 kV). X-ray diffractometer  with Cu K4 was mixed with 10 mL of DI water, and the obtained solution was boiled at 100 °C. Under vigorous stirring 300 µL of 1% TSC was then added to the solution. The color of the solution will change from colorless to reddish. After cooling at room temperature, the solution was kept in dark for 30 min for seeding crystallization of Au nanoseeds.Au nanoseeds were synthesized by the Turkevich method  with som4 were firstly added into 100 mL of DI water under vigorous stirring. Next, 20 µL of 1% TSC and 100 µL of 0.1 M EDTA were added to the mixture. The solution was kept in the dark for 30 min for stabilization. The whole procedure of AuNSps synthesis was illustrated in For the synthesis of AuNSps, 50 µL of the Au nanoseed solution and 100 µL of 25 mM HAuCl2SO4 and 30% H2O2 in 15 min, subsequently washed with DI water and dried in the air. Twenty microliters of AuNSps solution was gently dropped onto the clean substrate homogeneously, and the substrate was then dried in the air. Next, Twenty microliters of thiophanate methyl (in DI water) was gently dropped onto the AuNSps coated glass substrate, and the substrate was subsequently dried in the air for further SERS measurements. For comparison, Raman spectra of a clean substrate, AuNSps and thiophanate methyl solutions (10−4 M and 0.1 M) were also recorded. The procedure of SERS measurement was illustrated in The Raman spectra of thiophanate methyl were recorded on an AuNSps coated glass substrate in order to study the SERS effects. To prepare AuNSps coated glass substrate, the substrate was firstly rinsed with a 3:1 (v/v) mixture of concentrated HOptical properties of AuNSps and Au nanoseeds were studied through UV-Vis spectra. As shown in The solution of Au nanoseeds consisted of clusters of Au nanosized particles with the average diameter of 18.27 ± 0.08 nm a,b. Nanoθ = 38.1° (111), 44.3° (200), 64.5° (220) and 77.7° (311) [7° (311) . No impu−1, 726 cm−1, 779 cm−1, 898 cm−1, 959 cm−1 and 1538 cm−1, which are ascribed to -N-C=S deformation, N-H wagging of –(C=O)-NH-(C=S)-, C=S stretching, C-S stretching, C=S and C-N, respectively. Moreover, other signals of =C-H deformation, N-C-N asymmetric stretching, C-O, C-O-C, C=C, C=O and N-H were also observed at 1039 cm−1, 1154 cm−1, 1267 cm−1, 1298 cm−1, 1601 cm−1 and 1708 cm−1, respectively [−4 M , therefore, was estimated as 57 µM.To elucidate the sensitivity of the method, 20 µL of thiophanate methyl solutions with concentrations ranging from 11 to 30 µM were deposited onto AuNSps coated glass substrates, which serve as Raman probes. The corresponding Raman spectra of thiophanate methyl were collected in The reproducibility of the measurement was also evaluated by collecting SERS spectra of 30 µM thiophanate methyl at 10 random locations on the as-fabricated substrate, as shown in AuNSps were successfully synthesized from crystallization of Au nanoseeds through a facile seed-mediated method using TSC as a primary reducing agent and EDTA as a protective agent during the nucleation stage. The as-synthesized AuNSps showed SERS effect for thiophanate methyl with low LOD of thiophanate methyl down to 17 µM. The Raman signal intensity of thiophanate methyl covered on AuNSps was higher than that without AuNSps, which revealed the potential application of AuNSps as a surface plasmon resonance based sensor for in-situ detection of fungicides with high sensitivity and reproducibility.


Clostridiales and Bacilli, respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order Enterobacteriales. The most abundant proteins detected in the β-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community; furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source.The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex β-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex β-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on β-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders  Bacteroides fragilis is well known [Firmicutes and Actinobacteria due to medical and clinical implications have attracted more attention recently [Mycobacterium tuberculosis-derived EVs that contain iron-binding factors contribute to bacterial survival, thus promoting infection, while Staphylococcus aureus-derived EVs that contain β-lactamase have a role in the spread of antibiotic resistance [The distal gut microbiota has a profound influence on host health through the degradation of dietary fibers, production of metabolites and vitamins, maturation of the host immune system, and protection against pathogen invasion . The gutll known , while trecently . For inssistance ,17. NevePicea abies) β-mannan. Furthermore, we show that the proteome of EVs from these intestinal bacteria is modulated by the available carbohydrate source.Herein, we describe a proof-of-concept study to assess the feasibility of isolating EVs produced by a complex bacterial community in vitro. Before EVs isolation, the community derived from pig fecal samples was enriched using spruce .Acetylated galactoglucomannan (hereafter referred to as β-mannan) was produced in-house from steam-exploded Norway spruce, as described previously . Mannoperibed in . Glycan 2HPO4 0.04 g/L, KH2PO4 0.04 g/L, MgSO4·7H2O 0.01 g/L, CaCl2·6H2O 0.01 g/L, NaHCO3 2 g/L, Tween 80,2 mL/L, hemin 0.05 g/L, vitamin K 10 μg/L, L-cysteine hydrochloride 0.5 g/L, and bile salts (sodium glycocholate and sodium taurocholate at 0.5 g/L each)) was left overnight in an anaerobic chamber  under an atmosphere containing 85% N2, 5% CO2, and 10% H2. β-mannan, kept overnight in anaerobic conditions, was added to a final concentration of 2% (w/w) just before adding the fecal slurry at 0.01% v/v. The negative control consisted of a flask with a basal nutrient medium, but no added carbohydrate source. Before starting the experiment, pH was adjusted to 6.2–6.4 and the temperature of the anaerobic cabinet was set at 39 °C to resemble conditions in the porcine large intestine [We evaluated the ability of β-mannan to promote the growth of extracellular vesicle-producing bacteria by using an in vitro, static batch culture fermentation system inoculated with fresh pig fecal inocula. To obtain anaerobic conditions, a 1 L conical flask with 800 mL of sterile basal medium .Porcine fecal samples were collected directly from the rectum of four healthy, weaned pigs (with bodyweight ranging from 18 to 22 kg) from three different pens; anaerobiosis was maintained using an anaerobic jar  equipped with a gas pack system . To prepare the inoculum, equal amounts of feces (1.5 g wet weight) from each animal were pooled together and then diluted 1:5 in pre-reduced (oxygen-free) phosphate-buffered saline (PBS) as described in . The fecv/v) following incubation for 15 min at room temperature. The fixed cells were washed three times in PBS (pH 7.4) and dehydrated in a gradient series of ethanol , followed by critical point drying with liquid CO2. Samples were coated with gold–palladium (Au–Pd) and examined in a Zeiss Evo 50 EP scanning electron microscope .After 24 h fermentation, cells from three samples per condition were pelleted and pre-fixed in 50 μL of formaldehyde  and indexed with the Nextera XT Index Kit v2  according to the Illumina protocol for 16S metagenomic sequencing library preparation. Next, equal volumes from each indexing reaction were pooled together, quantified with a Qubit Fluorimeter , diluted, denatured, and mixed with 5% PhiX Control v. 3  according to the aforementioned Illumina protocol. The denatured library was sequenced on the Illumina MiSeq platform using the MiSeq Reagent Kit v. 3 (600 cycles). Data were retrieved from the sequencing machine as FASTQ files of the sequences for each demultiplexed sample.DNA was extracted from samples taken at 6 and 24 h with a MagAttract PowerMicrobiome DNA/RNA Kit , according to the manufacturer’s instructions, except for the bead beating step where we used a FastPrep-96 Homogenizer  at maximum intensity for a total of 3 min in three pulses of 60 s with a 5 min cooling period between each pulse. The extracted nucleic acids were quantified with a Qubit Fluorimeter and the Qubit dsDNA BR Assay Kit  and stored at −80 °C. SSU rRNA sequences were amplified using the primers Pro341F (5′-CCT ACG GGN BGC ASC AG-3′) and Pro805R (5′-GAC TAC NVG GGT ATC TAA TCC-3′), to which the MiSeq adaptors were additionally incorporated on the 5′ ends. The 25 µL PCR reactions consisted of 12.5 µL of iProof High-Fidelity Master Mix , 0.5 µL of a primer mix wherein each primer was at a 10 µM concentration, 2 µL template DNA, and 10 µL HQuality filtering, merging, denoising, and chimera detection were conducted to yield an amplicon sequence variant (ASV) table by using the DADA2  package g, 4 °C) and the supernatant was then filtered sequentially through 0.45 and 0.22 μm pore filters to remove the remaining bacterial cells. Then, the supernatant was concentrated from 1 L down to 0.1 L using a VivaFlow 200 cartridge with a 10 kDa cutoff  and further down to 4 mL using a centrifugal concentrator with a 100 kDa cutoff . The retentate was ultracentrifuged at 114,000× g for 2 h using an SW50.1 rotor in an Optima L-80 XP ultracentrifuge . Pellets were resuspended in 5 mL PBS and centrifuged again at 114,000× g. After washing, the pellets containing EVs were resuspended in 1 mL PBS followed by purification on an Optiprep  density gradient with centrifugation at 100,000× g for 18 h. To obtain a discontinuous density gradient, Optiprep solution was diluted with homogenization buffer  to obtain 40% (1.215 g/mL), 20% (1.111 g/mL), 10% (1.058 g/mL), and 5% (1.037 g/mL) iodixanol solutions. Aliquots of 4 mL with decreasing density were loaded sequentially into a 16.8 mL ultracentrifuge tube. Twelve 1 mL fractions were collected from the top. The protein concentration in the different fractions was determined using the Bradford protein assay  with bovine serum albumin as the standard. The EVs’ purity, approximate size, and morphology were verified by nanoparticle tracking analysis with a Zetasizer Nano ZS . Ultrapure water at 25 °C was used as a constant parameter for viscosity and refractive index. The particle size  were expressed as the z-average. Data were analyzed using Zetasizer Software (v. 6.20) to calculate the hydrodynamic diameters of the particles.EVs were isolated through a centrifugation protocol ,27 FiguA. BrieflEVs samples were subjected to negative staining for TEM analysis. Formvar- and carbon-coated copper grids were incubated on a drop of EV suspension for 5 min. The grids were then washed three times with PBS and the adherent EVs fixed with 1% glutaraldehyde  for 4 min. Next, the grids were washed three times with PBS, two times with Milli-Q (MQ) water, stained for 20 s with 4% uranyl acetate  in MQ water, washed once with MQ water, and finally incubated on a solution of 1.8% methyl-cellulose  and 0.4% uranyl acetate for 10 min on ice. The grids were then dried and viewed in a Philips CM200 transmission electron microscope. Images were taken using a Quemesa camera and iTEM software .w/v) SDS polyacrylamide gel. The proteins were separated through SDS-PAGE and stained with Coomassie Blue, and the image was acquired and evaluated using Gel doc™ XR+ with Image Lab™ software . Protein molecular weight standards were obtained from Bio-Rad.A standard SDS-PAGE procedure was used . Brieflym/z 200, after accumulation to a target of 1 × 105. The maximum allowed ion accumulation times were 60 ms. The proteomic analysis was performed by the Proteomic core facility of the University of Oslo, Norway. The acquired raw data were analyzed using MaxQuant [Sus scrofa (40.708 sequences). Peptide identifications were filtered to achieve a protein false discovery rate (FDR) of 1% using the target-decoy strategy. Only proteins detected in both biological replicates for at least one of the conditions were considered valid EVs proteins. The mass spectrometry proteomics data were deposited with the ProteomeXchange Consortium via the PRIDE [Two biological replicates of EVs per treatment were diluted to 20 µg of total protein in PBS, and the pH was adjusted to 8 by adding ammonium bicarbonate . Subsequently, the proteins were digested with 1 μg trypsin  overnight at 37 °C. The digestion was stopped by adding 5 μL 50% formic acid and the generated peptides were purified using a ZipTip C18  according to the manufacturer’s instructions, cleaned with micro-SPE, and dried using a Speed Vac concentrator . The tryptic peptides were dissolved in 10 µL 0.1% formic acid/2% acetonitrile and a 5-µL aliquot (~10 µg) was analyzed using an Ultimate 3000 RSLCnano-UHPLC system connected to a Q Exactive mass spectrometer  equipped with a nanoelectrospray ion source and loading capacity of 3.3 µg. For liquid chromatography separation, an Acclaim PepMap 100 column  was used. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 400 to 2000) were acquired with the resolution R = 70,000 at MaxQuant  v.1.4.1.he PRIDE  partner v/v)) and 1 µL of the sample, which was mixed directly onto an MTP 384 ground steel target plate . Droplets were dried under a stream of warm air.Enzymatic assays were conducted by adding 2 μL of either EVs or culture supernatant to 100 μL of a 1 mg/mL solution of the glycan substrates described in 600 of the cultures (Firmicutes (83%), which increased to 90% abundance in the presence of β-mannan when compared to 63% observed in control at 24 h. Proteobacteria, on the other hand, were present at only 4% in β-mannan vs. 30% in the control culture at 24 h , while Proteobacteria play a role in carbon cycling [Lactobacillales (40% abundance) and Selemonadales (39%) were dominant, whereas, at 24 h in control, they were 5% and 29% abundant, respectively . Enterobacteriales represented 5% abundance in the presence of β-mannan at 24 h  in a variety of gut microbiomes ,33,34. Tcultures B, while cultures C. The chcultures . The taxcultures D–F. Afte at 24 h D. Some F cycling . In the ectively E. Howeve at 24 h E. Recentectively F, while n et al.  could bermicutes .Next, we studied the capacity of commensal bacteria to secrete EVs in vitro, whereby EVs were detectable after 24 h cultivation. The density of bacterial EVs was analyzed using Optiprep density gradient ultracentrifugations and ranged between 1.08 and 1.20 g/mL. The results from SDS-PAGE demonstrated that while a minor amount of EVs were present in the control culture A, EVs weFirmicutes, from the orders Clostridiales and Bacilli, respectively  of proteins revealed that the most abundant proteins belonged to MAGs 53, 272, and 343, and are involved in translation, ribosomal structure, RNA metabolic processes, and nucleotide-binding . InteresClostridiales, and MAG343, which corresponds to Escherichia coli, a classical, intestinal, Gram-negative commensal ; however, the EVs did not contain polysaccharide degrading enzymes, contrary to previous observations for Fibrobacter [We succeeded in isolating and analyzing the protein content of EVs secreted from a complex microbial community. β-Mannan induced the secretion of EVs from robacter . None of


Lactobacillus plantarum is a popular probiotic species due to its safe and beneficial effects on humans; therefore, novel L. plantarum strains have been isolated and identified from various dietary products. Given that bacteria-derived extracellular vesicles (EVs) have been considered as efficient carriers of bioactive materials and shown to evoke cellular responses effectively, L. plantarum-derived EVs are expected to efficiently elicit health benefits. Herein, we identified L. plantarum APsulloc 331261 living in green tea leaves and isolated EVs from the culture medium. We performed quantitative lipidomic analysis of L. plantarum APsulloc 331261 derived EVs (LEVs) using liquid chromatography-mass spectrometry. In comparison to L. plantarum APsulloc 331261, in LEVs, 67 of 320 identified lipid species were significantly increased and 19 species were decreased. In particular, lysophosphatidylserine(18:4) and phosphatidylcholine(32:2) were critically increased, showing over 21-fold enrichment in LEVs. In addition, there was a notable difference between LEVs and the parent cells in the composition of phospholipids. Our results suggest that the lipidomic profile of bacteria-derived EVs is different from that of the parent cells in phospholipid content and composition. Given that lipids are important components of EVs, quantitative and comparative analyses of EV lipids may improve our understanding of vesicle biogenesis and lipid-mediated intercellular communication within or between living organisms. Helicobacter pylori infection, inflammatory bowel disease, and type 2 diabetes are often linked to compositional changes in the microbiota + ions, phospholipids except phosphatidylglycerol (PG), which was detected as [M + NH4]+ ions, and sphingolipids were detected as [M + H]+ ions , and 66.82 ± 13.64 for LEV, but this extraction efficiency was overall lower than that from solvent control  by LC-MS analysis   LPG, ceramide-1-phosphate (Cer1P), dihydroceramide-1-phosphate (dCer1P), sphingosine-1-phosphate (SO1P), sphinganine-1-phosphate (SA1P), and MG  control, 69.60% ± 11.64 for the other strain  control , possibl% of PC3 . A totalanalysis . Of the lantarum . We deteSkyline)  and presSkyline) . A summa, and MG , which wL. plantarum using the quantified value with a corresponding internal standard. According to a volcano plot depicting the distribution of detected lipid species by the magnitude and significance of their differential signal intensities, many lipid species were significantly fold-increased in LEVs compared to L. plantarum  (p < 0.01) and 19 (22.1%) showing decrease  in LEVs compared to L. plantarum  and PC(32:2) were markedly increased in LEVs, showing the highest enrichment of greater than 21-fold, and three PC species including PC(32:2), PC(34:2), and PC(30:1) were ranked in the top five  A. Of the = 0.01) , 86 lipilantarum A. These lantarum B and thelantarum . The larlantarum . More thlantarum . Comparetop five . In additop five . These rL. plantarum by accessing the proportion of each lipid class in lipid category (mol% of lipid category). LEVs showed a relatively high ratio in TG (46.5%) and DG (40.7%) but low in MG (12.8%), while L. plantarum showed similar ratios in composition  of healthy persons or patients  and Avanti Polar Lipids, Inc. .w/w) of green tea leaves weight, and left at room temperature for 3 h. The salted green tea leaves are mixed with 1000 mL of a 1% fructooligosaccharide solution and incubated for three days at 32 °C. The pH change in the solution was monitored and when the solution showed acidic pH (below pH 5.0), it was inoculated on to a de Man, Rogosa and Sharpe (MRS) agar plate  and incubated for two days at 32 °C in an anaerobic chamber  connected with a gas cylinder containing 5% H2, 5% CO2, and 90% N2 for putative LAB growth. After monitoring the morphology of colonies, putative LAB colonies were picked and reinoculated onto a MRS agar plate, and grown in MRS broth  at 32 °C under anaerobic conditions. To identify the obtained isolates, fifteen colonies were selected for partial 16S rDNA sequencing. Chromosomal DNA was purified using Wizard genomic DNA purification kit . PCR were performed on a thermal cycler using universal primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-GGATACCTTGTTACGACTT-3′) [L. plantarum was verified through the analysis of neighbor-joining tree using partial 16S rDNA sequencing.Green tea leaves were harvested from Dosun green tea garden in Jeju, South Korea. Two hundred grams of green tea leaves were washed twice with distilled water and mixed with salt equivalent to 8% (ACTT-3′) . The ampACTT-3′) . The phyLactobacillus plantarum APsulloc 331261 (deposit number: KCCM11179P) was grown in MRS broth for 24 h at 37 °C under anaerobic conditions, and was then subcultured under the same condition following 1 to 100 dilution. When the culture reached an optical density of 1.0–1.5 at 600 nm (O.D.600), cells (≥ 2.7 × 109 colony-forming units (cfu)/mL) were pelleted by sequential centrifugation at 3000× g for 10 min at 4 °C and 10,000× g for 20 min at 4 °C and frozen for lipid extraction. Lactobacillus plantarum APsulloc 331261-derived EVs (LEVs) were purified from culture supernatant according to purification methods for Gram-positive bacterial EVs [g for 3 h at 4 °C . After centrifugation, the pellet was diluted in HEPES-buffered saline (HBS), and the total protein concentration was determined using a Bradford assay . For lipidomic analysis, LEVs were further purified by OptiPrep density gradient  according to the manufacturer’s instructions and a previously described method [TM (60 w/v% iodixanol in distilled water) was diluted to 50%, 35%, and 20% in 0.25 M sucrose buffer . A bottom-loading and discontinuous gradient was formed by layering 2.5 mL of each solution in 10 polypropylene centrifugation tubes  and centrifuged at 200,000× g at 4 °C for 2 h . The EV fraction at density between 1.17~1.24 was collected using a 1 mL-syringe. To remove iodixanol, the fraction was diluted in 60 mL of HBS, ultracentrifuged at 150,000× g at 4 °C for 3 h, and then resuspended with 200 μL of HBS.d method  with somThe purified LEV size was measured by dynamic light scattering using a Zetasizer Nano ZS  and analyzed using Dynamic V6 software. The particle numbers of purified LEVs were determined by tunable resistive pulse sensing using a qNano-Gold . Cryo-TEM analysis of LEVs was performed as described previously . BrieflyL. plantarum KCTC 3108 and L. plantarum APsulloc 331261 which were washed three times with phosphate buffered saline (PBS) to remove media residue, and LEVs were treated or lysed by adding 5 μL of 0.1% sodium dodecyl sulfate (SDS) in PBS into 45 μL of each sample in PBS  and incubating for 20 min at 37 °C. After sonication  for 3 min on ice using a probe sonicator , the lysed samples were centrifuged at 13,800× g, 20 min at 4 °C. The supernatants were collected and protein concentration was determined using a Pierce bicinchoninic acid (BCA) protein assay kit . Samples set to contain the same amount of 30 μg protein in 50 μL of 0.01% SDS in PBS were added to methanol/chloroform  with 20 μL of the above-mentioned solution as internal standard (IS) lipids (a mixture of IS lipids). Samples were subsequently vortexed 3 × 30 s and incubated for 10 min at room temperature. After centrifugation , the supernatants were transferred to new 1.5 mL tubes. The remaining pellets were dissolved in chloroform/methanol/37% HCl  and incubated for 15 min at room temperature with vortexing three times for 30 s. Then, 250 μL of chloroform and 450 μL 0.1 M HCl were added to samples. Next, the samples were vortexed vigorously for 1 min and centrifuged at 6500× g for 2 min at 4 °C. The bottom of the organic phase was collected and pooled with that extracted before. Subsequently, all samples were split in half and dried by Speed Vac concentrator. One of the dried sample pairs was then dissolved in 100 μL of solvent A/solvent B  for neutral and positive lipid analysis, and the other half of the pair was dissolved in 100 μL of methanol for trimethylsilyldiazomethane (TMSD) methylation [Each lipid standard was dissolved in methanol or chloroform and diluted to 100 ng/μL for extraction. A two-step extraction method was performed for high efficiency extraction of polar and nonpolar lipids . Medium v/v) with 20 mM ammonium formate and 0.1% (v/v) formic acid and solvent B consisted of isopropyl alcohol with 20 mM ammonium formate and 0.1% (v/v) formic acid. The flow rate was 0.3 mL/min and the injection volume was 4 μL for each run. A 33 min gradient was performed as follows: 0–5 min, B 5%; 5–15 min, B 30%; 15–22 min, B 90%, and maintained for 5 min. Finally, the column equilibrated at B 5% for 5 min before subsequent analysis. All lipid analysis of samples was performed by QTRAP 5500 (AB Sciex) hybrid, triple quadrupole, linear ion trap mass spectrometer equipped with a Turbo V ion source, together with the Analyst 1.5.1 software package . Ultra-high-purity nitrogen gas was used for the collision. The parameters of operating source conditions were as follows: capillary voltage 5500 V; nebulizer gas (GS1) = 30–50 psi, heater gas (GS2) = 30–50 psi, collision gas setting (CAD) = high, source temperature (Temp) = 400–500 °C. All lipids were analyzed using optimized conditions and achieved in the multiple reaction monitoring (MRM) mode using computed transitions for each lipid class. For the MRM scan mode, the parameters are follows: different MRM transitions, run time, 22 min, entrance-potential, 10; collision cell exit potential (CXP), 15. The information for precursor and product ions per lipid target, the MS/MS collision energy, and the declustering potential, which was optimized for each lipid transition, is presented in L. plantarum APsulloc 331261 and LEV was repeated nine times, respectively .The HPLC analysis was performed on Waters Acquity UPLC instrument . A Hypersil Gold column  was used for separation of lipids. Solvent A consisted of methanol-acetonitrile-water . Targeted lipids on the MRM list, including precursor and product ions, were assigned by comparisons of retention times with internal standards. Skyline software package  was used with an in-house database to determine the peak area of each assigned lipid and all data were normalized by respective internal standard. The retention times, linear ranges, and limits of detection (LOD) for internal standards were summarized in L. plantarum KCTC 3108, L. plantarum APsulloc 331261, and LEVs at the same protein amount (30 μg), as described in Quality control (QC) experiments were performed to monitor the overall quality of the lipid extraction and MS analyses ,32. In aL. plantarum. In addition, there seems to be a notable difference in the composition and proportion of phospholipids between EVs and the parent cells. Given that lipids are important components of bioactive vesicles, EVs, quantitative and comparative analyses of EV lipids may improve our understanding of vesicle biogenesis and lipid-mediated intercellular communication within or between living organisms.In conclusion, our study using quantitative and comparative LC-MS analysis demonstrated that the lipidomic profile of LEVs is different from the parent cells, showing significant increase in 67 lipid species but decrease in 19 species compared to


The phytochemical mixture TriCurin  and resveratrol) eliminates human papillomavirus (HPV) (+) cancer cells in vitro and in vivo. In this study, we further evaluate TriCurin.The activity of TriCurin and its individual compounds was assayed on W12 cells, derived from a cervical precancer containing episomal and integrated HPV16 DNA, using MTT -2,5-diphenyltetrazolium bromide) assays, microscopy and reverse transcription-polymerase chain reaction (RT-PCR), and on HeLa cells by gene expression analysis. The stability and toxicity of TriCurin microemulsion were tested in an organotypic cervical tissue model.TriCurin and its individual compounds inhibit the growth of W12 cells, episomal, type 1 and 2 integrants; the relative order of activity is TriCurin, EGCG, curcumin, or resveratrol. RT-PCR shows that TriCurin activates p53 and suppresses HPV16 mRNAs E1, E2, E4, E6 and E7 at 24 h in W12 cells. Gene expression analysis shows that TriCurin activates pro-apoptotic genes and represses anti-apoptotic genes in HeLa cells. TriCurin in a microemulsion is stable and non-toxic to cervical tissue. The combination of TriCurin and tanshinone IIA exhibits additional synergy against HeLa cells.TriCurin, and the combination of TriCurin with tanshinone IIA, are effective against HPV (+) cells. The phytochemical mixture, in the microemulsion-based cream, is a promising therapeutic for the prevention and treatment of cervical cancer. Although the incidence has been considerably reduced in developed regions, such as the United Kingdom, millions of women are identified to harbour high-risk HPV infection. About 10% of HPV (+) women will progress to high-grade cervical dysplasia within 2 years.2 Based on the number of HPV-infected women worldwide, the global prevalence of pre-cancerous lesions of the cervix is estimated to be 30–40 million.3 Surgical and ablative interventions are the only available options to treat pre-cancerous lesions of the cervix once they develop. However, these modalities increase the potential for future adverse complications during pregnancy.Despite advances, human papillomavirus (HPV) continues to cause significant morbidity and mortality, as evidenced by 570,000 cases of cervical cancer and 311,000 deaths globally in 2018.4 tanshinone IIA,7 ellagic acid,8 epigallocatechin gallate (EGCG)9 and TriCurin10 inhibit the growth of cervical cancer cells. We further show that these plant-derived polyphenols, either alone or in combination, can inhibit HPV genes and eliminate HPV (+) cancer cells . These compounds target multiple cellular pathways, are available in developing countries, are inexpensive and thus provide an opportunity for drug development.It is imperative to develop a therapeutic agent for high-risk HPV infections and the associated lesions. The HPV vaccines are effective at preventing disease, but access to these vaccines may be limited in developing countries and these vaccines do not benefit women who are already HPV-infected. Studies have shown that curcumin,11HPVs are small non-enveloped double-stranded DNA viruses that contain several early viral genes coding for proteins E1, 2, 4, 5, 6 and 7, and two late proteins L1 and L2. The viral proteins E1 and E2 control viral amplification. E6 represses p53 and activates telomerase, while E7 promotes degradation of the Rb protein family; both actions result in increase in cell growth and viral replication. E4 and E5 facilitate viral replication. L1 is the major capsid protein.12A role for high-risk HPV’s in the development of cervical cancer has been shown by epidemiological and laboratory studies. Establishing and maintaining the malignant state requires the overexpression of high-risk HPV E6 and E7 proteins, which can occur if HPV DNA becomes integrated disrupting the regulatory effects of E2 on these two oncogenes.Curcuma longa L., Zingiberaceae), resveratrol, from grapes  and EGCG, from green tea (Camellia sinensis (L.) Kuntze, Theaceae)  chimeric mouse model of cervical cancer, both TriCurin and curcumin suppressed E6 and induced p53, acetyl-p53 and caspase-3. The response to TriCurin was several folds higher than the response to curcumin, while having better stability in an aqueous environment.10 In addition, TriCurin inhibited growth, clonogenic survival and tumoursphere formation in head and neck squamous cell carcinoma (HNSCC), and inhibited tumour growth in a preclinical model of HPV (+) HNSCC.13TriCurin is a combination of three purified plant-derived polyphenols mixed in specific ratios, curcumin, from the spice turmeric . In addition, the W12 cells generated clones 20861 and 20862, which contain 30 or 60 recombined viral genomes , respectively, most of which are repeated in tandem or as concatemeres (type 2 integrated clones).14 The W12 type 1 and 2 clones resemble the HPV DNA integration state of cancer-derived cells lines, SiHa (1–2 HPV16 copies) and CaSki (500 HPV16 copies), respectively.14To further evaluate the chemo-preventative potential of TriCurin, we explored the growth inhibitory effects on a unique set of cell lines derived from a cervical pre-cancerous lesion (CIN grade 1).Salvia miltiorrhiza Bunge, Lamiaceae).To advance towards the clinical setting, in this study we optimise the TriCurin mixture and develop a microemulsion-based cream appropriate for use in a future clinical trial. Since combination of phytochemicals, in previous studies, produced the most potent and stable mixtures, we tested the growth inhibitory effects of various TriCurin formulations combined with active phytochemicals, including turmeric (with 90% or 95% curcuminoids) and tanshinone IIA. Tanshinone IIA is the most abundant diterpene quinone present in the root of red sage , strongly inhibits the growth of W12 cells containing either episomal or integrated HPV16 DNA. Genome-wide analysis indicates that TriCurin reverses HPV-induced gene alterations. The phytochemical mixture in the microemulsion-based cream is a promising therapeutic candidate to prevent and treat cervical cancer.2O was distilled and deionised. Tanshinone IIA was purchased from Sigma  and was dissolved in ethanol (100%). The two turmeric preparations contained the curcuminoids curcumin, desmethoxycurcumin, and bis-demethoxycurcumin as follows: (1) Naturex #140500 ; 95% curcuminoids . Both were dissolved in dimethylsulfoxide (DMSO) , prior to addition to cell cultures. Resveratrol  and EGCG  were obtained from PCCA , and were dissolved in ethanol. All solutions were sonicated for 10 min. The source of the compounds for the gene expression studies was as follows: curcumin (≥98% curcuminoid content) (CAS number 458-37-7) , (−)-epicatechin gallate, and resveratrol (Thermo Fisher).All solvents and reagents were reagent grade; HTo prepare TriCurin, the following volumes of stock solutions were added to 5 mL Dulbecco’s modified Eagle’s medium (DMEM): resveratrol, 2.5 µL of 200 mM stock; EGCG, 1.6 µL of 25 mM stock; curcumin (turmeric (90% curcuminoids), except where noted), 4 µL of 40 mM stock; mixed briefly after each addition, and sonicated for 10 min. The concentration of DMSO is 1/1250 in TriCurin and this is further diluted when the cells are treated, DMSO < 0.08%.15 were kindly sent to us by Dr. Dennis Slamon . These cells were cultivated in RPMI supplemented with 10% FBS plus glutamine (1%) and PSF (penicillin G-streptomycin-fungizone solution) (1%) (Gibco). Cells were maintained at 37 °C, 5% CO2 and used at passages 1–4.HeLa human cervical cancer cells were a kind gift of Dr. Bettie Steinberg  and were obtained from ATCC  (combination assays). MDA-MB-453  cells were obtained from ATCC . HeLa and MDA-MB-453 cells were grown in DMEM  containing 10% (v/v) foetal bovine serum (FBS) (Gibco-BRL), plus Pen-Strep (Gibco). MCF7 and MCF7/Her2–18 16The online ATCC brochure Maintaining High Standards in Cell Culture describes the accessioning process, during which all ATCC cell lines undergo authentication tests.Human EpiVaginal tissues were purchased from MatTek .14W12 subclones  and J2 3T3 mouse embryo fibroblasts were a kind gift of Dr. Paul Lambert . W12 clones were cultured, as previously described.17 or the EZQUANT assay. For the EZQUANT assay, absorbance was read at 420 nM and corrected for the background value.Cells were seeded in 96-well plates at 1000 cells per well. Twenty-four hours after plating, phytochemicals were added and cells were incubated for 72, 96 or 120 h. The sensitivity of the various cell lines to herbal agents was assayed using the MTT -2,5-diphenyltetrazolium bromide) assay, as previously described,18 The median effect principle was used to analyse the combination assays.18 We used variable ratios of drugs and assumed mutually exclusive equations to determine the combination index (CI).18 Half-maximal inhibitory concentration (IC50) values determined from the combination graphs were used to calculate CI values; for agents 1 and 2: CI = [IC50 (agent 1 + agent 2)/] + [IC50 (agent 2 + agent 1)/IC50 ]. CI and its corresponding effect are as follows: >1.3, antagonism; 1.1–1.3, moderate antagonism; 0.9–1.1, additive effect; 0.8–0.9, slight synergism; 0.6–0.8, moderate synergism; <0.6, synergism.To assess possible synergistic effects, cells were treated with all combinations of four concentrations of each of the agents tested and a solvent control.5 cells per well. Each drug concentration was performed with triplicates. At 0, 6 and 24 h, cells were collected, and total RNA extraction was performed using Trizol reagent . RNA concentration was measured using NanoDrop 2000. cDNA was synthesised using ProtoScript® First Strand cDNA Synthesis Kit (NEB Biolabs). GAPDH  was used as a housekeeping gene control for mRNA analysis. Quantitative real-time PCR (qPCR) was performed using SYBR GreenER™ qPCR SuperMix (Life Technologies) on a Bio-Rad system. Fold-change 2−∆∆C method was used to calculate mRNA abundance. All primers are listed in Supplementary Table t test was run to calculate significance of triplicates.Cells were seeded and cultured on 12-well plates at 2 × 1019 HeLa cells were treated with TriCurin (32 µM) or its individual phytochemicals, curcumin (32 µM), EGCG (8 µM), resveratrol (100 µM) or control (DMSO) for 8 h. DMSO in all the results refers to the control condition, which has DMEM culture medium containing 0.28% of DMSO. The cells  were treated in triplicate in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS). At the end of treatment, 200 µL of RLT buffer (Qiagen RNA isolation kit) was added to each well and samples were frozen at −80 °C. The p values are calculated using ANOVA  KaleidaGraph with post hoc tests performed using Tukey. Differentially expressed genes were selected from the KEGG pathway database (https://www.genome.jp/kegg/). Data were analysed using the Genesifter software . Data were extracted to show genes significantly affected by TriCurin compared to individual compounds . All RefSeq and UniProt references were taken from genecards.org.Gene expression analysis was performed, as previously described.The effect of phytochemicals on human cervical cells was examined by microscopy using the NIS Elements software paired with a Nikon Eclipse TI-S microscope. Images were captured using bright-field channel at magnifications of ×10, ×20 and ×40; EpiVaginal cervical tissues, ×100.2, in a T75 flask. Two thousand cells were seeded in 96-well plates and were cultured overnight. After 96 h, the effect of the drug at different concentrations on the plated cells was tested by MTT assay. The absorbance was recorded at 570 nm. Background correction was achieved using separate wells without cells, but containing all other reagents.Manufacture of TriCurin cream was done at PCCA . TriCurin components included curcumin , epigallocatechin gallate , resveratrol . A proprietary oil-in-water microemulsion, called Enpen (developed by PCCA and Innovene Therapeutics), was used to make 20% TriCurin-Enpen cream (w/w)  tissue .To evaluate if TriCurin-Enpen and Enpen creams are non-toxic on Human EpiVaginal tissues, EpiVaginal cervical tissues were treated with (1) Enpen cream or two positive control creams known to be toxic Gynol II (nonoxynol-9 (3%) and 1% Triton X-100) and (2) TriCurin-Enpen and Enpen creams compared to Vagisil  and Gynol II (nonoxynol-9 (3%)), for 24 h.For histologic examination, EpiVaginal cervical tissues were treated with either Control (negative control), TriCurin-Enpen, Gynol II or Vagisil for 4 h. Entire EpiVaginal tissue implants were fixed in 10% formalin (overnight at room temperature), paraffin-embedded, sectioned (5–6 µm thick) using a microtome and stained with haematoxylin and eosin, according to standard procedures.t test assuming two samples with equal variance, and two-factor analysis of variance (ANOVA)-based t test for two different means; comparison standard error (SE) was estimated by ANOVA; theoretically, the ANOVA based t test is more accurate than the simple Student’s t test. Statistical significance was defined as p < 0.05 (Supplementary information).We performed two-tailed Student’s To gain insight to the action of TriCurin  and its individual compounds, we assayed the growth inhibitory effect of TriCurin on W12 cervical cells, which contain copies of HPV16 in different states of integration (five W12 cell lines), as well as 3T3 feeder cells.The W12 cultures contained irradiated 3T3 (mitomycin C-treated J2 3T3 feeder) cells that are metabolically active, but not replicating. The ratio of feeder to W12 was ~1:20 to 1:100 ; for the 20850 cells, the ratio was ~1:10 to 1:50. The feeder cells exhibited resistance to TriCurin and its individual compounds Fig. . Thus, t50 values on 20861 cells were as follows ; 4 μM on 20850 (p = 0.0108); 0.5 μM (p = 0.0245) or 4 μM (p = 1.68E − 05) on 20822; 1 μM on 20862 (p = 0.0106). The lowest concentrations of TriCurin and its individual compounds that induced significant effects on 20850 cells are: TriCurin (4 μM), p = 0.0108; curcumin (20 μM), p = 0.00171; EGCG (12.5 μM), p = 0.0252; resveratrol (25 μM), p = 0.0173.The lowest concentrations of TriCurin that induced significant growth inhibitory effects on W12 cells are as follows: 8 μM on 3T3 ; TriCurin (4 μM) on 20850 vs. 20822 (p = 2.06E − 06); TriCurin (2 μM) on 20850 vs. 20862 cells (p = 0.0350).For TriCurin, in this experiment, the order of sensitivity of the three cell types is as follows: 20861, 20862, 201402, 20822, 20850 and 3T3. The lowest concentrations of TriCurin that induced significant effects on 20850 vs. the other cell lines are: TriCurin (4 μM) on 20850 vs. 3T3  vs. 20822 (0.07) and 20862 (0.19) cells. This is consistent with an episomal state for the viral genome in the 20850 cells. E2 mRNA is also reduced in 20822 and 20862 vs. 20850 cells, about 2-fold. The level of E4 and E7 is reduced (~2-fold) in 20822 cells and slightly reduced in 20862 cells. The level of E6 is reduced in 20822 cells and increased in 20862 cells. For the tumour suppressor gene p53, the level is many folds higher in 20850 vs. 20862 (0.16) or 20862 (0.38) cells. Thus, the 20850 cells contain the highest level of viral mRNAs, with the exception of E6 in 20862 cells. The differences between the relative levels of p53 and HPV16 mRNAs in the three cell types  at 0 time are all significant (p < 0.01).The relative level of expression of the genes in 20850 cells is set to one. The level of expression of the early gene To gain insight into the action of TriCurin, we examined the effect of TriCurin (90% curcuminoids) on p53 and HPV16 mRNAs in W12 cells, using RT-PCR. The results are displayed in Supplementary Table p < 0.01); for 6 vs. 0 h, except for 20822 cells (p < 0.05)  Fig. . The difMDM2 (MDM2 proto-oncogene). In preliminary experiments on W12 20822 (type 1 integrant) cells , TriCurin activated the expression of p21 at 6 h (2.00-fold) and 24 h (2.04-fold); and the expression of MDM2 at 6 h (2.55-fold) and 24 h (1.2-fold) (data not shown). MDM2, which encodes a nuclear-localised E3 ubiquitin ligase, is the major negative regulator of p53. The effects on MDM2 are the inverse of the effects on p53. To explore if the biphasic effects of TriCurin on p53 could be related to effects on nuclear factor-κB (NF-κB), we examined the effect on the expression of NF-κB1, in type 1 20822 cells; TriCurin activated the expression of NF-κB1 at 6 h (1.68-fold) and 24 h (1.19-fold) (data not shown). The effects on NF-κB1 are the inverse of the effects on p53.It is of interest to monitor the effect of TriCurin on the mRNA levels arising from genes responsive to p53, such as p21 (cyclin-dependent kinase inhibitor 1A) and E1, E2, E4, E6 and E7) was assayed, using RT-PCR.To evaluate the action of TriCurin, the effect of TriCurin on the expression of HPV16 early viral genes (E1 and E2 were the most sensitive as follows: (20822) E1 and E2 at 6 h; (20862) E2 at 6 h; (20850) E1 and E2 at 24 h. At 6 h, the relative reduction for E1 is: 20822, 0.07; 20862, 0.25; 20850, 0.46. The 20822 cells at 6 h are as sensitive as the 20850 cells at 24 h. For 20822 cells, at 6 h, the sensitivity decreases 6.25-fold from the early to the late genes. At 24 h, for 20850 cells, there is a 4.26-fold decrease in sensitivity from the early to the late genes; for 20862 cells, there is a 1.57-fold decrease. There is thus a greater effect on early genes in episomal and type I integrants, and on later genes in type II integrants. The differences between the relative level of HPV16 mRNAs at 0, 6 and 24 h in the three cell types are significant for 0 vs. 24 h (p < 0.01); for 6 vs. 24 h, for E6 (p < 0.05); for 0 vs. 6 h, except for E7 on 20850 cells (p < 0.01)  20822 > (type 2) 20862 >  20850 Fig. . The ear50 value within parentheses): 20850 (2.7 μM); 20862 (6.25 μM); 20822 (8.25 μM) (data not shown). The lowest concentrations of TriCurin that induced significant effects on the three cell lines are: 2 μM on 20850 (p = 0.00650); 2 μM on 20822 (p = 0.0283); 2 μM on 20862 (p = 0.0549) cells , but the reverse is true for type 1 and 2 integrants . The lowest concentrations of the two compounds that induced significant effects are: TriCurin (2 μM) (p = 0.00650); tanshinone IIA (4 μM) (p = 0.0196)  in TriCurin with turmeric (95% curcuminoids) and found that turmeric (95% curcuminoids), as predicted, enhanced the activity. When the growth inhibitory activity of the two TriCurin preparations (95% and 90% curcuminoids) was assayed in the same experiment, the respective values were ~1.2 and 4.3 µM; the fold difference was ~3.5-fold.Since TriCurin and tanshinone IIA appear to alter different pathways in the W12 cells, we assayed the effect of combinations of these phytochemicals.50 values for tanshinone IIA and TriCurin (90% curcuminoids) alone were ~13 and 6 μM on HeLa cervical cancer cells.The ICp < 0.01) , indicating slight synergy.When increasing concentrations of both TriCurin (90% curcuminoids) and tanshinone IIA were combined, at a dose of TriCurin 2.0 μΜ, the percent viable cells decreased from 107.84% with TriCurin alone to 87.64% with tanshinone IIA 0.2 μM, to 98.69% with tanshinone IIA 0.8 μΜ, to 104.29% with tanshinone IIA 2.0 μΜ, to 66.22% with tanshinone IIA 8 μΜ (01) Fig. . Thus, t50 values for tanshinone IIA and TriCurin (95% curcuminoids) alone were ~8 and 0.2 μM, respectively, on HeLa cervical cancer cells. Increasing concentrations of tanshinone IIA were then combined with increasing concentrations of TriCurin (95% curcuminoids) . Thus, tanshinone IIA enhances the growth inhibitory effect of TriCurin (95% curcuminoids) on the human cervical cancer cell line HeLa. The CI for the combination of TriCurin (95% curcuminoids) (0.8 μΜ) and tanshinone IIA (0.2 μΜ) was ~0.84 (p < 0.001), indicating slight synergy (Supplementary Table The ICds) Fig. . At a do50 values for tanshinone IIA and TriCurin (95% curcuminoids) alone were ~6.7 and 3.5 μM, respectively, on HeLa cervical cancer cells. When increasing concentrations of both TriCurin (95% curcuminoids) and tanshinone IIA were combined, at a dose of TriCurin 1.6 μΜ, the percentage of viable cells increased from 92.22% with TriCurin alone to 94.88% with tanshinone IIA 0.2 μM, decreased to 36.10% with tanshinone IIA 0.8 μΜ, to 44.29% with tanshinone IIA 2.0 μΜ and to 19.46% with tanshinone IIA 8 μΜ (data not shown). Thus, tanshinone IIA enhances the growth inhibitory effect of TriCurin (95% curcuminoids) on the human cervical cancer cell line HeLa. The CI for the combination of tanshinone IIA (0.8 μM) and TriCurin (95% curcuminoids) (1.6 μΜ) was ~0.34, indicating strong synergy (percent viable cells: tanshinone IIA (0.8 μM), 81.73%; TriCurin (95% curcuminoids) (1.6 μΜ), 92.22%; combination, 36.10%).In a repeat experiment, the IC50 value was: 1.91 μg/mL (8.32 μM)  (Supplementary Table 50 of 2.7 μg/mL (9.0 μM) in our assay (data not shown).To further evaluate the anti-cancer activity of the compounds, we assayed the growth inhibitory activity of resveratrol, since it is the most abundant component of TriCurin, on the oestrogen receptor (ER)-negative, Her2-overexpressing human breast cancer line MDA-MB-453; the ICμM) Fig. . The low15 The IC50 values were: MCF7, 18 μg/mL; 78 μM; MCF7(Her2), 15 μg/mL; 66 μM, indicating that sensitivity is slightly related to Her2 expression, but the Her2 pathway is not the major pathway  and 0.1 μg/mL (p = 0.0130), respectively  and MCF7/Her2–18 .way Fig. . The lowIn prior studies, we examined the effects of TriCurin on the protein levels in HeLa cells. In addition, HeLa cells were used to derive TriCurin’s synergistic molar ratio. Therefore, to better understand the mode of action of TriCurin, we examined the effect of TriCurin on HeLa cells, using microarray analysis. Cells were treated with 32 μΜ TriCurin or equal amounts of individual compounds  and collected for RNA extraction at 8 h.IL1R1, SMAD2 and PRKARIA. The functions of these genes are as follows: IL1R1 (Interleukin-1 receptor type 1), a receptor for IL1A, IL1B and IL1RN, functions in interleukin-1-dependent activation of NF-κB, MAPK and other pathways; SMAD2 ) is an intracellular signal transducer and transcriptional modulator activated by TGF-beta (transforming growth factor) and activin type 1 receptor kinases; it may act as a tumour suppressor in colorectal carcinoma; PRKAR2A  binds to anchoring proteins, such as MAP2 kinase.Genome-wide expression analysis indicates that TriCurin alters the expression of p53, apoptosis and cell cycle pathway genes. The most highly altered genes, in these pathways, after treating with TriCurin, 32 µM for 8 h, were: (up) (fold > 10) TNF (tumour necrosis factor), STEAP, BCL2, APAF-1 (apoptosis-activating factor-1), CCNG2 (cyclin-G2), APAF1, SESN2 and CDC14A. The functions of these genes are: TNF is a cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumour cell lines: STEAP, endosomal ferrireductase, functions in erythroid iron homeostasis required for efficient transferrin-dependent iron uptake in erythroid cells; BCL2 (apoptosis regulator), represses apoptosis in various cell systems; inhibits caspase activity; binds to APAF-1. CCNG2, functions in growth regulation and in negative regulation of cell cycle progression; APAF1, oligomeric Apaf-1 functions in activation of pro-caspase-9 (Apaf-3), and thereby caspase-3 and apoptosis; SESN2, functions as an intracellular leucine sensor; prevents TORC1 signalling; CDC14A, dual specificity protein phosphatase.The top down-regulated genes are (fold < −5): To reveal altered protein connections, we subjected the results of gene expression analysis (significant top up- and down-regulated genes) to STRING  analysis, as displayed in Fig. Since TriCurin activated the expression of p53, it is of interest to examine the effect of TriCurin on the mRNA levels of genes responsive to p53. Gene expression analysis indicates that TriCurin activated the expression of LRDD, TSC2 and SIAH1 and repressed the expression of El24, PTEN, ZMAT3, SESN2, CCNG2 and STEAP3.HMGB1, CD30, Prosome, ABL1, OBSCN, ARHGEF17, MAPT, CAPN10, Prune2, ID3, BMF, FASTK, IREIa, PPP2R5B) and cell cycle  genes, and repressed the expression of anti-apoptotic  genes. Some of the genes that showed the most pronounced response to TriCurin vs. the individual compounds are shown in Fig. p value): CD30 (0.0059); HMGB1 (0.0469); Prosome (0.0026)) and Fig. CER vs. vehicle : ROCK (0.0018); PEG10 (< 0.0001); NOTCH1 (0.0307)).Data were extracted to show genes significantly affected by TriCurin compared to its individual compounds, Curcumin (C), EGCG (E) or Resveratrol (R). TriCurin activated the expression of apoptotic  at time 0 is comparable to the cytotoxicity of TriCurin when dissolved in solvents , on HeLa cells, measured by a tetrazolium-based assay  (Supplementary Table p = 0.00483).TriCurin-Enpen cream (20%) was made based on the maximum amount of dry compounds able to be emulsified, producing a homogeneous cream mixture confirmed by fluorescence microscopy  generated from human donor VEC cells; the model is used by industry to test feminine hygiene products  and 1.87% (Enpen vs. Triton X-100: p < 0.001) viability, respectively, at 24 h ) or Triton X-100. Treatment with Enpen resulted in 98% viability at 24 h, whereas treatment with Gynol II or 1% Triton X-100 yielded 3.64% , while TriCurin resulted in 32.51% decrease in viability  and Gynol II (nonoxynol-9 (3%)). Treatment with Enpen resulted in 20.25% decrease in viability , TriCurin-Enpen, Gynol II or Vagisil for 4 h and examined using histology. TriCurin-Enpen appears to be non-toxic in comparison to Gynol II and Vagisil Fig. .To our knowledge, we are the first to show that the phytochemicals in the TriCurin mixture inhibit the growth of various HPV (+) cells, including W12 cells derived from a pre-cancerous cervical lesion, harbouring extrachromosomal or integrated HPV16 DNA. Moreover, the synergistic mixture of phytochemicals when formulated as TriCurin inhibits viral transcripts and alters key cellular genes used by HPV to transform keratinocytes, especially the p53 pathway. This preclinical efficacy data support the possibility that the TriCurin mixtures may target the spectrum of epithelial diseases HPV induces.10 indicating that this agent possesses specificity and may have limited toxicity in patients, especially if delivered locally. The present studies confirm that TriCurin has selective cytotoxicity for HPV (+) W12 cells and cancer cell lines. Our preliminary data using donor VEC cells in the 3D tissue implants show no toxicity on H&E examination after TriCurin-Enpen is administered topically.It is important that previous studies show that TriCurin is selective for malignant vs. non-malignant cells,In addition, we showed that TriCurin displays potent growth inhibitory activity on W12 cells. The order of activity of the TriCurin mixture and phytochemicals is: TriCurin > EGCG > resveratrol or curcumin. TriCurin and EGCG possess comparable activity on 20850 cells containing episomal HPV DNA; curcumin and resveratrol are less active. TriCurin and EGCG alone may, in particular, be useful to prevent the development of cervical cancer. However, EGCG rapidly degrades in aqueous media.It is interesting to note that resveratrol strongly inhibits the growth of Her2-overexpressing MDA-MB-453 cells. To gain insight into the role of Her2 in the action of resveratrol, we tested the effect of resveratrol on the genetically matched pair of cells MCF7 and MCF7(Her2) and found that the activity partially, but not strongly, correlates with Her2 expression.10 we found that, in HeLa cells, TriCurin activates the expression of p53, while suppressing the expression of NF-κB, in HPV16 (+) TC-1 cells. HeLa cells contain integrated and amplified HPV18 DNA missing 2–3 kb sequences from the E2 to the L2 region.21 Further, in mouse TC-1 epithelial cells that express c-Ha-ras and HPV16 E6 and E7, TriCurin and curcumin increase the level of p53, acetyl-p53, the activated form of p53, and active caspase-3. It is important that TriCurin is several folds more active than curcumin: 4.7-fold for E6 reduction and 6-fold for acetyl-p53 induction.In prior studies,E6 and E7 at 24 h. In addition, we found that TriCurin represses the expression of p53 at 6 h and of HPV16 E1, E2 and E4 at 6 and 24 h in W12 cells. It is important to highlight that TriCurin repressed the expression of E1 and E2. Both these genes have been difficult to target pharmacologically, yet are essential for viral replication.22Our present studies confirm that in W12 cells, TriCurin activates the expression of p53 and represses the expression of HPV16 viral genes In regards to p53, TriCurin slightly reduced the expression at 6 h and activated the expression at 24 h in the three W12 cell types. The order of activation was type 2 integrant (20862), type 1 integrant (20822) and episomal (20850), which relates to the state of integration.23 induce two phases of the integrated stress response depending on the dose and duration of treatment; they first stimulate the expression of NF-κB and the survival phase, and second, repress the expression, leading to cell death. The biphasic effect of actein may relate to effects on calcium metabolism. Calcium regulates all IP3 receptors in a biphasic pattern.23 Modest increases enhance responses to IP3, whereas higher doses inhibit the response. An increase in intracellular calcium may lead to an increase in NF-κB activity. Actein induces a transient release of calcium; this release may cause a transient increase in NF-κB activity, which may result in anti-inflammatory activity, as shown for S-[6]-gingerol.24 TriCurin may induce a similar pattern of effects on NF-κB and the integrated stress response. Preliminary experiments indicate that TriCurin induced a biphasic effect on the expression of NF-κB1 in W12 type 1 integrant cells. In future experiments, we will assay the effect on NF-κB protein levels.The biphasic effect on p53 may relate to an effect on NF-κB. We have observed a biphasic effect on growth, mRNA and protein levels after treatment with various phytochemicals. Actein and an extract of black cohoshE6 and E7. The inhibition of NF-κB and resulting suppression of E6 result in induction of p53.10 The magnitude of the activation of p53 may relate to the magnitude of inhibition of NF-κB.The present studies indicate that p53 is differently reduced or induced in the three W12 cell types. This finding may also relate to an effect on NF-κB. Curcumin has been shown to inactivate the transcription factors AP-1 and NF-κB, which may induce transcription of HPV E1 and E2 were the most sensitive as follows: (20822) E1 and E2 at 6 h; (20862) E2 at 6 h; (20850) E1 and E2 at 24 h. The repression was greater on early genes in episomal and type 1 integrants; and on later genes in type 2 integrants. TriCurin could exert a direct effect on viral gene expression or an indirect effect. One possibility is that TriCurin could alter the HDAC pathway, which could suppress the expression of HPV16 E6 and E7.25Concerning an effect on HPV16 mRNAs, the order of sensitivity of the cell lines was: (type 1) 20822 and (type 2) 20862 integrated clones, then  20850 cells. The early genes In future experiments, we will confirm the effect of TriCurin on the level of important proteins, NF-κB, p53, acetyl-p53, cleaved-caspase-3, poly (ADP-ribose) polymerase and HPV16 E6, and on the mRNA levels of genes responsive to p53, at 6 and 24 h in W12 cells. In addition, we will examine the possibility that TriCurin may stabilise and extend p53 half-life.The relative order of sensitivity of the cells as measured by PCR is: 20822, 20862 and 20850. The relative order of sensitivity by MTT assay is the inverse: 20850, 20862 and 20822. Thus, the effect on mRNAs cannot account for the growth inhibitory effect; other factors may contribute to the growth inhibitory effect observed. It is notable that for this experiment, TriCurin is more active than tanshinone IIA on 20850  cells, whereas the inverse is true on type 1 and 2 integrant cells. This suggests that TriCurin may be especially effective to prevent the development of cervical cancer.E1 and E2, the level of E1 in 20850 cells was 10 or more times the level in 20822 and 20862 cells. This finding is consistent with an episomal state for the viral genome. The level of all the viral mRNAs tested was reduced in 20822 and 20862 cells vs. 20850 cells, except for E6 in 20862 cells. These findings are consistent with earlier findings on the level of E6 proteins in 20850 and 20862 cells26 and are consistent with an episomal state for the viral genome in the 20850 cells. However, it is possible that the DNA integrated in some percent of the cells. In future experiments, we will examine 20850 cells, using Southern blot analysis, to further confirm the effects on extrachromosomal and integrated forms of HPV16 DNA.We passaged the 20850 cells two more times than the 20822 and 20862 cells to generate adequate cells for the RT-PCR experiments. To gain insight into the state of the DNA  in the W12 cells, we assessed the level of p53 and the HPV16 mRNAs at 0 time. For the replication genes To better understand the mode of action of TriCurin, we examined the effect of TriCurin and its individual phytochemicals on HeLa cells, using gene expression analysis. TriCurin activated the expression of apoptotic, p53 pathway and tumour suppressor genes and repressed the expressions of anti-apoptotic genes.BCL2, APAF1, TNF and SMAD2. The top up-regulated genes alter the NF-κB and TGF pathways and function as tumour suppressor genes; the top down-regulated genes repress apoptosis and prevent Torc2 signalling and play a role in gene regulation. In addition, gene expression analysis indicates that TriCurin altered the expression of genes responsive to p53: (up) LRDD, TSC2 and SIAH1 and (down) El24, PTEN, ZMAT3, SESN2, CCNG2 and STEAP3.To reveal connections among the altered pathways, we subjected the results of gene expression analysis  analysis Fig. . The gen23 Concerning p53 and PTEN, Stambolic et al.27 have shown that p53, via PTEN, functions in negative regulation of cell survival. In addition, the study of Feng et al.28 indicates that miR-130a induces cervical cancer cell proliferation by targeting PTEN, dependent on NF-κB. It will be important to assess the effects of TriCurin, at early and later times, 6 and 24 h, on cell proliferation, NF-κB, miR-130a and PTEN.TriCurin inhibits cell proliferation, yet reduces the expression of PTEN at 8 h. A possible explanation for these findings is that TriCurin may induce a transient increase in PTEN and a stimulation of cell proliferation, which may be related to the effects we have observed for NF-κB1 (increase) and p53 (decrease) at early times (6 h). The action of TriCurin may resemble that of the phytochemicals black cohosh and actein, which induce two phases of the integrated stress response (iSR), a transient stimulation of proliferation and NF-κB activity, followed by a decrease.HMGB1, CD30, Prosome) and Fig. ROCK, ADAM17, NOTCH1).Further, TriCurin activated the expression of apoptotic genes and repressed the expression of anti-apoptotic genes more than its individual phytochemicals. The genes that showed the most pronounced response to TriCurin vs. its individual compounds are shown in Fig. Since cancer has been associated with mutations in multiple genes, we assessed the growth inhibitory effects of combinations of active phytochemicals. When we substituted turmeric containing 95% curcuminoids, for turmeric containing 90% curcuminoids, the activity of TriCurin increased >3.5-fold.50 of TriCurin was 3.55 µM and the combination with tanshinone IIa (0.8 µM) was 1.2 µM. Thus, the combination of TriCurin (95% curcuminoids) and tanshinone IIA may be useful for HPV (+) cancer therapy. The use of drug combinations may allow the use of lower doses, which may potentially lessen toxic side effects. These botanicals are attractive since, for the most part, they have a history of safe use and are inexpensive,Tanshinone IIA enhanced the growth inhibitory effect of TriCurin containing 90% and 95% curcuminoids. Tanshinone IIA synergised with TriCurin . In addition, in a repeat assay, the IC10Concerning in vivo activity, we previously showed that intralesional injection of TriCurin into tumours formed after HPV16 (+) TC-1 cells were implanted subcutaneously resulted in 80–90% decrease in tumour size. Further, TriCurin injected subcutaneously in tumour-naive mice yielded no adverse effects.29 The authors argue that TriCurin activates an innate and adaptive immune response that drives immune clearance.A recent study found that when TriCurin is injected into mice bearing tumour xenografts from a tumorigenic cell line expressing HPV16 E6 and E7 oncoproteins, it suppresses the growth of the tumours at concentrations that are not adequate to kill tumour cells in vitro.Specifically, injection of TriCurin had two effects: it repolarised tumour-associated macrophages from the M2-like form, which promotes tumour growth, to the M1-like phenotype, which produces an anti-tumour phenotype. In addition, TriCurin affected IL-12 signalling to recruit activated NK cells and cytotoxic T-lymphocytes (CTLs) to the tumour. It will be worthwhile to determine if the substitution of turmeric containing 95% curcuminoids, instead of 90%, in TriCurin, or the combination of TriCurin with tanshinone IIA enhances immune response.10 This finding suggests that our combinatorial approach can overcome many of curcumin’s limitations seen in prior clinical trials. To advance towards clinical testing, a microemulsion-based cream was developed to specifically deliver TriCurin locally. Our findings indicate that TriCurin in a microemulsion is stable and non-toxic. The major advantage of topical drugs is that significant tissue concentrations can be achieved while reducing any systemic side effects. HPV initially infects basal epithelial cells, creating a reservoir in keratinocyte stem cells along the basement membrane of epithelial tissue. An effective therapeutic will need to reach these basal cells to eradicate HPV. Our prior studies show topical application of TriCurin dispersed in a cream ) resulted in complete permeation of curcumin (fluorescent) across the full thickness of the skin at 20 h.10 Further, when TriCurin was injected subcutaneously in mice not bearing tumours, no adverse effects were observed. TriCurin is, therefore, a safe and promising therapy against HPV-induced diseases.The use of curcumin in clinical trials has shown limited utility due to its rapid degradation in an aqueous environment, poor systemic bioavailability and overall low target tissue delivery. Compounds with antioxidant activity that also developed intermolecular interactions with curcumin, leading to close π–π stacking of aromatic molecules in an aqueous environment, could stabilise curcumin. Our previous studies indicate that EGCG and resveratrol stabilise curcumin in aqueous media.Our findings show that TriCurin is effective against a variety of HPV (+) cells, including the unique cell line W12, derived from a cervical pre-cancerous lesion containing cells with extrachromosomal or integrated HPV16 DNA. The growth of three W12 cell clones, episomal, type 1 and 2 integrants can be inhibited and the relative order of activity is TriCurin, EGCG, curcumin or resveratrol. RT-PCR indicates that TriCurin activates the expression of p53 and reduces the expression of HPV16 mRNAs E1, E2, E4, E6 and E7 at 24 h in the three W12 cell clones. Genome-wide expression analysis shows that TriCurin activates the expression of pro-apoptotic genes and represses the expression of anti-apoptotic genes, more than its individual compounds, in HeLa cells. The combination of tanshinone IIA with TriCurin (95% curcuminoids) exhibits additional synergy on HeLa cells. An organotypic tissue culture model shows that TriCurin microemulsion is stable and non-toxic. The phytochemical mixture TriCurin in the microemulsion-based cream is a promising therapeutic candidate for the prevention and treatment of HPV-induced cancers.Supplementary Information


Bacterial community (16S rRNA metabarcoding), Microcystis specific 16S rRNA, microcystin synthetase gene E (mcyE) and microcystins (MCs) were assessed. Radionuclide measurements  were used to date sediments. (3) Results: Bacterial community, based on relative abundances, differed significantly between surface sediment sites (p < 0.001) but the majority of bacterial amplicon sequence variants (88.8%) were shared. Despite intense MC producing Microcystis blooms in the past, no Microcystis specific 16S rRNA, mcyE and MCs were found in surface sediments but occurred deeper in sediment cores (approximately 1950′s). 210Pb measurements showed a disturbed profile, similar to patterns previously observed, as a result of earthquakes. (4) Conclusions: A single sediment core can capture dominant microbial communities. Toxin producing Microcystis blooms are a recent phenomenon in Lake Rotorua. We posit that the absence of Microcystis from the surface sediments is a consequence of the Kaikoura earthquake two years prior to our sampling.(1) Background: Paleolimnological studies use sediment cores to explore long-term changes in lake ecology, including occurrences of harmful cyanobacterial blooms. Most studies are based on single cores, assuming this is representative of the whole lake, but data on small-scale spatial variability of microbial communities in lake sediment are scarce. (2) Methods: Surface sediments (top 0.5 cm) from 12 sites ( Lake ecosystems worldwide are experiencing dramatic changes in water quality, nutrient concentrations and phytoplankton community composition due to global warming, eutrophication and introductions of non-native species ,2. ConsePlanktothrix, Oscillatoria, Dolichospermum and most prominently Microcystis [mcy gene cluster (mcyA-J) encoding these biosynthetic enzymes has been employed as the target region for the molecular detection of potentially toxin producing cyanobacteria strains in water and sediment [MCs are potent inhibitors of eukaryotic protein phosphatases resulting in a wide range of organ toxicities, but primarily causing liver damage . Cyanobarocystis . At leasrocystis . MCs arerocystis . The mcysediment ,14,15.mcy gene cluster) identified in sediment samples [Microcystis sp. that overwinter in surface sediments [Considerable effort has been invested in developing management and restoration plans for water bodies experiencing cyanobacterial blooms ,17,18. B samples ,7. Accum samples . This isediments ,22.Microcystis spp., are highly dependent on wind and current drift, hence there is often high spatial variability across a lake surface [Both cyanobacterial DNA and MCs are thought to be well preserved in sediments, and both have been detected in sediment layers dating back over 200 years ,7,8,9,10 surface ,26,27. C surface ,29. Ther surface  to globa surface ,32,33. HDolichospermum, Aphanizomenon and MC-producing Microcystis present in high abundances. However, there is uncertainty as to whether these species have always been present in the lake and when blooms begun. The aims of this study were to: (i) determine the spatial variability in bacterial and cyanobacterial species in the surface sediment of Lake Rotorua, with a focus on the abundance of Microcystis, MC synthetase gene E (mcyE) copy numbers and MCs; and (ii) explore the historical bacterial (in particular cyanobacteria) composition in two sediment cores.This study focuses on Lake Rotorua, a small hypertrophic lake in the northeast of the South Island of New Zealand . The lak−1 and was highest in the embayment  and at one littoral site  , low  in samples S01–02, S06–07, S10 and S12, and no copies were detected in samples S08, S09 and S11  after quality-filtering and pre-processing was 1011. Negative controls contained 372 reads assigned to 12 ASVs. These were excluded when ASV that accounted for >0.01% of reads were removed. A total of 546 ASVs (54%) were shared between embayment, littoral and mid-lake sampling sites  and 65 Ayanobium b. Contradetected b. Furthet = 3.142, p = 0.0001; emb.-mid.: t = 4.011 p = 0.0007; mid.-littoral: t = 2.285, p = 0.0001; t = 2.153, p = 0.001; emb.-mid.: t = 5.577, p = 0.0006; mid.-littoral: t = 3.026, p = 0.0001; The NMDS ordination showed clear grouping of the total bacterial community by sampling site . The com−1 or <2 µg g−1 wet weight in a 10 g sample) in any of the surface sediments collected. Recovery rates of the spiked MC congeners ranged between 25% for MC-RR, 73% for MC-LR and 126% for MC-LA (average result for the four spiked samples), indicating absorption to the sample matrix for certain congeners  were shared among both cores . An addiyanobium b. In theyanobium . In deepyanobium .Microcystis was detected only in traces in the uppermost layer  but increased from 4 to 17 cm. A peak concentration of 18,217 mcyE copies g−1 sediment was observed at 10–11 cm. Beyond 17 cm, mcyE copy numbers were not detected (<100 copies g−1 sediment).The vertical profile of  results b. Copy n137Cs chronomarker profile showed a typical increase in activity over sediment depth. The increase was from 4 cm sediment depth down to a depth of 14–15 cm and was below background level for the two deeper layers  and the first below background measurement  no Microcystis cells have been recorded in water column samples . In November 2016, an earthquake of magnitude 7.8 struck the Kaikoura region. This was the second biggest earthquake recorded in New Zealand since European settlement. The earthquake triggered an estimated 100,000 landslides in the region, although no major ones were visible directly around the lake [The absence of the lake ,45. It ithe lake  and eartthe lake ,48. Eartthe lake . These sthe lake .137Cs profile of the sediment core was as expected, 210Pb did not display its typical linear decay profile known from undisturbed sediments. Non-linear 210Pb profiles where previously shown for Lake Antern in the French Alps [210Pb depleted material [While the nch Alps  and the nch Alps  as a resmaterial , or frommaterial , therebymaterial ,51, in amaterial  and in amaterial . This deCyanobium and Snowella are common freshwater picocyanobacteria [Aphanizomenon and Dolichospermum both produce akinetes, thick walled cells that remain dormant until environmental conditions are favorable. It is possible that these were more resilient to sediment mixing than Microcystis vegetative cells or survived in shoreline terrestrial habitats, allowing these genera to rapidly recolonize following the sediment disturbance.The 2016 Kaikoura earthquake had a magnitude of 7.8, which was higher or equivalent to the seismic event related changes in the sediments described for the European Alps, Chile and earlier seismic events in New Zealand. Thus, it is likely that the disturbed upper 3–4 cm in Lake Rotorua resulted from a mixing effect as well an increased deposition of erosion material . The finbacteria . They arbacteria ,55 and mMicrocystis 16S rRNA and mcyE gene copies and MCs in surface sediments, in addition to the disturbed 210Pb profile, suggests that allochthonous processes may have impacted the surface sediment, changing the cyanobacterial communities. The 2016 Kaikoura earthquake may have played a role in this, but further research is required to confirm this hypothesis. The absence of Microcystis 16S rRNA and mcyE gene copies in the sediment layers prior to about 1950′s indicates that toxin producing cyanobacterial blooms in Lake Rotorua are a relatively recent occurrence.In this study, we demonstrate that bacterial communities of Lake Rotorua surface sediments significantly differed between lake sites . However, the majority of ASV were shared between the mid and littoral sites, indicating that in small to medium sized lakes, the sampling approach used by most paleolimnological studies will also capture dominant microbial communities. The absence of 2), shallow (max. depth 3 m), hypertrophic lake in the northeast of the South Island of New Zealand [−1 [Dolichospermum and Aphanizomenon) to Microcystis later in summer [Microcystis blooms, peak concentrations of >2000 µg L−1 total MC and 4 × 106 cells mL−1 have been measured [Lake Rotorua  is a small  were screened in duplicates for inhibition using an internal control quantitative PCR (qPCR) assay [Subsamples of sediment (0.2–0.4 g) were weighed into the first tube of a PowerSoil DNA Isolation Kit  and DNA extracted according to the protocol supplied by the manufacturer. A random selection of DNA samples (R) assay .Microcystis cells present was determined by amplifying the mcyE gene (a single copy gene) and quantifying the number of amplicons, using a standard curve generated from MC-producing Microcystis sp. strain CAWBG617 [mcyE open reading frame  [mcyE probe [R2 > 0.99) and PCR efficiency was >0.9. The results of copies per μL DNA were converted to copies per gram of sediment using: (D × 80)/E, with D being the number of copies per μL, × 80 since 1 μL DNA of the elution volume (80 μL) was used, and E being the weight of sediment (g) used for DNA extraction.The number of toxin producing CAWBG617 . The qPCMcyE-R8) , 0.2 µL yE probe  and 1 µLMicrocystis sp., a region of the 16S rRNA gene was amplified using Microcystis sp. specific primers MICR184F [mcyE enumeration.To detect and quantify MICR184F  and MICRMICR184F . qPCR anmcyE copy numbers in sediment cores was carried out with droplet digital PCR (ddPCR) using a BioRad QX200 system. Each ddPCR reaction included 2 μL of 900 nM of each primer and 0.55 μL of 250 nM of primer probe (primers and probe as described for qPCR), 11 μL of BioRad ddPCR Supermix for probes, 1.5 μL DNA, and 4.95 μL of sterile water. The reaction mixture was divided into nanodroplets by joining 20 μL of the reaction mixture with 70 μL of BioRad droplet oil. After processing, the nanodroplet volume resulted in a total of 40 μL, which was transferred to a PCR plate for amplification at 96 °C for 10 min, followed by 40 cycles at 94 °C for 30 s, 60 °C for 60 s and a final extension step of 96 °C for 10 min. The ddPCR was run on one plate, with four negative controls  and four positive controls (genomic DNA extracted from a sample known to contain the mcyE gene) included. The results were converted to copies per gram of sediment using: (D × 66)/E, with D being the number of copies per μL, × 66 since 1.5 μL DNA of the elution volume (100 μL) was used, and E being the weight of sediment (g) used for DNA extraction.Quantification of the ® 360 master mix , 12.5 μL GC inhibitor , 2 μL of each primer , 2 μL template DNA (between 60 and 220 ng) and 6.5 μL Milli-Q water. PCR cycling conditions were: 95 °C for 10 min, followed by 27 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 45 s, and a final extension of 72 °C for 7 min. PCR products were visualized with 1.5% agarose gel electrophoresis with Red Safe DNA Loading Dye  and UV illumination to ensure amplification of a single 400 bp product. PCR products were purified , quantified , diluted to 10 ng µL−1 and submitted to New Zealand Genomics Limited  for library preparation. Sequencing adapters and sample-specific indices were added to each amplicon via a second round of PCR using the NexteraTM Index kit . Amplicons were pooled into a single library and paired-end sequences (2 × 250) were generated on a MiSeq instrument using the TruSeqTM SBS kit . Sequence data were automatically demultiplexed using MiSeq Reporter (v2), and forward and reverse reads were assigned to samples.Surface and core sediments were analysed via Illumina™ sequencing. A region of the 16S rRNA gene approx. 400 bp long was amplified using bacteria-specific primers 341F: 5′-CCT ACG GGN GGC WGC AG-3′ and 805R: 5′-GAC TAC HVG GGT ATC TAA TCC-3′, modified to include Illumina™ adapters ,64. PCR Analysis of sequencing data was performed using the QIIME 2 2019.1 . Raw seqTo visualize bacterial community dissimilarities between sampling sites and depth increments, non-metric-multidimensional-scaling (NMDS) was performed, using the R vegan package . Filtereg, 10 min, 10 °C). The extracts were pooled and dried at 40 °C under a stream of nitrogen gas and dissolved in 1 mL 100% MeOH. Extracts were stored at −20 °C until measurement.Surface sediment samples (6–10 g wet weight) were freeze-dried and extracted with three consecutive extractions using 100% MeOH (5 mL each). During each extraction step, samples were vortexed for 1 min, sonicated for 15 min and centrifuged  as described previously . To asseongeners . The MC-210Pb and 137Cs at the Institute of Environmental Science and Research . Activities were quantified using a gamma counter with a high-purity germanium well detector. Activities are reported in Bq per kg. Uncertainties are based on the combined standard uncertainty multiplied by a coverage factor (k) = 2, (providing a level of confidence of 95%) as described before [210Pb and 137Cs measurements were calculated using the constant rate of supply (CRS) model in R by means of the mudata2 package [210Pb to the sediment is the same for each time interval, the CRS model is calculated as follows:For age-dating of the mid-lake sediment core, subsamples representing six increments  were analyzed using gamma spectrometry of the long-lived radionuclides d before ,77. Core package . Assuminrval (z) .


The remarkable reliability of the PVA–PANI–MLG HDG was demonstrated by 92.6% retention after 3000 cycles of galvanostatic charge–discharge. The advantages of this HDG mean that a coin cell supercapacitor assembled using it is a promising energy storage device for mobile and miniaturized electronics.Electroactive polymer hydrogel offers several advantages for electrical devices, including straightforward synthesis, high conductivity, excellent redox behavior, structural robustness, and outstanding mechanical properties. Here, we report an efficient strategy for generating polyvinyl alcohol–polyaniline–multilayer graphene hydrogels (PVA–PANI–MLG HDGs) with excellent scalability and significantly improved mechanical, electrical, and electrochemical properties; the hydrogels were then utilized in coin cell supercapacitors. Production can proceed through the simple formation of boronate (–O–B–O–) bonds between PANI and PVA chains; strong intermolecular interactions between MLG, PANI, and PVA chains contribute to stronger and more rigid HDGs. We identified the optimal amount of PVA (5 wt.%) that produces a nanofiber-like PVA–PANI HDG with better charge transport properties than PANI HDGs produced by earlier approaches. The PVA–PANI–MLG HDG demonstrated superior tensile strength (8.10 MPa) and higher specific capacitance (498.9 F/cm With the growing development of supercapacitor devices, there have been great improvements in electroactive polymer (EAP) nanomaterials with high specific capacitance ,2,3. Pol2) that resulted, PANI HDG needs further development to improve its electrochemical, electrical, and mechanical properties for use in high-performance supercapacitor devices. Especially, there have been few reports on the PVA content needed for the optimal performance of PANI HDG.Recently, electroactive hydrogels (HDGs) have attracted interest as supercapacitor materials because of their high flexibility, durability, and remarkable structural stability during many charge/discharge cycles ,10,11,12sp2-hybridized carbon nanomaterial having a one-atom-thick planar structure that is predicted to have a large specific surface area of 2640 m2/g . ThTo confirm the effects of MLG on the mechanical properties of HDG, the stress–strain curves of PVA–PANI–MLG HDGs are shown in 2SO4 in the voltage range of 0–1.0 V at a scan rate of 20 mV·s−1. The rectangular shape of the curves indicates that MLG enhances capacitive behaviors and reduces response times [To evaluate the effects of the MLG content on the supercapacitor performance of PVA–PANI–MLG HDGs, CV, GCD, EIS, rate capability, cycling stability tests, and Ragone plots were utilized . The PVAse times ,19,20,21se times ,6,7,8,9.2 in the voltage range of 0–1.0 V. The IRs of the HDGs with different MLG contents were evaluated from the voltage drop at the onset observed in the discharge curves [2). These results reconfirm that the π–π stacking interaction between MLG and PANI chains significantly enhanced the charge transport within the HDGs, resulting in smaller voltage drops [e curves ,19,20,21ge drops . Howeverge drops ,9,17,25.To further investigate the effect of MLG on the electrochemical properties of the HDGs, Nyquist plots of the assembled samples employing PVA–PANI–MLG HDGs with different MLG contents were measured using EIS in the frequency range of 1–10 MHz c. Until AC, lC, and mC of the PVA–PANI–MLG HDGs with different MLG contents were estimated from the GCD analyses (AC (498.9 mF/cm2) was realized at an MLG content of 3 wt.%, which was superior to those of 4.5 wt.% (479.3), 1.5 wt.% (449.5), 0.5 wt.% (382.7), and 0 wt.% (344.8 mF/cm2) samples. The same trends were observed for lC and mC  < 0.5 wt.% (127.6) < 1.5 wt.% (149.8) < 4.5 wt.% (159.8) < 3.0 wt.% (166.3 F/cm3). The values of mC at a current density of 0.3 mA/cm2 for the 0, 0.5, 1.5, 4.5, and 3 wt.% samples were 210.1, 233.2, 273.9, 292.1, and 304.0 F/g, respectively. As the current density increased, the Faradaic reactions of HDGs became sluggish, and the effective thickness of the electrode material decreased [2, as used for all five samples) decreased more slowly as the current density increased than those of the 1.5 wt.% (90.1%), 0.5 wt.% (85.9%), 0 wt.% (82.0%), and 4.5 wt.% samples  of these materials are shown in E and P of the MLG HDG were 5.20 × 10−2 Wh/cm3 and 2.55 W/cm3, respectively, and these values were significantly superior than those of the HDG without MLG . Furthermore, the MLG HDG showed more gradual reductions in energy density than that without MLG. These results reaffirm that the synergistic effects of PANI chains and MLG were advantageous for combining the benefits from both the pseudocapacitor and EDLC mechanisms, resulting in remarkable improvements in both the energy density and power density of the coin cell supercapacitors [To compare further the practical applicability of coin cells employing PVA–PANI–MLG and PVA–PANI HDGs, the Ragone plots  and retention rate (84.3% retention after 3000 cycles of GCD) than that with 10 wt.% PVA . The FTIR spectrum of PVA–PANI–MLG HDG containing 3 wt.% MLG showed that it had successfully formed –O–B–O– bonds and enhanced conjugation paths. Due to the strong intermolecular forces between MLG, PANI, and PVA chains, remarkable tensile strength (8.10 MPa) and specific capacitances  were realized by introducing 3 wt.% MLG into the PVA–PANI HDG. More importantly, remarkable retention rate (92.6% retention after 3000 cycles of GCD) and improved energy density (5.20 × 10−2 Wh/cm3) and power density (2.55 W/cm3) ensure the practical applicability of the HDG. Thus, our work on a coin cell supercapacitor based on PVA–PANI–MLG HDG will accelerate the application of this material as a promising power source for various mobile and miniaturized electronic devices.In this comparative study, PVA–PANI–MLG composite HDGs with high scalability and remarkable mechanical, electrical, and electrochemical properties were demonstrated for use in a coin cell supercapacitor. The optimal amounts of PVA and MLG sheets to achieve the best performance were 5 and 3 wt.%, respectively. In FE-SEM images of these HDGs, HDG composed of PANI nanofibers could be achieved by choosing a PVA content of 5 wt.%, resulting in a higher aspect ratio for delocalizing electrons compared to those of the 10 and 15 wt.% samples. PVA–PANI HDG based on 5 wt.% PVA showed superior specific capacitance (344.8 F/cm