Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production. Auxin is a key regulator in vascular cambium development. This study shows that gibberellins promote polar auxin transport along the root, which leads to broadening of high auxin signalling domain in cambium, and thus, to increased xylem formation. Arabidopsis thaliana. The vascular cambium consists of meristematic cells that undergo periclinal cell divisions . Cambium cells that leave the meristem ultimately differentiate into parenchymatic or conductive cells, with secondary xylem being produced inwards and secondary phloem outwards3  growth of plant stems and roots. This process is particularly prevalent in tree species but also occurs in non-woody species such as 8. Mutations in genes encoding components of auxin signalling, including those associated with perception and polar transport of the hormone, cause defects in cambium development9, vascular patterning11, leaf venation12, and xylem and phloem formation in planta14, in tissue culture15 and during vascular regeneration16. Recently, we showed that ectopic clones with high levels of auxin signalling force non-xylem cells to differentiate into secondary xylem vessels, while cells adjacent to such clones divide periclinally and gain expression of cambial markers4. The ectopic clone thus behaves as an organizer that causes adjacent cells to specify as vascular cambium stem cell-like cells. In agreement with this, an auxin maximum is normally located on the xylem side of the vascular cambium, and stem cell divisions occur adjacent to this maximum4. These data raise the question of whether the location of the auxin maximum within the cambium has a role in stem cell fate decisions.A major regulator of cambium development is the phytohormone auxin8. For example, gibberellins  promote secondary xylem production in both Arabidopsis17 and poplar20. In Arabidopsis, this occurs during flowering, when GA levels rise17. Recently, AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 have been shown to mediate auxin-dependent xylem production that is downstream of GA signalling21. Interactions between auxin and GA also occur in other biological processes. For example, in Arabidopsis roots, GA directly promotes abundance of PIN-FORMED (PIN) polar auxin transporters in the root meristem, thus regulating polar auxin transport (PAT)22.Other phytohormones also influence cambium development alongside auxinArabidopsis thaliana roots. This results in an expanded auxin signalling maximum within the root vascular cambium, which forces cambial stem cell daughters to preferentially specify as xylem cells. Our data show how GA influences the position of the auxin maximum in cambium, therefore determining stem cell fate decisions between xylem and phloem.In this Article, we show that GA promotes PIN1-dependent PAT in Arabidopsis hypocotyls during flowering17. To understand the role of GA on cambial growth dynamics, we analysed GA’s effect in Arabidopsis roots at a cellular resolution. To reach that goal, we analysed roots during the early stages of secondary growth, when cell division and differentiation dynamics are easier to follow. At these stages, only two types of xylem cell are produced: secondary xylem vessels and xylem parenchyma ), had a reduced number of xylem vessels and parenchymatic cells 24 to determine whether GA affects the production of conductive phloem cells. We observed that the ratio of APL-positive cells to total phloem cells was increased after GA treatment and decreased in ga1 27, result in increased xylem area21 within the hypocotyl and could therefore also have an effect in root secondary growth. Indeed, we found that pRGA:GFP-RGA27 showed broad expression in the root cambium, appearing in both the xylem and the phloem. While 3 day GA biosynthesis inhibitor  treatment increased green fluorescent protein (GFP)-RGA signal, 24 h GA application led to strong reduction of the signal in all cell types  results in the formation of a dominant, non-degradable version of the protein28. By driving this dominant inhibitor of GA signalling under three different cell type-specific inducible promoters, we investigated where GA signalling is required for its effect on cambium development. Inhibition of GA signalling under the promoter of the early phloem gene PHLOEM-EARLY-DOF 1 (PEAR1)29 did not inhibit xylem production 4, and especially under the promoter of the early xylem gene HOMEOBOX GENE 8 (AtHB8) (ref. 4) significantly reduced xylem production -based version of the auxin response reporter, DR5v2 (ref.30)  and even reached the cell in position −2, but it was rarely seen in the phloem-side daughter (1). In ga1, a smaller proportion of stem cell daughters showed DR5v2 expression (expression in positions 1 or −1 was seen in 29% of ga1 roots and 48% of control (Col-0) roots; Fig. DR5v2 expression   , we obseref.30) , matchin-0) Fig. . Altoget4, this GA-induced broadened auxin response gradient could explain how GA promotes vessel production , ARF7 and ARF19 (ref. 4). We therefore treated two different allelic arf7,19 mutant combinations and the conditional triple mutant amiMP ; Since auxin drives xylem vessel formationion Fig. . To the 32 to promote xylem identity in the root cambium4. A representative member of the family, AtHB8, is expressed specifically in the early xylem cells4. Since ga1 has a narrow auxin signalling maximum  analysis  act downstream of auxin signallingmum Fig. , AtHB8 esis Fig. . Inducibion Fig. . Taken t34. Since GA has previously been reported to regulate PIN levels in the root apical meristem35, we investigated whether GA also regulates auxin accumulation, and thus auxin signalling, through PIN activity in the vascular cambium. Of the five plasma membrane-localized PINs  (ref. 34), only PIN1-GFP showed consistent expression on the xylem side of the vascular cambium  than induction of PIN1 (24 h), we hypothesized that GA-mediated PIN1 upregulation is independent of ARF-mediated auxin signalling. Supporting this hypothesis, GA treatment leads to spread in PIN1 expression in arf7,19, similarly to the GA treatment in WT  medium to grow for an extra 1 or 2 days. For the PAT assay, tritium-labelled indole-3-acetic acid (3H-IAA) was applied to the root–shoot transition zone, and radioactivity was measured in either the upper part of the root  auxin transporters, ABCB19 and ABCB21, also contribute to maintenance of PAT streams in the vasculaturepin1 mutants, pin1-1 and pin1-7. GA treatment led to an increased number of secondary xylem vessels in WT but not in either of the pin1 mutants 41. ANT was typically expressed in both stem cell daughters . After 6 days, under normal growth conditions, stem cell sectors spanned almost equally towards both the xylem and the phloem  oem Fig. , similaroem Fig.  and whatoem Fig. . Unexpec29) Fig. . Under n29) Fig. . HoweverArabidopsis hypocotyls21 to provide mechanical support for the growing inflorescence stem. Our study shows that GA regulates the balance between xylem and phloem production , gene of interest or a tag (second box) and a tag or terminator (third box) with Gateway-compatible binary destination vectors in a MultiSite Gateway LR clonase reaction. All of the expression vectors generated in this study are listed in Supplementary Table RGAΔ17 lines, five lines with PEAR promoter (five out of five behaved as shown), three lines with ANT promoter  and eight lines with AtHB8 promoter (six out of eight behaved as shown) were analysed. A previously published inducible microRNA (miRNA) against MP (amiMP)4 line was dipped into the arf7-2,19-5 background due to silencing issues in the earlier arf7-1,19-1 background. Seeds published in this study, as well as the already published lines, are listed in Supplementary Table pHS:Dbox-CRE x 35S:lox-GUS4, p35S:XVE»miR165a4, pANT:XVE-CRE x 35S:lox-GUS4, pPIN1:PIN1-GFP55, pPIN1:PIN1-GFP x ga122, pPIN2:PIN2-GFP55, pPIN3:PIN3-GFP56, pPIN4:PIN4-GFP57, pPIN7:PIN7-GFP57, pRGA:GFP-RGA27, DR5rev:GUS58, arf7-1,19-159, arf7-2,19-560, pin1-7 (SALK-047613)61, pin3,4,762, pin1-111, ga1 (SALK-109115)22, abcb19-10163 and abcb21-140.All of the expression vectors were dipped in the Col-0 background, and single insertion lines were screened based on Mendelian segregation of the selection marker. Several single insertion lines were screened for each construct to observe the most consistent phenotypes or expression patterns. For inducible 2O and then plated on a half-strength growth medium , 1% sucrose, 0.5 g l−1 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8 and 0.8% agar) and vernalized at 4 °C for 2 days. In the case of ga1 (SALK-109115), after sterilization the seeds were soaked in 100 µM GA3 for 5 days and covered at 4 °C. Before plating, seeds were washed five times with H2O. The age of the plants was measured from when the plates were vertically positioned in the growth cabinet. The temperature in the cabinets was 22 °C, and they had long-day conditions (16 h of light). To get seeds from ga1 plants, plants growing in soil were sprayed with 100 µM GA3 twice per week until they had seeds.Seeds were surface sterilized first with 20% chlorine and then with 70% ethanol, washed twice with H4,7 (Duchefa) and GA3 (Duchefa)  were prepared in 100% EtoH and stored at −20 °C. A 10 mM stock of GA biosynthesis inhibitor paclobutrazol (Sigma) was prepared in 100% EtOH and stored at −20 °C. A 10 mM stock of EdU, a thymidine analogue (Thermo Fisher), was made in dimethyl sulfoxide (DMSO) and stored at −20 °C. A synthetic derivative of oestradiol,17-b-oestradiol (EST) (Sigma), was prepared as a 20 mM stock solution in DMSO and stored at −20 °C.Stocks of GA3 (100 μM) was used for ga1 seed germination and seed production. The working concentration for GA4,7 was 2 µM, and paclobutrazol working concentration was 10 µM. XVE-based gene induction was achieved by transferring plants onto plates containing 5 µM 17-b-oestradiol or an equal volume of DMSO as a mock treatment. For EdU incorporation, plants were placed in liquid ½GM containing 10 µM EdU for the time stated in each experiment.GA64. Samples were fixed with 90% acetone on ice for 30 min, washed two times with a sodium phosphate buffer  and then vacuum infiltrated with the GUS-staining solution . Samples were placed at 37 °C until the staining was at the desired level (the required time varied between different lines).The protocol was modified from Idänheimo et al.After staining, the samples were fixed overnight in 1% glutaraldehyde, 4% formaldehyde and 0.05 M sodium phosphate pH 7.2. Fixed samples were dehydrated in an ethanol series , with 30 min for each step, and then incubated for 1 h in a 1:1 solution of 100% ethanol and solution A (Leica Historesin Embedding kit). After 2 h in solution A, samples were placed in plastic chambers and filled with 14:1 mixture of solution A: hardener.Sections of 5 or 10 μm were prepared on a Leica JUNG RM2055 microtome using a microtome knife (Leica Disposable blades TC-65). The sections were imaged without staining or after staining with Safranin O (Sigma-Aldrich)  or double staining with 0.05% ruthenium red (Sigma-Aldrich) and toluidine blue (Sigma-Aldrich) . Sections were mounted in water and visualized with a Leica 2500 Microscope.4, samples were vacuum infiltrated with 4% paraformaldehyde solution  in 1× phosphate-buffered saline (PBS) pH 7.2. After fixation, the samples were washed with PBS and embedded in 4% agarose. Embedded samples were cut with a vibratome into 200 µm sections for confocal analysis. Agarose slices were placed into PBS with SR2200  for cell wall staining. For root tip visualizations, we fixed the samples with 4% PFA, cleared them with CLEARSEE and stained the cell walls with SR2200, as in Ursache et al.65.Using a protocol modified from Smetana et al.41. Samples were incubated in the detection mix for 1 h on ice and then transferred into PBS with SR2200 (1:1000).To visualize EdU-positive nuclei, EdU detection was performed on the agarose sections before cell wall staining. The Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher) was used for detection with a modified EdU detection mixLight microscopy images were taken with a Leica 2500 microscope (20× and 40× objectives). Fluorescent markers were imaged with a Leica Stellaris 8 confocal microscope (63× objective). Confocal images were obtained with Leica Las AF software using PBS or water as the imaging medium. All confocal images with multiple channels were imaged in sequential scan mode. Confocal settings may have varied between experiments but always stayed the same for the experimental sample and the respective control. To better optimize the SR2200 cell wall staining, the signal was sometimes adjusted during imaging and may thus vary between the sample and control.The Leica Stellaris 8 has a Tau-gating mode that makes it possible to separate GFP signals from background signals. GFP markers were always imaged with this Tau-gating mode, gathering signals between 1.3 ns and 9 ns.Contrast was adjusted with Abobe Photoshop for the whole GUS images in the following figures: Figs. 123466), and image region of interest (ROI) area extraction was done using several different libraries, including OpenCV2 (ref. 67), Pillow68, Matplotlib v2.2.1 (ref. 69) and NumPy70. More detailed documentation is available on GitHub (https://github.com/LMIVainio/PolarUnwrap/find/main).For image projections  cell division, so this also sometimes includes the stem cells and stem cell daughters  and 1.8–2.6 cm . 3H-IAA was measured by liquid scintillation counting. The 1.8–2.6 cm segments contained lateral root primordia and emerged lateral roots. Data shown are mean ± standard deviation (s.d.) (three independent pools of ten seedlings).Six-day-old seedlings on half MS agar plates were treated by applying a thin surface drench of 3 µM GA75, to the reference genes ACT2, UBQ10 and TIP41. Three biological replicates were used for each line and treatment, as well as three technical replicates.RNA was collected from 2-cm-long pieces starting just below the hypocotyl of 10-day-old roots where lateral roots had been removed. RNA was isolated using the GeneJET Plant RNA Purification Mini kit (Thermo Fisher) and treated with DNase. Complementary DNA was synthesized from 100 ng of RNA using Maxima H Minus reverse transcriptase (Thermo Fisher) and oligodT primers (Thermo Fisher). The PCR reaction was done on a Bio-Rad CFX384 cycler using EvaGreen qPCR mix (Solis Biodyne) and the following program: 10 min at 95 °C, 50 cycles . All of the primers used in qRT–PCR are listed in Supplementary Table 76.For ABCB21 expression, 7 day seedlings were surface drenched with MS solution containing solvent control, 1 µM GA or 10 µM GA for 15 min. Solutions were decanted then plates returned upright in light for 24 h. Total RNA was isolated with TRIzol (Thermo Fisher) followed by lithium chloride precipitation. Total RNA (1.5 µg) was reverse transcribed with Superscript III (Thermo Fisher). PCR reactions were performed on a Bio-Rad CFX96 cycler using SYBR Green master mix (Applied Biosystems) and the following program: 3 m at 95 °C, 45 cycles (15 s at 95 °C and 1 min at 60 °C). Expression was normalized to the reference genes ACT2 and PP2A. Primers used were from Jenness et al.4,7 or ethanol (EtOH) plates and allowed to grow for 2 days. After this, they were fixed for agarose sections and confocal analysis.A short 6 h pulse of 10 µM EdU in liquid ½GM was used, after which the EdU was removed by washing twice for 15 min with liquid ½GM. Washed plants were transferred into 2 µM GAHSdCR plants, plates were placed at 37 °C for 14 or 17 min. They were then immediately cooled at 4 °C for 15 min (ref. 4). The plants were then transferred to 2 µM GA4,7 or EtOH plates for 6 days. For the oestradiol (EST)-inducible lineage-tracing lines, 14-day old plants were incubated in 5 µM EST in liquid ½GM for 2 h (pPEAR1:XVE»CRE) or 30 min (pANT:XVE»CRE), washed three times for 15 min and then transferred to 2 µM GA4,7 or EtOH plates for 6 days. For the HSdCR experiments, we considered for the analysis only the sectors that proliferated. Some of the sectors, especially after GA treatment, ended on xylem vessels, which are dead and thus cannot be observed with GUS staining. Therefore, the length of these sectors towards the xylem is an underestimation of the actual length.Lineage-tracing experiments were performed in 16- or 14-day-old plants. For the 16-day old n in figures or figure legends. The fraction in the corner of some images indicates the frequency of the observation. All statistical analyses were performed using R version 4.1.2 (http://www.r-project.org/).The number of individual plants, cross-sections or clones analysed is displayed as All measurements were taken from distinct samples and the same sample was not measured repeatedly.t-test and other data with a two-tailed nonparametric Wilcoxon test. When comparing multiple means with each other, a two-way ANOVA with Tukey post hoc was performed. In the case of single variable and not normally distributed data, we used Kruskall–Wallis test with Bonferroni adjustment. Categorical data were analysed with a chi-squared test.Before comparing means, the normality of the data was confirmed with the Shapiro–Wilk test. When doing pairwise comparisons, normally distributed data were analysed with a two-tailed In all of the box plots, the centre line represents the median, and the upper and lower box limits indicate the 75th and 25th percentiles, respectively. Whiskers show the maximum and minimum values, and outliers are shown as circles. Filled circles represent individual data points. In violin plots, the white dot shows the median and the thick line the interquartile range. The thinner line represents the rest of the distribution. Each side of the line is a kernel density estimation that shows the distribution shape of the data. Filled circles represent individual data points.Leica LAS x, Leica LAS x lite, Bio-Rad CFX Manager, Fiji 1.53, R 4.1.2, R-studio 2022.07.2, Adobe Illustrator, Adobe Photoshop, Python 3.8.10 and MS Office Professional Plus 2016: Excel, WordFurther information on research design is available in the Supplementary InformationSupplementary Tables 1–4.Reporting SummarySource Data Fig. 1Source data and statistics for Fig. 1.Source Data Fig. 2Source data for Fig. 2.Source Data Fig. 3Source data and statistics for Fig. 3.Source Data Fig. 4Source data and statistics for Fig. 4.Source Data Fig. 5Source data and statistics for Fig. 5.Source Data Extended Data Fig. 1Source data and statistics for Extended Data Fig. 1.Source Data Extended Data Fig. 2Source data and statistics for Extended Data Fig. 2.Source Data Extended Data Fig. 3Source data and statistics for Extended Data Fig. 3.Source Data Extended Data Fig. 6Source data and statistics for Extended Data Fig. 6.Source Data Extended Data Fig. 7Source data and statistics for Extended Data Fig. 7.


Perilla frutescens var crispa f. purprea) is a traditional medicinal herb that exerts anti-inflammatory effects and alleviates lower urinary tract symptoms. In this study, we examined the effects of rosmarinic acid, a major polyphenol in shiso, on urinary function and the bladder in a rat hydrochloric acid-induced cystitis model. Sprague–Dawley rats were administered intravesically with hydrochloric acid or saline solution (control) to induce cystitis. Afterwards, the rats were administered orally with distilled water or rosmarinic acid for three days and then the intravesical pressure was measured, a stretch stimulation test was performed using the harvested bladder, and histological and biochemical analyses were performed. In addition, we investigated the effects of rosmarinic acid on the expression of inflammation-related molecules in normal human bladder epithelial cells. Rosmarinic acid ameliorated hydrochloric acid-induced shortening of micturition interval by 49%. In hydrochloric acid-treated bladders, significantly more prostaglandin E2 was released after stretching; however, rosmarinic acid suppressed its release to control levels. Rosmarinic acid also reduced hydrochloric acid-induced epithelial thickening and the levels of inflammatory molecules in the bladder. Furthermore, rosmarinic acid suppressed interleukin 1β-induced increases in Cox2 and Il6 expression in bladder epithelial cells. These findings indicate that rosmarinic acid can ameliorate hydrochloric acid-induced cystitis in rats and that these effects are due, at least in part, to its anti-inflammatory effects on the bladder and inhibition of stretch-induced prostaglandin E2 release.Shiso ( In part2 (PGE2) , 5, whic2 (PGE2) . In addi2 (PGE2) . Hunners2 (PGE2)  and the 2 (PGE2) , suggestithelium , it may  effects –14. In a effects . Therefo effects , 17.ad libitum and kept at 23 ± 2°C with a humidity of 55 ± 10% and a light-dark cycle of 12 h from 07:00–19:00. All animal experiments were approved by the Animal Care Committee of Kao Corporation  and were conducted according to its guidelines.Female Sprague-Dawley (SD) rats  were used in this study. All animals were provided with a standard diet  and water n = 7) with equal body weight [To create models of cystitis, SD rats were randomly divided into three test groups , threshold pressure (cmH2O), and maximum pressure during voiding (cmH2O) were measured. Threshold pressure was calculated as the pressure in the bladder just before urinary contraction. Afterwards, the rats were euthanized under isoflurane anesthesia by cutting the abdominal vena cava and the bladder was harvested to measure its weight.Three days after catheter implantation, intravesical pressure was measured in conscious rats. Briefly, the indwelling catheter in the bladder was connected to a transducer  and a syringe pump . Each rat was placed in a Ballman cage (W 46.5 mm (adjustable), D 160.0 mm, H 51.0 mm, Yamashitagiken Co. Ltd., Tokushima, Japan, #RTM2008) and urinary output was measured using an electromagnetic scale . Rats were not given food or water during intravesical pressure measurement. The bladder was infused with 0.9% saline using a syringe pump at a rate of 5 mL/h. After 20 min, the data for the 60 min following the first urination were analyzed. Micturition interval (sec), volume per voiding (mL), basal pressure (cmHn = 7) with equal body weights and a catheter was inserted through the urethra. The cystitis model was prepared as described above and the test solution was orally administered once daily for three days  until the day of euthanization. Approximately 1 h after the final oral administration, the rats were euthanized under isoflurane anesthesia by cutting the abdominal vena cava and were subjected to stretch stimulation as described previously [2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11.1 mM D-glucose) and the bladder was weighed. Briefly, a catheter was inserted into the bladder through the urethra and the urethra was ligated. After catheter insertion, the bladder was acclimatized in a 10 mL tissue bath  for 20 min under 95% O2, 5% CO2 conditions in Krebs solution at 37°C. After acclimation, 300 μL of Krebs solution was injected into the bladder through the catheter at a rate of 40 μL/s to measure baseline values. Ten minutes later, the injected solution was collected. After acclimation for 20 min, 600 μL or 900 μL of Krebs solution was injected into the bladder and the injected solution was collected after 10 min. After stretch stimulation, the bladder was cut into sections and were immersed in RNA later solution  or OCT compound . The collected Krebs solution and sections of the bladder were flash frozen in liquid nitrogen and stored at -80°C.SD rats were randomly divided into three experimental groups  and mean values were calculated under blind conditions.MPO activity in the bladder was measured using an MPO activity assay kit  and normalized to the homogenate protein concentration. Absorbance was measured using a Viento XS multispectrophotometer . Homogenate protein concentration was measured using a Pierce BCA Protein Assay Kit .2. Cells were seeded in 12-well plates at a density of 5.0 × 104 cells/well, pretreated with RA (Carbosynth) for 1 h at approximately 70% confluence, and then incubated with 10 ng/mL IL1β  for 4 h. After the supernatant had been collected, the cells were washed with phosphate buffered saline  and RNA was extracted using an RNeasy Mini Kit .Normal human bladder epithelial cells  were cultured in medium containing UroLife BM  using a Urolife Comp kit  at 37°C with 5% COActb (Rn00667869_m1), Cox2 (Rn01483828_m1), Il6 (Rn01410330_m1), ACTB (Hs01060665_g1), COX2 (Hs00153133_m1), IL6 (Hs00174131_m1). Results were normalized to Actb or ACTB.Bladder tissue sections (approximately 5 mm × 5 mm) were homogenized in ice-cold Tissue Protein Extraction Reagent  using a Physcotron NS-310EII . Total RNA was extracted from homogenized bladder samples using an RNeasy Mini Kit (Qiagen). Total RNA (0.1 μg) extracted from bladder homogenates and bladder epithelial cells was reverse transcribed using a High Capacity RNA to cDNA kit . cDNA was subjected to RT-PCR using the following TaqMan probes (Thermo Fisher Scientific) with an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific): 2 in the Krebs solution collected from the bladder stretch stimulation test was measured using a PGE2 Express EIA kit  and normalized using bladder weight. The amount of IL6 in culture medium collected from bladder epithelial cells was measured using a human IL6 Quantikine ELISA kit .Bladder tissue sections (approximately 5 mm x 5 mm) were homogenized in ice-cold T-PER (Thermo Fisher Scientific) using a Physcotron NS-310EII (Microtec). The homogenate protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Absorbance was measured using a Viento XS multispectrophotometer . Bladder IL6 protein levels were measured using a rat IL6 ELISA kit  and normalized to the homogenate protein concentration. The amount of PGEP values < 0.05 were considered significant.Data were presented as the mean ± the standard error of the mean (SEM). All data were analyzed using GraphPad Prism6 . Mean values were compared between groups using Dunnett’s test. p < 0.001 and p = 0.004, respectively). Conversely, the micturition interval was significantly increased by 49% in the HCl + RA group compared to the HCl group (p = 0.017). No significant changes in micturition volume, basal pressure, threshold pressure, or maximal pressure during voiding were observed between the different experimental groups.First, we examined changes in intravesical pressure in the rat model of cystitis . Micturi2 release from the luminal side after the bladder had been stretched by intravesical injection with Krebs solution. After stimulation with 600 μL of Krebs solution, PGE2 release was approximately 10-fold higher in the HCI group than in the control group (p = 0.026) but was suppressed to control levels in the HCl + RA group (p = 0.021). Although intravesical injection with 900 μL Krebs solution resulted in similar trends in PGE2 release, the differences between the three groups were not significant . However, bladder weight increased to 204.4 ± 10.1 mg in the HCl group (p < 0.001) and 169.4 ± 6.5 mg in the HCl + RA group compared to the control group (138.1 ± 4.3 mg). In addition, histological analysis revealed redness and thickening of the epithelial layer in HCl-treated rats compared to control rats. After HCl treatment, the percentage of the epithelial layer to the bladder wall increased from 21% to 34% (p = 0.003); however, epithelial layer redness and thickening were reduced in the HCl + RA group compared to the HCl group and the percentage of the epithelial layer to the bladder wall decreased from 34% to 24% (p = 0.026). Furthermore, we found that MPO activity [p < 0.001) but was decreased to control levels in the HCl + RA group (p < 0.001).The rats subjected to intravesical pressure measurement and the stretch stimulation test displayed no difference in body weight among groups ; however, this increase was significantly suppressed by RA administration . In addition, IL6 levels were higher in bladders from the HCl group than from the control group (p < 0.001), but were suppressed in the HCl + RA group (p = 0.008). In human bladder epithelial cells treated with IL1β, which is abundant in the bladder of rat model of cystitis and is known to induce IL6 and COX2 [COX2 and IL6 expression were significantly increased (p < 0.001); however, pretreatment with RA for 1 h significantly suppressed the IL1β-induced increase in COX2 and IL6 expression . Similarly, the addition of IL1β increased the release of IL6 from bladder epithelial cells into the culture medium and this increase was suppressed by pretreatment with RA .Finally, we examined the effect of RA on the expression of inflammation-related molecules in models of cystitis and COX2 , 13, COX2 release in response to stretch stimulation. Furthermore, we found that oral administration with RA improved HCl-induced shortening of micturition interval without affecting the volume per voiding, basal pressure, threshold pressure, or maximal pressure during voiding, suggesting that RA improves HCl-induced cystitis by acting on the bladder epithelium rather than bladder smooth muscle.RA has been reported to exert anti-inflammatory and anti-oxidant effects; however, its effects against cystitis with bladder inflammatory changes and dysfunction remain unclear. In this study, we showed that RA improved HCl-induced cystitis in rats, possibly by acting against the bladder epithelium to suppress the production of inflammatory mediators and inhibited PGEAlthough the detailed pathogenesis of cystitis including IC remains unclear, it is thought to involve inflammation –8. Rat mDuring the acute phase of inflammation, neutrophils infiltrate tissues and produce various inflammatory mediators, including IL1β and IL6 . In addiIL6 expression and IL6 production by IL1β were significantly inhibited by RA. Together, these results suggest that RA can exert a series of effects against bladder epithelium, including inhibiting IL6 production. Previous studies in models of inflammatory diseases, including colitis, pancreatitis, and asthma, have reported that RA can suppress pro-inflammatory cytokine production and the activation of NF-κB, an upstream transcription factor [IL6 and COX2 expression, both of which have NF-κB consensus sequences in their promoter regions [The bladder epithelium is thought to be the lesion responsible for cystitis since intravesical DMSO injection  and elecn factor . Here, w regions , in blad2, which is synthesized from arachidonic acid by phospholipase A2 and COX, has vasodilatory effects and plays an important role in the inflammatory response [2 levels are increased in patients with IC [2 administration decrease bladder capacity and increase the frequency of urination [2 is released from the bladder in response to stretch stimulation [2 production may be a cause of bladder dysfunction. In the rat model, the amount of PGE2 released from the harvested bladder during stretch stimulation was significantly increased by HCl treatment, whereas RA suppressed PGE2 release. Furthermore, RA suppressed HCl-induced Cox2 expression in the bladder and IL1β-induced COX2 expression in cultured bladder epithelial cells, suggesting that RA inhibits PGE2 synthesis and release, and subsequent mechanosensitive urinary transmission. It has been reported that parecoxib, a COX2 inhibitor, can suppress COX expression and improve cystitis in rats [2 release.PGEresponse . Urinary with IC  and intrrination . In addimulation  and is imulation . These f in rats . This alIn this study, RA was administered three days after HCl treatment; therefore, our findings reflect the effects of RA during the acute phase of cystitis. Consequently, future studies should clarify the efficacy of RA on cystitis in animal models with chronic as well as acute inflammation and the detailed mechanisms involving NF-κB inhibition.Since lower urinary tract obstructions such as benign prostatic hyperplasia are often associated with urinary retention in older adults, it is necessary to improve urinary retention symptoms without reducing the contractility of bladder smooth muscle . ConsideTaken together, the findings of this study demonstrate that RA ameliorates HCl-induced cystitis in rats by exerting anti-inflammatory effects in the bladder. Thus, RA, which can be consumed on a daily basis, could be an effective way to improve lower urinary tract symptoms, including cystitis.S1 TableData represent the mean ± SEM ; HCl, hydrochloric acid; RA, rosmarinic acid.(DOCX)Click here for additional data file.S2 TableData represent the mean ± SEM (n = 7); HCl, hydrochloric acid; MPO, myeloperoxidase; RA, rosmarinic acid.(DOCX)Click here for additional data file.S3 TableActb mRNA levels and expressed relative to the control group. HCl, hydrochloric acid; RA, rosmarinic acid.Data represent the mean ± SEM (n = 3); The amount of each mRNA was normalized to (DOCX)Click here for additional data file.S4 TableData represent the mean ± SEM (n = 7); HCl, hydrochloric acid; RA, rosmarinic acid.(DOCX)Click here for additional data file.S5 TableACTB mRNA levels and expressed relative to the control group. IL1β, interleukin 1β; RA, rosmarinic acid.Data represent the mean ± SEM (n = 3); The amount of each mRNA was normalized to (DOCX)Click here for additional data file.S6 TableData represent the mean ± SEM (n = 7); IL1β, interleukin 1β; RA, rosmarinic acid.(DOCX)Click here for additional data file.


Beer consumption has been identified as a risk factor for osteoarthritis (OA), a rheumatic disease characterised by cartilage degradation, joint inflammation, and eventual joint failure. One of the main isoflavonoids in beer is formononetin (FNT), an estrogenic compound also found in multiple plants and herbs. In this study, we aimed to investigate the effect of FNT on chondrocyte viability, inflammation, and metabolism. Cells were treated with FNT with or without IL-1β for 48 h and during 7 days of differentiation. Cell viability was determined via MTT assay. Nitrite accumulation was determined by Griess reaction. The expression of genes involved in inflammation and metabolism was determined by RT-PCR. The results revealed that a low concentration of FNT had no deleterious effect on cell viability and decreased the expression of inflammation-related genes. However, our results suggest that FNT overexposure negatively impacts on chondrocytes by promoting catabolic responses. Finally, these effects were not mediated by estrogen receptors (ERs) or aryl hydrocarbon receptor (AhR). In conclusion, factors that favour FNT accumulation, such as long exposure times or metabolic disorders, can promote chondrocyte catabolism. These data may partially explain why beer consumption increases the risk of OA. Osteoarthritis (OA) is the most common rheumatic disease and one of the most disabling pathologies worldwide ,2,3. It It is well-known that inflammation plays a key role in OA catabolic process and, therefore, in the progression of the pathology ,4,6,7. SDiet has been proposed as one of the potentially modifiable risk factors in OA’s development. Specifically, beer consumption has been linked to OA progression . HoweverSeveral studies have suggested the impact of isoflavonoids’ consumption on health ,22,23,24Because of its structural similarity to mammalian estrogen and its ability to bind to estrogen receptors (ERs), such as ER alpha (ERα) and beta (ERβ), it has been suggested that FNT could exert its actions through binding to these receptors ,35,36,37For this reason, in this in vitro study, we focus on the role of FNT in inflammatory and catabolic chondrocyte responses. Moreover, we tried to elucidate whether FNT actions in chondrocytes are due to its capacity to modulate estrogenic pathways. The results obtained in this study showed that FNT does not alter chondrocyte viability and has moderate anti-inflammatory activity at the lowest concentration tested. However, FNT promotes chondrocyte catabolic responses by itself and in combination with the inflammatory stimuli in an estrogen-independent way.ATDC5 murine chondrogenic cell line was obtained from RIKEN Cell Bank . Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with Ham’s F-12, Fetal Bovine Serum (FBS), antibiotics Penicillin-Streptomycin, Transferrin, Sodium Selenite, L-Glutamine, Trypsin, TRI Reagent, FNT, 3′methoxy-4′nitroflavone (3-MF), mouse IL-1β, MTT reactive, chloric acid, SDS, and Griess reactives  were purchased from Sigma Aldrich . ZK164015 was acquired from Cienytech . Insulin (Actrapid 100 UI/ML) was kindly donated by the Pharmacy Service of the Clinical Hospital of Santiago de Compostela. E.Z.N.A. Total RNA Kit I was bought from Omega BIO-TEK . Digestion kit was purchased from Lucigen . High-capacity cDNA Reverse Transcription Kit was bought from Applied Biosystems, Life Technologies . MasterMix PCR was purchased from ThermoFisher Scientific . IL-6, neutrophil gelatinase-associated lipocalin (LCN2), C-C motif chemokine 2 (CCL2), COL2A1, ACAN, type X collagen (COLX), SRY-Box transcription factor 9 (SOX9), and MMP13 primers were purchased from Sigma Aldrich. Cell culture plates were purchased from ThermoFisher Scientific.4 cells/well) in DMEM/Ham’s F-12 medium supplemented with 5% FBS, penicilin-streptomicyn (2%), L-glutamine (2%), transferrin (10 μg/mL), and sodium selenite (0.03 nM) . Once cells adhered to the plates, they were serum-deprived. Then, 24 h after seeding, an FBS-supplemented medium was added, and cells were treated with 12.5 or 25 μM FNT in the presence or absence of IL-1β. Then, 48 h after FNT treatment, cells were lysed, RNA was obtained, and RT-PCR was performed.ATDC5 cells were seeded on six-well plates (25 × 105 cells/plate for the day 0 plate and 3 × 104 cells/plate for day 7 plate). Once they adhered to the plate, the medium was changed for differentiation medium, which consisted of basal medium with insulin (10 μg/mL), to promote cell differentiation [Cell differentiation was performed for 7 days. Cells were seeded in 100 mm culture plates ; FNT 5, 12.5, and 25 μM; ZK164015 (ZK) 0.1 μM; and 3′methoxy-4′nitroflavone (3-MF) 10 μM. IL-1β was diluted in water, whereas FNT, ZK, and 3-MF stocks were dissolved in DMSO, following the manufacturer’s instructions.3 cells were seeded on 96-well plates and treated with FNT 5, 12.5, and 25 μM in presence or absence of IL-1β (0.1 ng/mL). After 48 h of treatment, cells were incubated with MTT 5 mg/mL over 4 h at 37 °C. Then, 100 μL of solubilizing agent was added per well and cells were incubated overnight. After formazan salt was dissolved, absorbance was measured at 550 nm with one NanoDrop One® spectrophotometer (Thermo Fisher Scientific).Cell viability was examined using a colorimetric MTT assay, as previously described . Briefly4 cells/well). After cell adhesion to the plate, they were serum-deprived and treated with FNT 25 μM in the presence or absence of IL-1β, as aforementioned. After 24–48 h of treatment, nitrite accumulation in the culture medium was determined using the Griess reaction, as previously described [ATCD5 cells were seeded in six-well plates  in combination with specific primers in a QuantStudio 3 (ThermoFisher) . RelativERα, ERβ, and AhR structures were obtained from the RCSB Protein Data Bank (PDB)  and optit-test analysis using the Prism computerized package . A difference was considered significant when p < 0.05.Data were reported as mean ± standard error of the mean (SEM) for at least three independent experiments. Statistical analysis was performed by analysis of variance followed by the Tukey’s multiple comparisons test or Beer has been associated with anabolic actions in bone tissue ,11,12, aConsidering the key role of a pro-inflammatory environment on OA progression ,6,7, we As shown in OA is primarily characterised by progressive cartilage degradation, where chondrocyte’s anabolic responses are inhibited and catabolic ones are promoted ,51,52,53With this aim, ATDC5 cells were cultured over 48 h in the presence or absence of FNT and/or IL-1β. To test the anabolic/catabolic effects of FNT, we measured the expression of some relevant genes that play a key role in cartilage maintenance: COL2A1 and ACAN, both indispensable for ECM integrity ,55; SOX9The obtained results showed that 48 h of stimulation with 25 μM FNT discretely reduced the expression of COL2A1 and ACAN on ATDC5 cells in a significant way. Treatment with 12.5 μM FNT also reduced the expression of COL2A but not ACAN A,B. In aConsidering the effects of FNT on chondrocytes’ anabolism and catabolism and the possibility of infant exposure to this isoflavonoid through breast milk or infant formulas ,31,33,34To evaluate this, we carried out the differentiation of ATDC5 cells over 7 days in the presence or absence of 25 μM FNT (the concentration with the strongest catabolic effect) and/or IL-1β, and measured the expression of differentiation and catabolic markers by RT-PCR. Our results indicated that FNT significantly diminished ACAN A but notIt has been described that FNT could interact with estrogenic receptors ERα and ERβ ,15,35,36As an aim of the molecular docking results, we carried out the differentiation of ATDC5 cells over 7 days in the presence or absence of 25 μM FNT; 0.1 μM ZK, an ERα and ERβ receptor antagonist; and 10 μM 3-MF, an AhR receptor antagonist. The obtained data showed that the effect of FNT was not significantly modified by co-treatment with either ZK A–D or 3-In this study, we investigated the effect of FNT, one of the main isoflavonoids from beer, on chondrocyte inflammation and catabolism. We found that FNT does not affect chondrocyte viability and lowers the expression of inflammation-related genes at a low concentration (12.5 µM). However, at a high concentration (25 µM), it promotes chondrocyte catabolism independently of estrogenic and aryl hydrocarbon receptors. We aimed to investigate the effect of FNT in this context because beer consumption has been identified as a risk factor for OA , but theOur study shows that FNT has no effect on cell viability either alone or combined with IL-1β at the range of concentrations (5–25 μM) and time (48 h) tested. Other authors also found that 24 h FNT treatment did not affect chondrocyte viability, even at concentrations as high as 100 μM ,28. HoweSome isoflavonoids are known to have anti-inflammatory activity . In our Interestingly, it has been described that isoflavonoids may stimulate cell growth at low concentrations, but inhibit proliferation at higher concentrations ,35. FNT As a further matter, it has been suggested that FNT can exert its actions through binding to ERs ,15,35,36It is known that AhR activation can impact chondrocyte differentiation. In addition, AhR expression positively correlates with chondrocyte maturation during endochondral ossification . DespiteIn conclusion, even though FNT can be beneficial for cartilage homeostasis, exposure to FNT at certain concentrations or physiological/pathological status may promote chondrocyte catabolism. Factors that favour FNT accumulation, such as long times of exposure  or metabolic disorders, can lead to this catabolic effect, which is independent of both ERs and AhR. These data may provide a partial explanation for why high beer consumption increases the risk of OA .


The nanomotor achieves 82% of EPS biomass disruption anda 96% reduction in cell viability, which contrasts with a remarkablylower reduction in biofilm elimination when the components of thenanomotors are used separately at the same concentrations. Such alarge reduction in biofilm biomass in S. aureus has never been achieved previously by any conventional therapy.The strategy proposed suggests that engineered nanomotors have greatpotential for the elimination of biofilms.Development of bioinspirednanomotors showing effective propulsionand cargo delivery capabilities has attracted much attention in thelast few years due to their potential use in biomedical applications.However, implementation of this technology in realistic settings isstill a barely explored field. Herein, we report the design and applicationof a multifunctional gated Janus platinum–mesoporous silicananomotor constituted of a propelling element (platinum nanodendrites)and a drug-loaded nanocontainer (mesoporous silica nanoparticle) cappedwith ficin enzyme modified with β-cyclodextrins (β-CD).The engineered nanomotor is designed to effectively disrupt bacterialbiofilms via H Related to thisis the misuse and overuse of antimicrobials, which are the main driversin the development of drug-resistant pathogens. In fact, antimicrobialresistance (AMR) is a global health and development threat, the WorldHealth Organization (WHO) having declared that AMR is one of the top10 global public health threats facing humanity.5 In this context, the design of new approaches for the treatmentof infections caused by bacteria is of vital importance.Bacterialinfectious diseases have become a global public healthproblem in recent years, causing elevated morbidity and mortalityrates, particularly in immunocompromised patients.8 or metals with intrinsic catalytic activity, among which platinum9 (Pt decomposes hydrogen peroxide in water andoxygen) and zinc10 (Zn autooxidation generateshydrogen bubbles) are the most widely used. Regarding the shape, chemicalengines are usually based on nanowires,11 microtubes,12 and Janus-type particles.13 Interestingly, the mechanical force generatedby the self-propulsion of nano/micromotors can improve penetrabilitythrough different barriers. In addition, in relation to biotechnologicalapplications, nano/micromotors with the ability to release therapeuticpayloads on command are highly desired. Among materials used in cargodelivery, mesoporous silica nanoparticles (MSNs) can be highlighteddue to their biocompatibility, biodegradability, and the possibilityto functionalize the external surface with gatekeepers, which allowcargo delivery on command in the presence of selected stimuli.14 Although several publications describe the useof antimicrobial-loaded MSNs, these systems did not present self-motionand are mostly focused on the elimination of pathogens in a planktonicstate yet not embedded into biofilms.15 In this scenario, both characteristics, motion and cargo deliveryat-will, could improve the penetration of the antimicrobial therapeuticagent into difficult-to-access areas, making them a powerful toolfor active biofilm removal. To date, a few examples describe the useof microengines that are usually tubular in shape and chemically driven,19 employing high concentrations of H2O2 (upto 3%) for an effective EPS disruption. In addition, at the nanoscale,Vilela et al. designed drug-free urease-propelled MSN nanomotors forthe treatment of Escherichia coli (E. coli) in urinary tract infections inducing a 60%of biofilm biomass reduction.8 In addition,Ji et al.20 and Cui and co-workers21 designed nanomotors propelled by near-infrared(NIR) light, resulting in a photothermal penetration into the EPSand drug release. However, the described examples still show somedrawbacks such as the addition of high levels of toxic chemical fuel,poor mobility, and penetration that does not cause significant physicaldamage to the EPS or the use of external energy sources  not suitable for treating deep tissue infections.In addition, light-induced photothermal effects may cause collateraldamage to adjacent healthy tissues and cells due to the high temperature(≥45 °C) required to achieve complete ablation of pathogens.22Fromanother point of view, one ambitious objective of nanotechnologyis the development of nanodevices able to mimic intrinsic functionsof living systems, among which is an autonomous movement. In relationto this topic, synthetic nano/micromotors powered by chemical reactionsor external physical forces have attracted much attention recently.In particular, movements in chemically powered nano/micromotors arerelated to the transformation of chemical substances into mechanicalenergy. These motors usually contain highly active enzymesStaphylococcus aureus, whichis one of the main pathogens responsible for chronic biofilm-associatedinfections in tissues such as the skin, the respiratory tract, andin medical devices.23 Due to the difficultyin preventing the formation and/or eradicating S. aureus biofilms, different approaches have been studied, such as the combinationof conventional antibiotics,24 passivenanoparticles,15 or their association withDNAases25 or proteases26 However, these therapies usually fail to effectively penetratethe intricate EPS of biofilms . PtNDs are responsiblefor self-propelled motion, acting as catalysts for the local decompositionof H2O2 (l) into H2O (l) and O2 (g). The vancomycin-loaded ficin-functionalized nanomotoris expected to achieve a deep penetration and disruption of EPS, bythe synergistic effect of self-motion and the protease activity officin, and effective bacterial killing related to the specific antibioticdelivery inside the acidic pH of the biofilm.The nanomotor we havedesigned for biofilm eradication 1, bottom29 Due to their well-known characteristics, MSNs were selected as cargonanocontainers, while PtNDs were chosen as the motion system on accountof their high surface roughness and catalytic activity toward H2O2 reduction. MSNs were synthesized by alkalinehydrolysis and condensation of tetraethyl orthosilicate (TEOS) employingcetyltrimethylammonium bromide (CTAB) as the structure-directing agent.Concurrently, PtNDs were synthesized by the autocatalytic chemicalreduction of H2PtCl6 with ascorbic acid in thepresence of poly(vinylpyrrolidone) (PVP).29 A toposelective synthesis using a Pickering emulsion formed by paraffinwax (oily phase) and water–ethanol (aqueous phase) was usedto link both nanoparticles. Briefly, MSNs were partially embeddedat the interface of the paraffin/water emulsion allowing the unmaskedMSN surface decoration with reactive thiol groups, by reaction with(3-mercaptopropyl) trimethoxysilane. Then, PtNDs were subsequentlychemisorbed by thiol bonds. By removing the paraffin with chloroform,the starting Janus nanoparticle NMs were obtained.NanomotorsJanus Pt–MSN (NMs) were made following a synthetic method inwhich two types of independent nanoparticles, PtNDs and MSNs, wereconjugated in a single anisotropic nanodevice .28,29 DuVF) the MSN facewas functionalized with iodopropyl trimethoxysilane to which benzimidazolegroups were attached and the mesopores were loaded with the antibioticvancomycin. Finally, cyclodextrin-modified ficin (F-βCD) wasemployed as the capping ensemble by the formation of inclusion complexeswith the benzimidazole moieties that decorated the MSN surface. Similarnanodevices to NMVF were prepared yet deprived of someof the components, i.e., lacking platinum (NMMSN), vancomycin(NMF), or ficin (NMV) or lacking the Pt motor(MSNVF). In addition, a complete nanomotor loaded withrhodamine B-functionalized vancomycin was also prepared (NMV-RhF). Studies were also carried out with a system containing in solutionall of the unassembled components of the nanomotors (NA). A pictureof the nanomaterials used in our study is depicted in Table S1.To synthesize the final nanomotor . Individual nanoparticles presenteda spherical morphology with an average diameter of 102 ± 13 nmfor MSNs and 31 ± 9 nm for PtNDs. In addition, PtNDs are constitutedby the union of Pt seeds 1b. FigurFigure S2)at high angles show the characteristic Bragg peaks of Pt indexed as(111), (200), and (220) planes, confirming the HR-TEM results. Inaddition, the spectrum at low angles reveals a diffraction peak at2.4°, relative to mesoporous structures of type MCM-41 and indexedas a plane (100). This characteristic peak is preserved from NMs toNMVF confirming that surface modification and pore loadingdo not affect the mesoporous arrangement scaffold. Moreover, N2 adsorption–desorption isotherm studies on NMs showedan adsorption step at intermediate values (0.1–0.3 P/P0) attributed to the nitrogen condensation inside the mesoporousordering. Applying the Barret–Joyner–Halenda model (BJH),33 NMs’ pore diameter and pore volume arefound to be 2.2 nm and 0.3 cm3 g–1, respectively.The specific surface area of the NMs was calculated by the Brunauer–Emmett–Teller(BET)34 model to be 551 m2 g–1. For NMVF the N2 volume adsorbedwas dramatically reduced as expected due to the loading with vancomycinand gating functionalization, and a decrease in the pore volume (tonearly zero) and surface area to 5.84 m2 g–1 was observed .The PXRD spectra of NMsascribed to the bond stretching vibrations of Si–O–Sibonds.35 The NMVF spectrum showsthe bond stretching of CH2 groups at 2945 cm–1, as well as a broad band at 1060 cm–1 correspondingto the cyclic oligosaccharide β-CD. Moreover, the presence officin was confirmed by the amide I absorption band at 1651 cm–1 and quantified by the bicinchoninic acid assay (BCA)in 18.6 μg per mg of NMVF. The amount of β-CDattached to ficin was calculated to be 4 wt % by the phenol–sulfuricacid method. Additionally, the F-βCD content was confirmed bythe thermogravimetric analysis and the loaded vancomycin amountedto 22.4 μg per mg of NMVF. Contents of the cappingunit and cargo were also completed for control nanodevices . In addition, the protein degradation capacity of ficin was evaluatedby running a protease activity assay against casein,36 with a result of 1.02 × 10–5 U mg–1 of NMVF.The construction of NM by FTIR 1e. The nVF, whereas ζ potentialschanged from −24.5 and −28.7 mV in MSN and NMs, respectively,to −7.5 mV in NMVF due to the presence of the gatekeeperF-βCD, which has an isoelectric point of 9 and therefore ispositively charged at neutral pH.37 Bothresults indicated the success of gatekeeper surface functionalizationprocesses. In addition, DLS studies were also completed for controlnanodevices .To complete the characterization,the nanomotors were studied bydynamic light scattering analysis (DLS) 1f,g. TheS. aureus biofilm (ca. pH 5).38 For these studies,nanomotors loaded with vancomycin modified with rhodamine B (V–Rh),i.e., NMV-RhF, were used . Nanodevices were dispersed in sodium phosphate buffer(PBS) or in acidic acetate buffer and the fluorescence signal fromdelivered V–Rh was monitored at scheduled times. At neutralpH, there is no significant cargo release to the medium, whereas,at acidic pH, maximum cargo delivery occurs in only 30 min (Ka = 5.55)39 that results in a disassembly of the benzimidazoleand F-βCD inclusion complex. The benzimidazole and β-CDcomplex formation constant is reduced from 104 ± 8 M–1 (KβCD-Bnz) for neutralbenzimidazole to 42 ± 12 M–1 (KβCD-Bnz+) for the protonated benzimidazoleform.40After the characterizationof the nanodevices, we tested the nanomotor ability to deliver vancomycinin acidic conditions mimicking the ere used S6. Nanod2O2. The reactionrate of ABTS oxidation by PtNDs in NMVF at different H2O2 concentrations displayed a typical Michaelis–Mentenbehavior , demonstrating thatthe nanomotor exhibits a peroxidase-like activity, with an apparentaffinity value .41Previous to characterizing the nanomotormotion, the catalytic activity of PtNDs in NMVF was demonstrated followinga spectrophotometric assay based on the oxidation of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) in the presence of Halue KM, 1 for H2OVF basedon the catalytic decomposition of H2O2 (2O2 (concentrations from 0 to 0.2%)in PBS. For each sample, 30 s videos were registered and analyzed.The x–y coordinates of 15nanoparticles (sizes between 100 and 400 nm) were used to obtain NMVF mean-square displacement (MSD) using VF were plotted vstime intervals (Δt) in each fuel concentrationto evaluate motion behavior, through an in-house developed R code.Results, summarized in t for all H2O2 concentrations tested.Applying D) were obtained(D0 for nonfueled nanomotors and theeffective diffusion coefficient (Deff)for H2O2-fueled nanomotors. Nonfueled nanomotorsdisplay a D0 of 1.1 ± 0.4 μm2 s–1, which is close to the theoreticalvalue (1.1 μm2 s–1), predictedby the Stokes–Einstein equation . Thesefindings indicate that NMVF acts as an autonomous catalyticnanomotor with remarkable diffusion coefficients in the presence ofrelatively low H2O2 concentrations. Enhancedeffective motion is attributed to the high density of active sitesand surface roughness of PtNDs, as well as to the asymmetry of thenanodevice NMVF that presents two very differentiated faces,i.e., PtNDs and MSNs. On the other hand, the propulsion mechanismfollowed by the NMVF is based on the periodic formation,growth, and collapse of O2 bubbles.45 In fact, O2 bubbleswere observed by the naked eye after only 30 s of fuel addition  (Video S1), noting at this point that due to theresolution of the microscope, aggregations of nanomotors are shown.The self-propelled motion of the nanomotor NM of H2O2 2a was inobtained2d expresequation 3 for nanarticles 2e and S8addition 2f. Furthled NMVF 2g. This VF contain active ficin protease and are capable of (i) vancomycindelivery triggered by acidic pH conditions simulating an S. aureus biofilm interior environment and (ii) displayingautonomous motion at low H2O2 concentrations.Accordingly, NMVF complies with ideal features, for beinga self-propelled antibiotic vehicle for the treatment of biofilms.Overall, these studies confirmed that our developed NMVF fueledby H2O2 on S. aureus biofilms was studied. Previously it was confirmed that althoughhigh fuel concentrations are slightly harmful to S.aureus bacteria, biofilm growth and viability arenot affected at low H2O2 concentrations . Based on these studies, a 0.15% H2O2 concentration was selected for antibiofilm assays.Biofilms were cultivated for 6 h and then either treated with 1 mgmL–1 NMVF in the absence or presenceof 0.15% H2O2 or with the combination of freevancomycin (23 μg mL–1) and ficin (25 μgmL–1) (VF),  values, which correlatewith the total biofilm mass.46 Administrationof nonfueled NMVF only caused a 34% decrease in the totalbiofilm mass at 24 h of biofilm growth, a value similar to that obtainedwith the VF treatment. On the contrary, treatment with H2O2-fueled NMVF caused an 82% biofilm disruptionin only 1 h, and up to 72% at 24 h, compared to the untreated control. Biofilm growthstudies were also performed for free vancomycin (V) and free ficin(F) with nonrelevant results compared to treatment with fueled NMVF .Encouragedby previous results, the effect of nanomotor NMtrations S9. Basedtrations S9. Basedtrations S9. BasedVF nanomotor, the next step was the evaluationof the antimicrobialeffect resulting from the controlled release of vancomycin. For thispurpose, the number of bacteria was determined by viable colony countsin both the remaining biofilm and the supernatant. VF treatment showeda low capacity (<1 log decrease) to reduce cell viability in thesupernatant, which was even weaker in bacteria embedded in the biofilm. On the other hand, the totalcell viability was reduced in both the biofilm and supernatant byalmost 2 orders of magnitude when biofilms were treated with NMVF in the absence of fuel .In contrast, NMVF activation with 0.15% H2O2 led to efficient killing of both biofilm-embedded and free-floatingbacterial cells, reaching over 3-logs reduction in viable cells comparedto the untreated control , . This suggests that, although the movement of the nanomotorcoupled with its functionalization with ficin presents a strong abilityto detach the biofilms (as shown by CI impedance results above), NMF was not able to kill the bacteria embedded in the biofilm.Similar results were observed for NMV, showing that inthe absence of ficin nanomotors fail to disrupt the biofilm. As aconsequence, NMV did not reach the acidic area and didnot release the antibiotic, so the bacterial cell viability remainssimilar to the control. Finally, we also tested the effect of freeV and F , the control MSNVF and the unassembled nanodevice, NA, on cell viability . Results showed no relevant antimicrobialproperties both in the presence or the absence of fuel. Taken together,these studies demonstrate that NMVF is adequate for biofilmdisruption and bacterial elimination, whereas, the lack of elementsin the nanodevice results in a lower performance.After confirming the efficient EPS disruption bythe fueled NM 0.001), 3d. This valuated3e. NMF iVF on biofilms,the EPS of the preformed 6 h S. aureus biofilm was labeled using an Alexa Fluor 647-dextran conjugate andthen the biofilm was treated with 1 mg mL–1 NMVF or with 1 mg mL–1 NMVF fueledwith 0.15% H2O2. A significant reduction inthe area covered by the EPS matrix (labeled in red) was observed afterthe treatment with H2O2-fueled NMVF, whereas the nonfueled NMVF did not show a significanteffect. The EPS degradation by NMVF in the presence ofH2O2 is clearly observed in the three-dimensional(3D) reconstruction of the biofilm top layer using ImageJ software(VF produced a 66% reductionin the EPS area of the top layer (vide ante).In addition,to visualize the effects of NMsoftware4a. Compaop layer 4b, in agS. aureus biofilms was also quantified by CLSM. Inthis case, preformed S. aureus biofilmswere incubated as above with 1 mg mL–1 NMVF or with 1 mg mL–1 NMVF fueled with0.15% H2O2 for 24 h and then stained with SYTO9green .In addition, the percentage of viable bacterial cells in O9green 4c.Imageol group 4d. RemarS. aureus biofilms by scanning electron microscopy (SEM) after 24 h of treatment.SEM images showed that both H2O2-fueled andnonfueled NMVF reduced biofilm biomass, yet the largerEPS destruction and the lowest levels of bacterial aggregates wereobtained for H2O2-fueled NMVF .To complete the in vitro study, we furthertested the ability ofthe nanomotor to eradicate mature led NMVF 4e. The cVF nanodeviceloaded with vancomycin and capped with ficin is able to destroy thecomplex EPS matrix of preformed and mature S. aureus biofilms and kill bacteria at low fuel concentrations. The combinationof synergistic capabilities  in asingle multifunctional nanodevice improves the results of previouslydeveloped nano/micromotors focused on the same application.In summary, our results highlight thatthe NMBiofilm eradication is a key issue in the successful treatmentof many severe infectious diseases. In addition, elimination of biofilmson surfaces used in the food industry and in medical devices is critical.However, nowadays this goal is not efficiently achieved with commonapproaches. In fact, the only treatment of biofilm-related infectionsin medical devices that cannot be surgically removed requires theadministration of high dosages of antibiotic combinations that maylead to multidrug resistance, failure of different organs, or evendeath. These issues have fueled the research of new concepts for biofilmeradication, a problem of significant importance.2O2. The pores in the MSN face are loaded with theantibiotic vancomycin and capped with inclusion complexes betweenbenzimidazole groups, decorating the MSN surface, and CD-functionalizedficin enzyme. Vancomycin is a common strategy to treat biofilm-relatedinfections caused by S. aureus. However, strains sensitive to thisantibiotic show extreme resistance when the biofilm is already formed,45 resulting in the emergence of vancomycin-resistantstrains.46In an attemptto contribute to this area, we report herein thepreparation of multifunctional Janus Pt–MSN nanomotors as anew approach for the eradication of biofilms. In the nanomotor, MSNsare selected as cargo nanocontainers, while PtNDs are used as themotion system considering the catalytic Pt ability to reduce HVF synergistically combines three strategiesin one single nanoparticle to disrupt biofilm EPS and kill bacteria:(i) self-propelled motion due to the catalytic decomposition of H2O2, (ii) ficin ability to degrade the extracellularpolymeric matrix of the biofilm, and (iii) controlled pH-triggeredvancomycin delivery at the acidic pH of the biofilm. Our engineeredNMVF nanomotor exhibits an autonomous catalytic motionin response to H2O2, displaying a remarkableincrease in the diffusion coefficient from 1.11 ± 0.42 μm2 s–1 for nonfueled nanomotors to 7.22 ±2.45 μm2 s–1 at low concentrationsof fuel (0.2%). In addition, NMVF allows controlled deliveryof vancomycin at acidic pH (inside the biofilm) due to protonationof the benzimidazole groups, which results in disassembly of the benzimidazoleand F-βCD inclusion complex. On the other hand, antimicrobialstudies on the real-time growth of S. aureus biofilms treated with H2O2-activated NMVF demonstrate an 82% disruption of the total biofilm massin only 1 h. As far as we are aware, this drastic reduction in biomasshas never been achieved with any conventional therapy against S. aureus.49 Remarkably, activatedNMVF exhibits highly effective biocidal activity, resultingin 96% reduction in viable bacterial cells embedded in the biofilmas a result of the controlled release of the antibiotic. This behaviorcontrasts with that shown by NMVF in the absence of fuelor when noncomplete nanomotors or free vancomycin or ficin were used.In these cases, biofilm disruption, as well as bacterial killing,is much less effective.The nanomotor NMin vitro results exposed in the currentmanuscript may provide a long-sought solution to the problem of howto approach the treatment of infections caused by bacterial biofilms.Although we are aware that the external addition of the fuel mightlimit some in vivo applications, our nanomotors powered with low concentrationsof H2O2 might find application in certain microenvironments.These would include tissues in which endogenous H2O2 is present or released, such as those that occur in the woundinflammatory response,50 or in vaginalinfections, where the presence of H2O2-producing Lactobacillus, tetraethyl orthosilicate (TEOS), dihydrogen hexachloroplatinate(H2PtCl6), poly(vinylpyrrolidone) (PVP), ascorbicacid, paraffin wax, (3-mercaptopropyl)trimethoxysilane, (3-iodopropyl)trimethoxysilane,benzimidazole (BENZ) triethylamine (TEA), vancomycin (C66H75Cl2N9O24), ficin fromfig tree latex, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), β-cyclodextrin (β-CD),rhodamine B (Rh), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonicacid (ABTS), trifluoroacetic acid (TFA), and casein from bovine milkwere purchased from Sigma Aldrich. Alexa Fluor 680-dextran conjugate,bicinchoninic acid assay (BCA) kit, hydrogen peroxide ,and SYTO9 were supplied by Thermo Fisher. Sodium hydrogen phosphatemonohydrate, disodium hydrogen phosphate heptahydrate, sodium acetate,sodium hydroxide (NaOH), ethanol, toluene, chloroform, dimethyl sulfoxide(DMSO), acetonitrile, phenol, sulfuric acid, and acetic acid wereprovided by Scharlau. Tryptic Soy Agar plates (TSA) and Tryptic SoyBroth (TSB) were used in this study.2 adsorption–desorption isotherms were recordedusing a Micromeritics TriStar II Plus automated analyzer. Nanomotorswere degassed at 90 or 120 °C under vacuum overnight. The specificsurface area was calculated from the adsorption data within the low-pressurerange using the Brunauer–Emmett–Teller (BET) model,while the pore size was determined following the Barrett–Joyner–Halenda(BJH) method. Thermogravimetric analysis (TGA) was performed witha TA Instruments SDTQ600 apparatus in an oxidizing atmosphere  and a heating rate program between393 and 1273 °C at 10 °C min–1 followedby an isothermal heating step at 1273 °C for 30 min. Fouriertransform infrared spectroscopy (FTIR) measurements were performedin a Tensor 27 instrument (Bruker). Dynamic light scattering (DLS),and ζ potential evaluations were carried out using a ZetasizerNano ZS . Nanoparticle tracking experiments were performedusing a Nanosight NS300 . UV–visible measurementswere performed with a JASCO V-650 spectrophotometer. Fluorescencemeasurements were carried out in a JASCO FP-8500 spectrophotometer.Elemental mapping was conducted using scanning transmission electronmicroscopy coupled with electronic energy-dispersive X-ray spectroscopy(STEM-EDX) using a JEM 2100F instrument. Nanomotor transmission electronmicroscopy (TEM) images were achieved using a JEOL TEM-2100F electronmicroscope. Optical density (OD) measurements were performed witha Tecan spectrophotometer . Bacterial cell index experimentswere realized with an xCELLigence real-time analyzer (Agilent). Confocalmicroscopy imaging was obtained with a Leica TCS SP8 AOBS invertedlaser scanning confocal. Scanning electron microscopy images of S. aureus biofilms were performed using a HitachiS-4800 field emission scanning electron microscope .Powder X-ray diffraction(PXRD) analysis was carried out using a D8 Advance Seifert 3000TTdiffractometer using Cu Kα radiation at low angles  and high angles (35⟨2θ⟩ 80 with steps of 0.04° and 1s for astep). N1 g of CTABrwas dissolved in 480 mL of deionized water. Then, 3.5 mL of 2 M NaOHwas added to the mixture, and the temperature was set at 80 °C.Then, 5 mL of TEOS was added dropwise to the solution that was stirredfor 2 h, giving a white precipitate as a result. Next, the solid productwas isolated by centrifugation, washed with deionized water, and driedat 70 °C. Finally, the solid was calcinated for 5 h at 550 °Cunder an oxidant atmosphere, to obtain the final nanoparticles, MSNs.2PtCl6 and 20 mg of PVP were mixed in 20 mL of deionizedwater. Concurrently, 350 mg of ascorbic acid was dissolved in 10 mLof deionized water. This mixture was poured dropwise to the H2PtCl4 solution. Then, the temperature was increasedat 45 °C and the reaction was magnetically stirred for 3 h. Thecolor change, from pale yellow to black reveals the successful synthesisof platinum nanodendrites, PtNDs.54164 mg of H29 180 mg of MSNs was dispersedin 10 mL of a 6.7% ethanol solution in water. 208 μL of CTAB(1 μM) was added and the temperature was set at 75 °C.Then, 1 g of paraffin wax was added. Once the paraffin was melted,an Ultra-Turrax T-8 (IKA) was used for 10 min to homogenize the sample.Later, the reaction was magnetically stirred for 1 h at 75 °Cto create a Pickering emulsion. To the resultant cooled mixture, 200μL of (3-mercaptopropyl) trimethoxysilane, and 10 mL of methanolwere added. Then, the mixture was magnetically shaken for 3 h. Next,the obtained solid was isolated by centrifugation and washed withmethanol twice. Afterward, the one-face mercapto-functionalized MSNsreacted with the previously synthesized PtNDs, under magnetic stirringat room temperature overnight, to yield the final Janus Pt–MSNnanomotors, NMs, the product was filtered, washed with chloroform,and dried.The synthesisof Janus Pt–MSN was performed following a method previouslydescribed by us.BENZ andMSNBENZ were centrifuged, washed with toluene, and driedat 37 °C.The mesoporousface in MSNsand NMs were functionalized with benzimidazole groups to further reactwith β-cyclodextrins attached to ficin creating inclusion complexesthat act as pH-triggered nanovalves. For that, 60 mg MSNs or NMs weresuspended in 4 mL of anhydrous ACN and treated with 60 μL of(3-iodopropyl) trimethoxysilane for 5.5 h. Solids were isolated bycentrifugation and washed with toluene. To prepare a saturated solutionof benzimidazole, 0.25 g was mixed with 330 μL of TEA and 20mL of toluene and heated at 80 °C until complete dissolution.Then, 10 mL of the benzimidazole solution was added over MSNs or NMsand the mixtures were magnetically stirred at 80 °C and refluxedfor 3 days. Finally, the intermediate solids NM2 (synthesized as described previously)55 was added to the mixture, which was magneticallystirred for 12 h to generate amide bonds between the amino groupsof the βCD-NH2 and the activated acid groups of theficin protease. Finally, ficin conjugated with β-cyclodextrins(F-βCD) was dialyzed vs cold 100 mM sodiumphosphate buffer (PBS), pH 7.5, using Amicon Ultra-05 centrifugalfilter units (3 kDa).56 F-βCD wasconserved at 4 °C until use.25 mg of ficin was dissolved in 4 mLof 100 mM sodium phosphatebuffer pH 6 at 4 °C and mixed with 21 mg of EDC and 21 mg ofNHS for 30 min to activate the protein acid groups. Later, 13 mg ofβCD-NHBENZ was mixed with 7.5 mg of vancomycin in 4 mL of 100 mMPBS pH 7.5 and stirred magnetically overnight. Then, the solid wasisolated by centrifugation and resuspended again in PBS. To attachthe molecular gate (F-βCD), vancomycin-loaded NMBENZ was reacted with 12.5 mg of F-βCD at 4 °C overnight.Lastly, NMVF was centrifuged and washed several times with100 mM PBS pH 7.5.NMs nanopores were loaded withthe antibiotic vancomycin. In a typical synthesis, 50 mg of NMV, 15 mg of vancomycin-loadedNMBENZ reacted overnight with 5 mg of β-CD in 3 mLof 100 mM PBS pH 7.5.To synthesizethe control nanomotor without ficin, NMF, 15 mg of NMBENZ reacted with3.75 mg of F-βCD in 3 mL of 100 mM PBS pH 7.5 overnight at 4°C.For the synthesis of the controlnanomotor withoutvancomycin, NMVF, was prepared following the same proceduredescribed forNMVF, employing MSNBENZ instead of NMBENZ.The control nanodevice withoutPtNDs, MSN57 5 mg of rhodamine B  was activatedwith an excess of EDC  and NHS  in 2 mL of 100 mM PBS pH 6 for 30 min. Then, the mixturewas added to 10 mg of vancomycin  and incubatedat 4 °C overnight.For UV–visible and CLM visualization purposes, vancomycinwas modified with the dye rhodamine B (V–Rh).V-RhF was prepared following the same steps employedfor the synthesis of NMVF. However, their pores were loadedwith V-Rh. All functionalized nanodevices were kept at 4 °C untiluse. Table S1 summarizes all of the nanodevicesemployed in the study.Solid NM58 The test is based on theplatinum capability to oxidize ABTS to ABTS* in the presence of H2O2 (H2O2 + ABTS →ABTS* + H2O). ABTS* is a blue-colored product detectableby UV–visible spectrophotometry. Reactant concentrations werethe following: 500 mL of ABTS (9 mM), 500 mL of H2O2 (from 0 to 30 mM), and 40 mL (1 mg mL–1) of NMVF. The variation of the absorbance (λabs = 405 nm) was kinematically recorded for 2 min at 25 °C.As can be observed in Figure S8, NMVF owns an intrinsic peroxidase activity with a typical Michaelis–Mentenbehavior, where the kinetic parameters, Michaelis–Menten constant(KM), and the maximum velocity (Vmax) were estimated from the Lineweaver–Burkplot. Results were obtained by the application of Vmax isthe maximum velocity, KM is the Michaelis–Mentenconstant, and S is the concentration of the substrate.To evaluatethe catalytic properties of NMVF, a peroxidase-like activity testwas performed.VF were dispersed in 100 mM PBS pH 7.5 at a concentration of 0.002mg mL–1, ultrasonicated, and introduced by a 1 mLsyringe in the Nanosight NS300 device chamber at a temperature of 25 °C, in the presence of several concentrationsof the fuel, H2O2 . Fuel solutions were already present in the chamber when nanodeviceswere added, to avoid any drift interference. Next, 5 videos of 30s at a speed of 30 frames s–1 were recorded by asCMOS camera coupled to an optical microscope. The NTA 3.0 softwarewas employed to analyze the x and y coordinates of 15 nanomotors (between100 and 400 nm in size) over time (Δt = 0.4s), to calculate, by applying Deff for nanomotors in the presence of H2O2 and D0 for nanomotorsin the absence of H2O2 were obtained by plottingMSD vs Δt, through the linearcomponent of the Stokes–Einstein equation, For the evaluation of the nanomotors’ motion capability,nanoparticle single tracking analysis (NTA) was applied. NMV-RhF movement in the presenceof S. aureus cells was corroborated.1 mg mL –1 NMV-RhF and 0.15% ofH2O2 were incubated with a preformed 6 h old S. aureus biofilm in an IBIDI μ-Slide I Luermicrofluidic device, and a 10 min time-lapse was recorded by CLSM.N is the number ofaveraged nanoparticles and xi and xt are thevector positions of particles at different times.D0 is thediffusion coefficient of nanomotors without fuel and Deff is the effective diffusion coefficient of nanomotorsin the presence of fuel.–3 Pa·s; kB is the Boltzmann constant; T is the temperature, 25 °C; and Rh is the hydrodynamic radius, 191.35 nm.In addition, NM59 100 mLof phenol (3% in DI water) was added to 100 mL of unmodified ficinor F-βCD (1 mg mL–1) and incubated for 10min at 25 °C. Later, 500 mL of H2SO4 wasadded. Moreover, a β-CD calibration curve was prepared at concentrationsof 350, 250, 150, 75, 30, and 20 μg mL–1,and the same procedure used for the modified enzyme was followed.The formation of a yellow-colored compound was determined by UV–visiblespectrophotometry (λabs = 490 nm). β-CD boundedto ficin was calculated to be 4% by interpolation on the calibrationcurve.A typicalphenol–sulfuric acid method for carbohydrate determinationwith some modifications was employed to calculate the quantity ofβ-CD attached to ficin.VF ficin enzyme content,the BCA method was applied following the procedure suggested by theprovider with some adaptations.60 200 mLof B/A solution (1:50) was mixed with 10 mL of NMVF (1mg mL–1) and incubated for 10 min at 60 °C.Then, samples were centrifuged at 4 °C, and the absorbance at570 nm was measured. The calibration line was prepared with differentconcentrations of ficin (from 7.8 to 1000 mg mL–1) solved in 100 mM sodium phosphate buffer pH 7.5 following the samesteps. Ficin attached to NMVF was calculated to be 18.7μg per mg of the nanomotor.For the determination of the NMVF protease activitywas studied by a casein degradationassay.36 1 g of casein was solved in 9.9mL of 100 mM PBS pH 7.5 at 60 °C for 15 min to ensure completedissolution. Then, 1 mL of 1% casein was mixed with 500 mL of NMVF (1 mg mL–1) and incubated at 40 °C.20 min later 3 mL of 5% trifluoroacetic acid was added to stop thereaction. Samples were centrifuged, followed by filtration  of the supernatant. Finally, the proteinconcentration was measured by the BCA method.60 The same process was repeated for control samples and standards. The activity was calculatedby applying the supplementary VF exhibited 1.02 × 10–5 U mg–1.A is the absorbance(λabs = 570 nm), the slope is the slope of the calibrationline, t is the incubation time with casein, and c is the concentration of the sample.NMV-RhF was suspended in 1 mL of 100 mM PBS pH 7.5 (blank), and 1 mg ofNMV-RhF was suspended in 100 PBS pH 4.5. Mixtureswere stirred in a shaker at 37 °C. At different times , samples were centrifuged  to remove NMV-RhF. Lastly, the fluorescenceemission of the V–Rh released to the supernatant was measured.To study the nanomotors’capability to deliver vancomycin, 1 mg of NMS. aureus strainSa240 (S. aureusssp.aureus Rosenbach 1884). The strain was plated on TSAand TSB and grown overnight at 37 °C with vigorous shaking at120 rpm.Experimentswere performed using 2O2 effect onplanktonic Sa240 growth, 100 μL (OD600 = 0.175) ofthe bacterial suspension was added into corresponding 96-well platesin triplicate for each condition. After that, 100 μL of H2O2 diluted in TSB supplemented with 0.25% of filter-sterilized d-glucose (TSB-glu) was added reaching final H2O2 concentrations of 0.35, 0.30, 0.25, 0.20, 0.15, and 0.1%(v/v). Continuous Sa240 growth was then monitored by means of absorbanceat 610 nm for 24 h at 37 °C.To assess the HS. aureus Sa240 biofilms, xCELLigence Real-Time Analyzer was used in accordancewith the manufacturer’s instructions. The preformed biofilmeradication experiments were performed, as previously described,61 where biofilm growth was expressed as the cellularindex (CI), which corresponds to a total biofilm mass. Briefly, S. aureus 240 was grown overnight in TSB media. Onehundred microliters of TSB-glu were used for background measurements(in triplicate for each condition). After that, 75 μL of theSa240 bacterial suspension diluted in TSB-glu to OD600 =0.153 was added into the corresponding E-plate wells, reaching a finalconcentration of OD600 = 0.0875. This OD corresponds toapproximately 1 × 107 cells. Subsequently, E-plateswere incubated in an xCELLigence system at 37 °C, and the biofilmgrowth was measured for 6 h with CI values registered every 10 min.After 6 h of biofilm growth, the experiment was stopped, and differenttreatments were added into the corresponding wells in E-plates. Followingthe treatments, biofilm growth was observed for an additional 24 h.Appropriate negative controls were included in each experiment intriplicate. CIs values  were obtained by subtractingtheir respective negative controls.To evaluate the effectof different nanoparticles on 6 h preformed S. aureus Sa240 biofilm treatments, the supernatant was collected, and serial10-fold dilutions were prepared. After that, 100 μL of eachdilution was plated on TSA plates. Three biological replicates wereplated for each condition. To assess the viable cell number in bacterialbiofilms, the biofilms were rigorously rinsed using PBS pH 7.4 toeliminate nonadherent cells. After that, the biofilms were collectedusing 200 μL of PBS and sonicated for 5 min to disrupt the biofilmmatrix and release biofilm-embedded S. aureus cells. Biofilms then were serial-diluted and plated in triplicate,as described above. TSA plates were incubated at 37 °C overnight.After that, viable colonies (CFUs) were counted, averaged, and expressedas log10.To assess the number of viableunattached planktonic bacteria after N-acetyl neuramic acid and polysaccharideadhesin, involved in biofilm formation was employed. Briefly, S. aureus 240 biofilms were cultivated, by adding175 μL of the bacterial suspension into the corresponding wellsof IBIDI 80827 μ-Slide 8-well plates and biofilms, and biofilmswere grown at 37 °C for 6 h as described above, in the presenceof 50 μL of the Alexa Fluor 488 fluorescent conjugate (0.13mg mL–1). Later, the supernatant was removed, andthe biofilm was washed with TBS several times. After that 1 mg mL–1 NMVF, 0.15% H2O2 NMVF, and 0.15% H2O2 were addedinto the corresponding wells and Sa240 biofilms were grown for anadditional 24 h.To show how nanoparticlesinteract with the biofilm EPS, the Alexa Fluor 488 fluorescent conjugate,which binds to –1 NMVF,0.15% H2O2 NMVF, and 0.15% H2O2, the SYTO9 fluorescent dye that binds to microbialDNA was employed. Biofilms were grown, as described above. Thereafter,treatments were added into the corresponding wells, and Sa240 biofilmswere grown for an additional 24 h. Subsequently, the supernatant wasdiscarded, and the biofilms were carefully rinsed with PBS to eliminateunattached cells and stained with 200 μL of SYTO9 solution (workingsolution 3 μL of SYTO9 in 2 mL of sterile water) for 20 minin the dark. After biofilm staining, the excess dye was removed bygently washing using water. After that, CLSM image acquisition wascarried out. To acquire the signals, a 679 nm excitation laser and702 nm emission filters were used for the Alexa Fluor 680-dextranconjugate and a 488 nm laser and 505–550 emission filters wereused for SYTO9.To quantify live cells within bacterial biofilmsafter treatment with 1 mg mLS. aureus 240 overnight culture was adjusted to OD600 = 0.0875,and the biofilms were grown on gold electrodes for 24 h. After that,biofilms were treated with NMVF in the presence and absenceof H2O2, VF, and V for 6 h. Then, supernatantswere discarded, and biofilms were gently washed with PBS to eliminatenonadherent cells. Prior to observations, biofilm samples were fixedwith Karnovsky’s fixative for 4 h, rinsed with PBS three times,and dehydrated using a gradual ethanol series (30–50–70%)twice. Then, the samples were dried using critical point drying withCO2. Subsequently, the samples were observed at SEM applyingan accelerating voltage range of 0.5 kV and a magnification rangeof ×2.50 k.Toevaluate the biofilm spatial structure after the treatments, scanningelectron microscopy (SEM) was performed. 62 Statisticaldifferences in the viable cell number were assessed using Student’s t-test. The data presented as the mean ± SDs from triplicatesof three independent experiments for each condition (n = 9). For comparisons between the means in CLSM analysis, ordinaryone-way ANOVA and Dunnett’s multiple comparison tests wereemployed (n = 3). p-values <0.05 were considered significant.In order tostudy the differencesbetween CIs, regression analysis was assessed by a linear model at24 h of biofilm growth, using the lm library in the R StatisticalPackage version 1.0.7.1.


AC010883.5) is a dysregulated long non-coding RNA (lncRNA) that has never been investigated in cervical cancer (CC). Thus, the potential function and molecular mechanism remain unclear. Our study explored the biological function of AC010883.5 to determine the underlying mechanisms in CC and provide potential therapeutic targets for improving the clinical treatment strategy. We used quantitative real-time polymerase chain reaction to measure mitochondrial RNA levels and western blot to measure the protein levels of target genes. Further, we used Cell Counting Kit-8 and 5‐Bromo-2'-deoxyuridine incorporation assays to evaluate cell proliferation in vitro. Cell apoptosis was analyzed by flow cytometry. Cell invasion was analyzed by wound healing and Transwell migration assays was ued to analyze cell migration. Finally, the biological function and mechanism of AC010883.5 in CC growth were evaluated by in vivo xenograft assay. AC010883.5 was enhanced in CC tissues and cell lines, and enhanced AC010883.5 expression accelerated CC cell proliferation, migration, and invasion and induced epithelial–mesenchymal transition in vitro and in vivo. AC010883.5 also activated the mitogen-activated protein kinase (MAPK) signaling pathway by promoting phosphorylation of extracellular signal-regulated kinase 1/2  and MAPK kinase 1/2 . Blocking the MAPK signaling pathway could counteract the pro-proliferative, pro-migrative, and pro-invasive effects of AC010883.5 over-expression. We found that the lncRNA, AC010883.5, is an oncogenic molecule involved in CC tumor progression via dysregulation of the MAPK signaling pathway, implying that AC010883.5 could be a tumor progression and therapeutic response biomarker.Homo sapiens chromosome 2 clone RP11-339H12 (The online version contains supplementary material available at 10.1186/s12885-023-10825-2. Cervical cancer (CC) is the second most common malignancy with the highest mortality in females worldwide, ranking just after breast cancer. Globally, there are almost 527 000 new cancer cases and 265 000 patient deaths per year . DespiteHOTAIR) gene expression is strictly controlled by the human papillomavirus (HPV) E7 protein  × 100 where the area (A) at time zero (0) and the area after incubation time (t) were used to calculate the percent wound healing  = length × width [2] × 0.5. Mice were sacrificed 33 days after implantation, and the specimen was collected for weight measurements and in vitro assays. All experiments involving animals were approved by the Animal Care and Use Committee at Tongji University and followed by Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35,892–2018.Female BALB/c nude mice  from Shanghai Slark Experimental Animal Company were housed in specific pathogen-free conditions. Animals were randomized into two groups (six mice per group). SiHa cells were suspended in phosphate-buffered saline at 2 × 10p < 0.05 was considered statistically significant.All data analysis was performed using GraphPad Prism version 6.0  and SPSS version 22 . The data were expressed as means ± standard deviations. Continuous data were analyzed using Student’s t-test or one-way analysis of variance. AC010883.5 expression was significantly enhanced in the cancer tissues compared with the adjacent tissues  dramatically decreased AC010883.5 expression in SiHa cells  and SiHa control cells ; we evaluated the efficiency by qRT-PCR  is one of the most important indicators of cancer metastasis. Thus, we explored the correlation between lls Fig. I, indicaAC010883.5 cells (transfection with an AC010883.5 overexpression vector) and control C-33A cells (transfection with an empty vector); we evaluated the efficiency by qRT-PCR  and extracellular signal-regulated kinase (ERK) 1/2 (p-ERK1/2), key biomarkers of MAPK signaling pathway activation, by western blot. p-MEK1/2 and p-ERK1/2 significantly increased in AC010883.5 overexpressing cells and decreased in AC010883.5 knockdown cells  and Ki67 expression were significantly down-regulated in the shAC010883.5 mice compared with the control mice  [GAS5) [CCHE1) [PVT1) [NKD1) [p‐GSK3β and β‐catenin expression and suppressing Wnt/β‐catenin pathway activation [AC010883.5 is located at chromosome 2:43.46, is 216626 bp long [AC010883.5 in cells for the first time. Our study showed that AC010883.5 expression was upregulated in CC tissues and cell lines. We examined the expression of AC010883.5 in 4 different cervical cancer cell lines, all of which were elevated compared with cervical epithelial cells (HcerEpic). However, the level of AC010883.5 was higher in SiHa and Caski cells than in HeLa and C33A, which was caused that cell lines derived from different individuals have different genetic backgrounds. In addition, we discovered that AC010883.5 was substantially enriched in the cytoplasm. AC010883.5 depletion suppressed CC cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT).In recent decades, the biological functions of lncRNAs have been widely investigated , 14. AbnHOTAIR , H19 [18) [GAS5) , cervica [CCHE1) , and Pvt) [PVT1) , are cru) [PVT1) –24. HOTA) [NKD1) . Differetivation . Furthertivation . These r bp long , and is  bp long . In thisAC010883.5 inhibited CC cell growth in vitro. The MAPK signaling pathway modulates the cell’s response to proliferation, migration, apoptosis, and stress. Further, ERK1/2 and MEK1/2 expression and hyperactivation play an essential role in the survival and development of tumor cell proliferation, migration, invasion, and EMT [lncRNA-CCHE1 promoted the proliferation, migration, and invasion in non-small cell lung cancer (NSCLC) cell line through the ERK/MAPK pathway [GINS2 also inhibits cell activity by interfering with the MAPK/ERK pathway and induces cell cycle arrest, thus promoting apoptosis of pancreatic cancer cells [SLC25A22 by the MAPK/ERK pathway could promote mitochondrial apoptosis, thus inhibiting the growth and proliferation gallbladder carcinoma cells [AC010883.5 knockdown on the ERK/MAPK pathway. In the present study, we demonstrated that upregulation of AC010883.5 promote the growth and invasion of tumor by activating this signaling cascade. It was observed that overexpression of AC010883.5 significantly increased the expression of phosphorylated ERK1/2. However, no detectable changes in the expression of total ERK1/2 protein were observed. To further verify the role of signaling activation and inactivation in AC010883.5-aberrant expressed CC, the addition of PD98059 was used to pretreat cells. Results showed that inhibition of MAPK signaling suppressed AC010883.5 high expression-induced levels of phosphorylated ERK1/2 and MEK1/2. Thus, we concluded that AC010883.5 promoted cell proliferation, invasion, migration, and epithelial-to-mesenchymal transition in cervical cancer by activating the MAPK signaling pathway.Furthermore, we explored the molecular mechanisms by which knockdown of  and EMT , 30. An  and EMT . It has  and EMT , 33. Lia pathway . Zhang eer cells . Moreovema cells . TherefoThis study had several limitations, including the low number of clinical samples, the lack of a reliable clinical characteristics analysis, and insufficient research on the regulatory mechanism. These limitations might contribute to unilateral results. Future research should include a larger sample size and involve more complex regulation processing to validate our results.AC010883.5 is markedly enhanced in CC tissues and cell lines. Further, it was involved in cell proliferation, invasiveness, and migration and reduced apoptosis, accelerating tumor growth and EMT in vitro and in vivo. We also found that enhanced AC010883.5 expression induced an ERK1/2 and MEK1/2 phosphorylation increase and activated the MAPK signaling pathway to promote tumor progression. The results demonstrate that AC010883.5 is a possible CC oncogene, and therapies targeting AC010883.5 may be of interest.In summary, in this study, we first found that Additional file 1.