Two pilot trials of powdered activated carbon (PAC)/(coagulation)/ceramic microfiltration were conducted to compare continuous 10–12 mg/L PAC inline dosing with 8–10 mg/L dosing to a 2 h-contact tank. Two low turbidity/low natural organic matter  surface waters spiked with 7.2–10.3 µg/L total-pesticides were tested and the dosing options were compared towards operational performance, average removal of pesticides and NOM and costs. Removal differences between the two PAC dosing options depended on pesticides’ amenability to adsorption and NOM characteristics . Waters containing low A254-absorbing NOM and only pesticides amenable to adsorption showed very high removals  and no significant differences between the two PAC dosing options. Waters containing higher A254-absorbing NOM and high loads of pesticides less amenable to adsorption  required higher inline PAC dose. Those or more severe conditions may require PAC doses higher than tested to comply with the Drinking Water Directive limits for pesticides. Cost analysis showed PAC inline dosing is more cost-effective than PAC dosing to the contact tank when identical PAC dose is sufficient or when the doses are low, even if 50% higher for inline dosing, and the plant is small. Agriculture is a major source of pesticides in European surface and groundwaters, important drinking water resources, but there is still limited information available on pesticide contamination and a lack of reliable and comparable data . A recenPowdered activated carbon (PAC) based solutions are amongst the best available technologies for controlling organic microcontaminants in conventional water treatment plants (WTPs) due to PAC high adsorption ability for a wide range of microcontaminants ,8,9,10, 3 for 100,000 m3/d. However, investment costs, that could have a significant share, would be saved if inline PAC dosing could provide a significant microcontaminants’ removal. Although several authors have approached PAC dosing modes, the focus has been on single versus step or continuous PAC dosing, and the advantages/limitations of inline PAC dosing have not yet been sufficiently studied or contradicting results were reported. For instance, our previous work [The existence of a PAC contact tank is seen as a requirement for increasing the contact time and achieve a maximum removal of contaminants with a PAC load as low as possible. Nevertheless, inline PAC dosing is an easier to implement solution that would allow cost savings and a smaller treatment footprint, which might be important for small plants or densely populated areas where space is a limiting factor. Cost functions earlier developed for PAC/(coagulation)/MF , with 10ous work  with pilous work  for remoous work  lab-scalous work ,27,28,29ous work ,28,29, tous work . For lowous work ,27. The ous work ,32, PAC This paper aims at comparing two PAC dosing modes during a pilot PAC/coagulation/ceramic MF study, in terms of operational performance, average removal of pesticides and organic matter, and costs. “PAC dosing modes” (or options) refer to inline versus tank dosing, both continuous modes but providing very different PAC contact time, and not to single versus step or continuous PAC dosing commonly analysed in literature. PAC continuous dosing into a contact tank vs. PAC continuous inline direct addition were compared during two short-term trials conducted with two surface waters of low turbidity/low NOM spiked with a mixture of pesticides of different amenabilities to PAC adsorption. This study is believed relevant and with novelty to the current state of the art since: (i) it approaches a pressurized PAC/MF process with ceramic membranes, less studied for hybrid adsorption/membrane processes; (ii) it is a pilot study with natural waters , while many studies were conducted at lab scale and/or with synthetic waters; (iii) it compares inline versus tank PAC dosing in different water matrix conditions and targeting the removal of pesticides with different amenabilities to PAC adsorption.European Union Member States monitor a considerable number of pesticides and metabolites in drinking water, which are selected at national level and are thus Member State specific. Nine pesticides, selected to be monitored in Portugal by the Portuguese Environment Agency, were studied. They were spiked into two surface waters feeding PAC/coagulation/ceramic MF pilot .According with our previous results with an identical pool of pesticides, though with other PAC in PAC/CFS application , differeConcentrated stock solutions (5 mg/L in deionized water) of the compounds (Sigma-Aldrich) were prepared and stored in the dark at around 20 °C and stirred until the trials. On the day of the trials, the stock solution was diluted in deionized water to 400 µg/L  and the diluted solution was then continuously added to the pilot feed tank with a peristaltic pump and mixed with the intake water.3) low NOM concentration  and low content of aromatic organic matter, the latter inferred by the low values of SUVA, the specific UV absorbance , below 2 L/(mg·m). W2 presents a somehow higher DOC content (+38%) and higher turbidity and A254 content (+167%), the latter parameter indicating a potentially higher pesticide-NOM competition for PAC adsorption, since PAC is known to preferentially adsorb A254-absorbing compounds [Trials were conducted with two surface waters from Alcantarilha WTP (Águas do Algarve S.A.), herein designated as W1 and W2. As shown in ompounds ,34.2 adsorption-desorption isotherms at −196 °C in an automatic apparatus Micromeritics ASAP 2010 , as detailed in Viegas et al. [pzc) of 11.3), presenting an average PAC particle of 15 μm, a surface area of 1126 m2/g and a total pore volume of 0.83 cm3/g. It has a high percentage of mesopores (mesoporous volume of 0.44 cm3/g and microporous volume of 0.39 cm3/g).PAC Norit SA Super (Cabot) was used, and its textural characterization was subcontracted to an external lab and performed by Ns et al. . PAC SA Two PAC options were tested: 10–12 mg/L inline PAC dosing into a loop before the membrane module, providing 1 min hydraulic retention time, or 8–10 mg/L PAC dosing to a stirred tank before the membrane, providing 2 h hydraulic retention time. A PAC slurry with 0.8 g/L was used, prepared with dechlorinated tap water.2O3 dose. Alum was only applied (inline dosing) in the runs with W2, hereafter referred as Alum/MF and PAC/Alum/MF.A widely used metallic coagulant, alum, was used in PAC/coagulant/MF pilot at a 3 mg/L Al2/TiO2) membranes  , with 19 channels each (3.5 mm diameter each), providing a total surface area of 0.75 m2. The membrane was operated in dead-end mode, at constant flux (133 L/(m2·h), in short lmh), with 60-min filtration cycles followed by backwash with 9.3 L permeate/m2 membrane area, at 1.4–1.5 bar (backwash time was varied to ensure this backwash specific volume). Chemically enhanced backwashing (CEB), either with sulphuric acid or sodium hypochlorite, was conducted after the trial end; it used approximately 22 L of permeate and comprised four steps: (1) flushing with water and cleaning agent; (2) 20–30 min. soaking with cleaning agent; (3) backwashing and (4) water permeation discarding the permeate. Based on the results of a long-term demonstration period conducted with similar waters [PAC/(Alum)/MF pilot is fully automated, remote controlled and with inline monitoring of pressure, flow rate, temperature, pH and turbidity. Pilot specific scheme may be found in . The maiTwo trials were conducted in PAC/(Alum)/MF pilot, with W1 or W2 intake water spiked with a mixture of 6 to 9 pesticides (single pesticide concentrations of 0.3–2.3 μg/L). Conditions used in trials 1 and 2 are summarized in In trial 1, W1 was spiked with six pesticides  gathered at 10 min, 20 min, 30 min, 40 min and 50 min of the filtration cycle were collected. To ensure stable conditions, sampling started after 1 or 2 stabilization (1h-filtration) cycles, depending on the systems’ hydraulic residence time . As indicated in −3 mbar. Pesticides’ limit of quantification (LOQ) was 0.06 µg/L. The inter-day assay precision and accuracy were determined by analysing two different quality control (QC) samples over the years, LOQ (0.06 µg/L) and high QC (0.50 µg/L). The accuracy data were accepted if the accuracy values were within ± 20% deviation (80–120%) from the nominal concentrations, whereas the precision was measured as percentage standard deviation (%RSD) within ± 20%. Accuracy of 98–107% (at 0.06 µg/L pesticides) and 91–112% (at 0.5 µg/L) was verified for the targeted pesticides, while precision was 13–20% (at 0.06 µg/L) and 3–10% (at 0.5 µg/L). Other regular water quality parameters were analysed in Águas do Algarve accredited laboratory using standard methods for the examination of water and wastewater—SMEWW [Samples were analysed for pesticides by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS-MS), subcontracted to an external laboratory certified for these parameters . Pesticides were quantified on a triple quadrupole mass spectrometer (Waters ACQUITY TQD) combined with a Waters ACQUITY UPLC system  operating in multiple reaction monitoring (MRM) mode. A 20 µL sample was directly injected. The chromatographic separation was performed with an Acquity BEH C18  column Waters Milford  set to 40 °C. The mobile phase components were (A) ultra-pure water + 0.1% formic acid + 0.05% ammonium and (B) acetonitrile + 0.1% formic acid. 8-min runs were conducted using 0.5 mL/min and the following elution gradient: 0 min, 95% A and 5% B; 5 min, 0% A and 100% B; 6 min, 95% A and 5% B; 8 min, 95% A and 5% B. The electrospray ionization (ESI) probe was operated in both negative (ESI-) and positive (ESI+) polarity modes. Instrument parameters were: capillary voltage at 0.5 kV; extractor voltage at 3.0 V; source temperature at 150 °C; desolvation temperature at 500 °C; the desolvation gas flow (nitrogen) at 1000 L/h and the cone gas flow at 50 L/h. Argon, used as the collision gas, was run at a collision gas pressure set at 3.5 × 10er—SMEWW . Turbidip-values) of differences in inlet and outlet concentrations of pesticides and in NOM and pesticides’ removal efficiencies in the three different configurations tested (MF vs. inline PAC/MF vs. tank PAC/MF or Alum/MF vs. inline PAC/Alum/MF vs. tank PAC/Alum/MF) was assessed through statistical tests using the Past 4.01 program. Briefly, one-way ANOVA (homogeneous variance with Levene’s test) or Welch F test  were conducted for normal distributions, and Kruskal Wallis test was used for not normal distributions . Significance levels of 0.1 were applied instead of the usual 0.05 due to the low sample size [The statistical significance  at 20 °C is summarized in 3/ceramic MF pilot for treating a secondary effluent [Results showed similar transmembrane pressure and membrane permeability for MF, inline PAC/MF and tank PAC/MF with W1 and for Alum/MF, inline PAC/Alum/MF and tank PAC/Alum/MF with W2 , indicateffluent . Moreoveeffluent  showed teffluent . The preeffluent  and takieffluent ,39, yet effluent . The useeffluent ,41,42.Concentrations of pesticides and NOM content  in the intake, and concentrations in permeate and removals after MF and PAC/MF filtration cycles are shown in Our previous work studying PAC adsorption of pharmaceutical compounds and a similar pool of pesticides in the same concentration range, though with a different PAC and conventional application , showed Microfiltration, either alone or combined with coagulation, did not result in effective removal of pesticides MF in a. These p-values ≥ 0.4) and outlet pesticides’ concentrations  between the two PAC dosing options. All pesticides were very near or below LOQ with both tank PAC/MF and inline PAC/MF  was observed with both tank PAC/MF and inline PAC/MF, reaching 15–24% for TOC, 18–26% for DOC and 29–35% for A254 .When 10 mg/L PAC was dosed to a 2h-contact tank or inline a major increase in pesticides’ removal from W1 was observed, without significant differences in pesticides’ removals  positively and directly, by adsorption of pesticides or pesticides-NOM complexes onto coagulation flocs, which may be retained by the MF membrane; (ii) positively and indirectly, by reducing NOM competition for PAC adsorption; (iii) negatively, by interfering with PAC adsorption.On one hand, pesticides’ removal by coagulation depends on their adsorption onto colloids or NOM, a phenomenon apparently relevant only for very highly hydrophobic compounds (Log Kow > 6) and waters with high turbidity/high molar mass NOM ,43,44, wOn the other hand, coagulant may also negatively affect the removal of pesticides by interfering with PAC adsorption ,50), a mp-values of 0.4–0.6, i.e., >0.1) nor in pesticides’ outlet concentrations  regardless of the 50% higher PAC dosed inline.In trial 2, a major increase in pesticides’ removal from W2 was also observed when 8 mg/L PAC was dosed to a 2-h contact tank, attaining ≥90% for all pesticides except dimethoate (81–82%) and bentazone (53–59%) , namely 47–50% (tank) vs. 45–47% (inline) for TOC, 41–48% (tank) vs. 41% (inline) for DOC and 50–51% (tank) vs. 53–54% (inline) for A254. Moreover, PAC addition, for both PAC dosing options tested, significantly  enhanced the TOC, DOC and A254 removals also verified with Alum/MF . Studies have shown coagulation and PAC adsorption to complement each other in DOC removal, the former removing mostly high molar mass and the latter lower molar mass NOM [As depicted in mass NOM ,33.Overall, and under the conditions tested, equivalent removals of pesticides and NOM were obtained with inline PAC dosing and to a contact tank in trial 1 using the same PAC dose, while in trial 2 a higher inline PAC dose was apparently required . Results from trial 1 are in agreement with those observed by Ivancev-Tumbas et al.  for p-ni−1/mg PAC in trial 2 (W2) vs. 0.03–0.04 m−1/mg PAC in trial 1 (W1)), suggesting higher pesticide-NOM competition effects.In the European Union, the pesticides’ levels in water intended for human consumption are addressed by the Drinking Water Directive  and theSumming up, very high removals of total-pesticides were verified in our study with PAC/(Alum)/ceramic MF , namely Atrazine removals in our study (81–90%) were far higher than those reported by Humbert et al.  with 20–Given the above results and considerations, a cost analysis was performed for three scenarios , built bA cost analysis of PAC dosing was conducted for the full-scale operation of PAC/(Alum)/MF. The (Alum)/MF operational conditions considered were previously optimized during the pilot operational demonstration and are shown in –Plant lifespan (PLS): 40 years –PAC slurry concentration: 30 g/L–PAC preparation residence time: 10 h–−1PAC preparation mixing gradient: 120 s–PAC contact time in the contact tank: 2 hTechnical assumptions–Finance rate (i): 1.87% Financial assumptionThe following assumptions were made for developing the cost functions:The cost elements  considered for the PAC dosing are described in Scenario 1 considered an intake water similar to W1, with low A254-absorbing NOM content, and all pesticides amenable to adsorption, i.e., conditions analogous to trial 1. Equivalent removals of pesticides and NOM were obtained with inline PAC dosing and to a contact tank in trial 1 using the same PAC dose (10 mg/L); therefore, in this scenario, identical PAC dose was considered for both PAC dosing modes. As 8 mg/L was sufficient to comply with the DWD limit for the more amenable pesticides in trial 2, with more severe competing conditions than trial 1, this PAC dose was assumed adequate for scenario 1.Scenario 2 considered an intake water similar to W2, with a bit higher content of A254-absorbing NOM, and containing a high load of pesticides amenable to adsorption, i.e., conditions analogous to trial 2 but disregarding the pesticides less amenable to adsorption (dimethoate and bentazone). Results of trial 2 showed the compounds amenable to adsorption to be highly removed (below the DWD limits); thus, those PAC dosing conditions were considered, i.e., 12 mg/L inline and 8 mg/L to tank.Scenario 3 considered an intake water similar to W2, with a bit higher A254-absorbing NOM content, and containing a high pesticides’ load, with some compounds less amenable to adsorption, i.e., conditions analogous to trial 2. Results of trial 2 showed that 50% higher inline PAC dose than tank dose (12 mg/L inline vs. 8 mg/L to tank) reached identical removals of pesticides and NOM. As the DWD limits were not complied for the less amenable compounds in trial 2, 50% higher PAC doses than those used in trial 2 were assumed in scenario 3, i.e., 18 mg/L (inline) and 12 mg/L (tank).Three scenarios were considered , combiniThe investment costs, the annualized CAPEX and the total (CAPEX+OPEX) production costs breakdown for the three scenarios are depicted below, as functions of the plant flow rate, in 3/d) with low PAC dosing needs.Analysing the investment needs a, it canConcerning the costs breakdown b,c, rega3  for scenario 1, 0.03–0.05 €/m3 for scenario 2 and 0.04–0.07 €/m3 for scenario 3, thus much depending on the nature of the pesticides (more or less amenable to adsorption) and on the A254-absorbing NOM.The total PAC dosing costs (CAPEX and OPEX) are estimated in 0.02–0.04 €/mWhen comparing PAC dosing options, it can be concluded that when PAC dose is the same in both options (as in scenario 1) or the required doses are low, though 50% higher in inline dosing (scenario 2), and the plant is small, inline PAC dosing would be more cost-effective. However, for larger plants under scenario 2 or if PAC doses are higher for both options and particularly for inline dosing, as in scenario 3, PAC dosing to a contact tank will be more cost effective.Two pilot trials of PAC/(Alum)/ceramic MF were conducted with two surface waters (W1 and W2) spiked with a mixture of pesticides to compare two PAC dosing modes: inline and to a contact tank. The short-term trials conducted with these low turbidity/low NOM waters showed no PAC-driven membrane fouling for both PAC dosing options.Differences of pesticides and NOM removal between inline PAC dosing and PAC dosing to contact tank depended on pesticides’ amenability to adsorption and NOM characteristics . In trial 1, with low A254-absorbing NOM and all pesticides amenable to adsorption, high removals of total-pesticides were reached, 95–100% (tank) and 94–100% (inline), with no significant differences in pesticides and NOM removal between the two PAC dosing options. In trial 2, with a bit higher NOM competition  and high concentration of some pesticides less amenable to adsorption, total-pesticides’ removals somewhat decreased, 85–88% (tank) and 83–87% (inline), with a higher inline PAC dose apparently being required to match pesticides’ and NOM removal with tank dosing. Pesticides with low hydrophobicity (low Log Kow) and low number of aromatic rings or negative charge presented the poorest removals.A considerable NOM removal was observed with the hybrid processes of PAC/Alum/MF or PAC/MF, 41–48% DOC and 50–54% A254 for the former, and 18–26% DOC and 29–35% A254 for the latter, which might be relevant for minimizing oxidation by-products after final disinfection and for water stability during distribution. Moreover, PAC complemented coagulation, adding 20–28% NOM removal.3 to 0.04 €/m3 for large-scale plants and from 0.03 €/m3 to 0.07 €/m3 for small-scale plants, the lower end of the intervals corresponding to waters containing low A254-absorbing NOM and only pesticides amenable to adsorption and the higher end of the intervals to waters containing higher A254-absorbing NOM and pesticides less amenable to adsorption. For the first scenario, PAC inline dosing shows to be more cost-effective, whereas PAC dosing to a contact tank proves to be advantageous for the latter.The cost analysis undertaken showed that, for controlling high loads of pesticides, total PAC dosing costs (CAPEX and OPEX) range from 0.02 €/mTo comply with the Drinking Water Directive limits for pesticides, the control of those less amenable to PAC adsorption or in waters with high NOM competition may require higher PAC doses than those herein tested. Moving towards mitigating pollution at source and regular monitoring are therefore extremely important measures and a multi-barrier treatment approach might be advisable if highly challenging waters/conditions are expected to be found in drinking water production.Further research comparing tank and inline PAC dosing in pressurized adsorption/membrane hybrid processes, at pilot scale and with natural waters, is recommended to test: (i) different PAC doses and confirm the effect above proposed; (ii) different water matrixes; (iii) the removal of other microcontaminants with different characteristics and amenabilities to PAC adsorption; iv) the removal of other key-parameters of water quality . Additional tests are also recommended to confirm the coagulant influence on pesticides removal by PAC/MF and its dependence on water quality.


CD49f+ cells possess self-renewal ability and express epithelial and pluripotent markers. Compared to CD49f negative cells, freshly isolated CD49f+ cells highly expressed inhibin beta A and beta B, which are components of activin that has anti-proliferative effects. Notably, an inhibitor of activin, follistatin was expressed in mechanically-damaged SGs, meanwhile no follistatin was expressed in normal SGs in vivo. Moreover, sub-cultured CD49f+ cells highly expressed both Follistatin and a series of proliferative genes, expressions of which were decreased by Follistatin siRNA. These findings indicated that the molecular interaction between activin and follistatin may induce CD49f+ cells proliferation in the regeneration and repair of mouse SGs.Salivary glands (SGs) are very important for maintaining the physiological functions of the mouth. When SGs regenerate and repair from various damages, including mechanical, radiological, and immune diseases, acinar and granular duct cells originate from intercalated duct cells. However, the recovery is often insufficient because of SGs' limited self-repair function. Furthermore, the precise repair mechanism has been unclear. Here, we focused on CD49f, one of the putative stem cell markers, and characterized CD49f positive cells (CD49f Through these effects, saliva can contribute to prevent oral infectious diseases, such as dental caries2 and periodontitis3, and to promote wound healing4. Therefore, salivary glands, the saliva secreting organ, is crucial for oral health. Salivary glands consist of many kinds of cells and secrete many kinds of proteins, such as amylase, kallikrein5, nerve growth factor, and epidermal growth factor6. Since salivary glands have a complex structure with various kinds of cells, the molecular mechanisms for embryonic development and postnatal remodeling remain unclear. Consequently, once they are damaged by Sjögren’s syndrome or radiation therapy for head or neck cancer, they cannot completely recover their abilities because they have a limited self-repair function7. This is the reason why we do not have effective means for their regeneration, so far.Saliva has many abilities, including the protection of mucous membranes, buffer capacity, digestion, and antibacterial activity8. Generally, it is believed that somatic stem cells are involved in the turnover and repair of mature tissues. As represented by hematopoietic stem cells9, keratinocyte stem cells11, and mesenchymal stem cells in bone marrow12, somatic stem cells are present in various organs, and have a limited potency to differentiate into mature cells only in the specific organ where they exist as one of the components. In salivary glands, although there is still room for consideration of the stem cells location, one of the hypotheses is that somatic stem cells may exist in intercalated ducts because it is known that acinar and granular duct cells originate from intercalated duct cells during remodeling15. However, somatic stem cells in salivary glands have not been specifically defined, because only few specific markers have been found as stem cells in salivary glands17.In general, during the regeneration and repair of mature organs, it is important to control the appropriate number and kinds of cells for the proper recovery of organ size and structure. To achieve these goals, there are two possibilities of cell replacement mechanisms: one is proliferation and differentiation from stem or precursor cell pools, and the other is self-duplication of existing mature cells10 and breast cancer stem cells18. Meanwhile, it is used as the putative marker for salivary stem cells20. The general molecular structure of CD49f allows its heterodimerization with integrin β1 chain (CD29) to form very late activation antigen 6 (VLA-6) and with integrin β4 chain (CD104) to form tumor surface protein 180 (TSP-180). The heterodimers can bind laminin, which is one of the major epithelial extracellular matrix proteins. Since CD49f is a putative marker for salivary stem cells20, it promotes submandibular glands morphogenesis and differentiation21. CD49f was expressed by mouse endothelial and epithelial cells on embryonic day 13, and branching morphogenesis of mouse submandibular glands were perturbed by an anti-CD49f monoclonal antibody in the organ culture22. CD49f was also observed in epithelial cells that compose the excretory duct of mouse submandibular glands23. Furthermore, CD49f positive cells (CD49f+ cells) isolated from mouse submandibular glands differentiated into the hepatic and pancreatic lineages24. However, the underlying mechanisms for the morphogenesis and repair/regeneration by CD49f+ cell populations still remain unclear.A differentiation marker, CD49f, also known as integrin subunit α6, is used as the marker for keratinocyte stem cells25. This character of GCT could be beneficial for the isolation of growth factors and their related factors required for the repair of salivary gland.It is well known that submandibular glands of male rodents have the specific organ, Granular convoluted tubules (GCT), and GCT secret many kinds of growth factors and bioactive substances+ cells are related to repair/regeneration in male mouse salivary glands as the fundamental research question. To reveal how CD49f+ cells are related to repair/regeneration in male mouse salivary glands, we confirmed whether CD49f+ cells in male mouse salivary glands have multi-potency as somatic stem cells firstly. Moreover, to identify the molecular functions regulated by CD49f in male mouse salivary glands, we investigated what kind of growth factors are highly expressed by CD49f+ cells isolated from male mouse salivary glands, compared to CD49f negative cells (CD49f- cells) of male mouse salivary glands, and how these growth factors are related to cell proliferation in regeneration and repair of salivary glands.In the present study, we hypothesized that CD49f+ fractions from salivary glands of five-week-old male C57BL/6 mice was 12.1 ± 1.1%  < 30) and 5 genes in the CD49f+ fractions had a threefold higher expression level than CD49f- fractions  expression increased by approximately fivefold in CD49f+ cells  and Growth differentiation factor 10 (Gdf10) showed a decrease greater than tenfold in CD49f+ . In addition, mRNA expression analysis of 3 inhbb-related genes, Inha, Inhba, and Follistatin, revealed that Inhba and Follistatin expressions increased along with Inhbb in freshly isolated CD49f+, compared to CD49f– , but Inha expression presented a similar level for both fractions , but follistatin presented an undetectable level in both cell fractions   of cultured CD49f+ cells were examined using enzyme-linked immunosorbent assay (ELISA) and western blotting. The secreted follistatin level in the supernatant of cultured CD49f+ cells was significantly increased on days 19 and 22 , Cyclin D2, and Proliferating cell nuclear antigen (Pcna). The CD49f+ cells were transfected by Follistatin siRNA , which is myoepithelial cells marker, may be useful for the isolation of somatic stem cells in adult salivary glands. Moreover, we need to characterize CD49f+ cells population more precisely by RT-qPCR using other stem cell markers; Sca-1, CD133, and lineage markers; CK14 , CK18 , or SMA for the separation of stem cells population. Future study in our research direction should incorporate these matters.Several studies indicate that CD49f could be stem cell marker+ cells, we examined their self-renewal and multipotent abilities in vitro. Several studies reported that CFU assay is carried out for checking self-renewal ability38 and the expression of intracellular laminin is used for verifying multipotent ability26. This study revealed that the number of CFUs in CD49f+ cells was remarkably higher than that in CD49f− cells 39. Inhibins, which are pituitary FSH secretion inhibitors and inhibit the activin effects by binding to activin’s receptor competitively40, are composed of inhibin α and βA or βB subunits, encoded by Inha, Inhba, and Inhbb, respectively. Follistatin has a similar effect as inhibin, by inhibiting the signaling pathway of activin through the inhibition of the binding of activin to the receptors, by forming an activin-follistatin complex41.Therefore, in mRNA and protein levels, we examined the expression levels of these 4 growth factors; inha, inhba, inhbb, and follistatin. Inhba, Inhbb, and Follistatin, and INHBA and INHBB expression levels in freshly-isolated CD49f+ cells were higher than those in CD49f− cells, but for Inha mRNA expression, there were no significant differences  superfamily and arrests cell proliferation in the G0-G1 cell cycle phase27. Regarding mRNA levels, we showed that those of P21 were increased and those of Cyclin D2 mRNA were decreased by Follistatin siRNA; however, those of P21 were decreased and those of Cyclin D2 and Pcna were increased by the addition of recombinant mouse follistatin  activity by forming a binary complex with CDK4; by contrast, P21 negatively regulates kinase activities of both CDK4 and CDK2 by binding with cyclin D-CDK4 and cyclin E-CDK2, respectively. From the above, we suggest that the control of the proliferation of CD49f+ cells is made through the interaction between activin and follistatin (activin-follistatin axis). The alteration of the molecular axis has a crucial role in the regulation of the regeneration of mouse salivary glands. However, it is unclear whether the interaction between activin and follistatin on cultured CD49f+ cells absolutely occurs in this study. We will examine whether CD49f+ cells express ACVR2A or ACVR2B in protein level by western blotting, and whether recombinant follistatin protein can affect phosphorylation of R-SMAD, that is activation of activin pathway, in the future research.Next, we examined mRNA expression of tin Fig. A–C. In c+ cells absolutely express these proteins in vivo. In the next step, we will perform multiple immunofluorescence staining. Moreover, lobules sometime have duct-like structure after ligation. Accordingly, we will carry out multiple immunofluorescence staining to clarify the distribution of CD49f+ cells using CK14, CK18, and SMA as our next stage study.To confirm the expression of activin and follistatin in vivo, we used acinar apoptosis models of mouse salivary glands induced by main duct ligation. INHBA and INHBB were expressed for all days and follistatin was expressed only at the 8-day point after releasing the main duct ligation Fig. . However43. Furthermore, activin is continuously produced in the liver and follistatin administration promotes liver regeneration in rat hepatectomy models45. On the other hand, the kidney is known to have a low regenerative ability, and activin expression in the damaged kidney increases notably and follistatin administration improves kidney function, based on histological findings in kidney46. These results suggest that the activin-follistatin axis may be associated with liver and kidney regeneration and restoration. Moreover, the activin-follistatin axis has important roles in other organs and early embryogenesis49. Therefore, further in vivo assays would be essential to investigate the role of the activin-follistatin axis in salivary glands.The liver is known to have high regenerative ability, and in vitro rat hepatocytes produce activin and follistatin for proliferation+ cells expressed inhba and inhbb, which are both activin components, and cultured CD49f+ cells began to express follistatin, which has the ability of activin inhibition in mRNA and protein levels. In vivo, follistatin was expressed in mechanically damaged salivary glands. These results suggest that CD49f+ cells express activin in non-damaged salivary glands, which may suppress a disordered proliferation of CD49f+ cells. However, when salivary glands are mechanically damaged, CD49f+ cells produced follistatin and may proliferate and supply progenitor cells through activin-follistatin axis . Animal care was performed in accordance with the Guidelines for the Treatment of Experimental Animals, and all experiments were approved by the Animal Care and Use Committee of Okayama University (# OKU-2014168).g for 5 min at room temperature (RT). Five-week-old male mice were sacrificed and both submandibular glands were excised, minced, and incubated in 1 mL of ethylene glycol-bis (β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) buffer at 37 °C for 20 min, and then centrifuged. The cell pellets were resuspended in 1 mL of Dulbecco’s modified Eagle medium/F12 1:1  containing 1.67 mg/mL collagenase  and 1.33 mg/mL hyaluronidase , and incubated for 40 min at 37 °C. After centrifugation, the remaining tissue was further treated with D-MEM/F12 1:1 containing 1.67 mg/mL Dispase II  for 60 min at 37 °C. The cell suspension was passed through a 70-μm Cell Strainer , and centrifuged. The pellets were resuspended in 10 mL of keratinocyte serum-free medium . Subsequently, the cells were fractionated using magnetic cell separation , as described below, and these freshly-isolated CD49f+ and CD49f− cells were immediately used for some experiments. For other experiments, CD49f+ and CD49f− cells were plated on a 60-mm culture dish  at 1.0 × 104 cells/cm2 with K-SFM containing 100 U/mL penicillin G and 100 μg/mL streptomycin (Life Technologies). The cells were cultured at 37 °C, 5% CO2, and the medium was renewed 7 days after cell seeding, and then renewed every 3 days. The shape of cultured CD49f+ and CD49f− cells was observed under a microscope . For induction of the hepatic lineage, CD49f+ cells were plated (1.0 × 104 cells/well) on a 1:3-thick Matrigel (BD Biosciences) in 12-well dishes (1.5 mL/well) in K-SFM medium with antibiotics, which was additionally supplemented with 10 mmol/L nicotinamide.For the isolation of cells from the cell suspension, all centrifugations were performed at 120 × 6 cells; clone: GoH3, AbD Serotec, Oxford, UK) for 5 min on ice, followed by reaction with goat anti-rat IgG MicroBeads (Miltenyi Biotec) for 5 min on ice. The cells were subsequently passed through a 35-μm Cell Strainer (BD Biosciences), and separated into CD49f+ and CD49f− cell fractions using an autoMACS (Miltenyi Biotec) and the separation program .The cells were incubated with an anti-CD49f antibody  and CellQuest (BD Biosciences).The cells isolated from salivary glands were reacted with the fluorescein-5-isothiocyanate (FITC)-conjugated CD49f antibody  for 10 min at 4 °C, and FITC-conjugated isotype control antibody  was used as a negative control. A total of 1.0 × 10+ and CD49f− cells from freshly-isolated and sub-cultured CD49f+ cells were respectively isolated using a RNeasy Mini Kit . RNA concentration and quality  were determined using a NanoDrop 2000 . One microgram of total RNA extracted from freshly-isolated CD49f+ and CD49f− cells was amplified using Applied Biosystems 7300 Real-Time PCR System (Life Technologies), and the RNA expression levels between these cells were compared using a Mouse Growth Factors RT2 Profiler PCR Array (QIAGEN). Obtained data were analyzed using the free Web-based PCR Array Data Analysis Software .Total RNAs of CD49fGlucuronidase beta (Gusb) and β-Actin were used as internal controls.cDNA was synthesized with 1 μg of the total RNA using SuperScript VILO MasterMix (Life Technologies). cDNA was quantified by using qPCR with specific primers . The total protein was measured using an RC DC Protein Assay Kit . After the separation of 30 µg of protein using a denaturing 12% polyacrylamide gel and transferal to a polyvinylidene difluoride membrane , the membrane was blocked with 5% skim milk in TTBS , and incubated with an anti-Inhba monoclonal antibody , an anti-Inhbb monoclonal antibody , an anti-Follistatin polyclonal antibody , and an anti-β-Actin monoclonal antibody , followed by horseradish peroxidase (HRP)-conjugated secondary antibodies . Membranes were reused after re-probing using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). The blots were scanned and analyzed using Image J . β-ACTIN intensity was used as an internal control.Total cell lysates were respectively extracted from CD49fAfter submandibular glands at both sides of five-week-old male mice were exposed, one main secretory duct was ligated with the clip , and another side was not ligated and was used as a control. Six days later, the submandibular glands were re-exposed, and the clips were removed. At 1, 2, 4, 8, 16 days after their removal, each salivary gland was excised, weighed, and photographed.Submandibular glands of duct ligation mouse model were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (4% PFA-PBS), and embedded in paraffin. Sections were sliced in 4-μm thick slices for immunohistochemical staining and hematoxylin–eosin staining. The sections were incubated with anti-Inhibin beta A antibody , anti-Inhibin beta B antibody , anti-CD49f antibody , and anti-Follistatin antibody , followed by the reactions with SuperPicture Polymer Detection Kit (abcam) and hematoxylin. The stained sections were observed under a microscope .+ and CD49f− cells were plated into 6-well plates (Corning) at a density of 2.5 × 104 cells/cm2. The cells were cultured for 8 days in K-SFM, as mentioned above. Colonies that consisted of more than 10 cells were counted under a microscope (ECLIPSE TS100). Their colony forming ability was the mean value of the total number of colonies counted in 3 wells, and the assay was performed 3 times independently.CD49f+ and CD49f− cells were cultured in Lab-Tek II Chamber Slide (Thermo Fisher Scientific) at a 1.0 × 104 cells/cm2 density for 7 days with K-SFM, as mentioned above. Subsequently, the cultured cells were fixed with 4% PFA-PBS for 15 min at 37 °C, followed by blocking with 10% Non-Immune Goat Serum (Life Technologies) or Donkey Serum (Sigma-Aldrich) for 30 min at RT. Slides were then treated in PBS containing 0.2% Tween-20 for 5 min at RT. On the other hand, control slides were washed only using PBS. Regarding CD49f+ cells after induction of hepatic lineage, the Matrigel Matrix was depolymerized using a Cell Recovery Solution (Corning), and spheres were solidified in iPGell , fixed in 4% PFA-PBS, embedded in paraffin, and sliced in 4-µm-thick slices. Subsequently, the cells were incubated with an anti-CD49f , an anti-Laminin , an anti-E-cadherin , an anti-pan Cytokeratin , an anti-Albumin , and an anti-alpha 1 Fetoprotein antibodies  (Bioss), for 30 min at RT, followed by incubation with Alexa 594-labeled anti-rat IgG (1:500), Alexa 488-labeled anti-rabbit IgG (1:500), Alexa 594-labeled anti-rabbit IgG (1:500), Alexa 594-labeled anti-mouse IgG (1:500), and Alexa 594-labeled anti-goat IgG  for 30 min at RT after washing 3 times with PBS. Subsequently, they were mounted using VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) . Cells were observed under a fluorescence microscope .CD49f+ cells were cultured for 16, 19, and 22 days. Follistatin in culture supernatants was measured using an ELISA kit .CD49f+ cells were plated on 6-well plates at 3 × 105 cells/well. After 48 h, CD49f+ cells were transfected with Follistatin siRNA  or Non-Targeting siRNA Pool , and Lipofectamine RNAiMAX Reagent (Life Technologies), when cells reached a 50–60% confluence. Subsequently, 0.2 μg/mL Recombinant Mouse Follistatin protein  was added to each well. The total RNA was isolated from cells cultured for 1, 2, 4, 8, 12, 18, 24, 36, and 48 h after rmFOLLISTATIN addition. Effects of the addition of siRNA and rmFOLLISTATIN were evaluated by using RT-qPCR. Total RNA from CD49f+ cells transfected with a non-target control siRNA (Thermo Fisher Scientific) was used as a negative control.CD49ft-test or one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test using the JMP 9 software . A P-value ≤ 0.05 was determined as statistically significant.All results are represented as means ± SD from at least 3 independent experiments, as shown in each figure legend. Statistical analysis was determined by Student’s Supplementary Information.


P. aeruginosa in sewage and water bodies in India is well documented.In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen  Pseudomonas aeruginosa are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of P. aeruginosa bacteriophages. In this study, we report the isolation and characterization of a novel P. aeruginosa N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10−4. Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like P. aeruginosa phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant P. aeruginosa clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy.Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen IMPORTANCE In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging P. aeruginosa strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem. Pseudomonas aeruginosa, an important Gram-negative opportunistic human pathogen, causes a wide range of infections, including ventilator-associated pneumonia, burn and wound infections, septicemia, and chronic pulmonary infection in cystic fibrosis patients (P. aeruginosa is included among the ESKAPE pathogens capable of rapidly developing multidrug resistance (MDR)  –4. WhileP. aeruginosa is more prevalent in Indian aquatic sediments that receive hospital effluents than those in other countries  of AM.P2 virions revealed phage head and tail dimensions of 716 Å and 235 Å, respectively , gentamicin (4 μg/ml), and ciprofloxacin (0.25 μg/ml) (600) characteristic of AM.P2 treatment was mirrored in the antibiotic-phage combination treatments, suggesting that supplementation with an antibiotic did not impact phage efficacy in the initial cycles of phage replication and infection  and three classes of antibiotics , each with different mechanisms of action . MIC5 μg/ml) , and fornfection . Howevereatments  and C, rP. aeruginosa bacteriophages. The AM.P2 genome has 110 coding sequences, of which 78 are for hypothetical proteins and 10 for rho-independent terminators  Early genes. The AM.P2 genome encodes many N4-like proteins involved in phage DNA replication, including a PD-(D/E)XK nuclease domain-containing protein/N4 gp42-like protein, putative DNA primase/N4 gp43-like protein, putative ATPase/N4 gp44-like protein, putative single-strand binding protein (SSB)-like protein/N4 gp45-like protein, and putative RuvC-like Holliday junction resolvase/N4 gp46-like protein.(ii) Middle genes. Genes required for transcription, including the RNA polymerase small subunit RNAP1/N4 gp15-like protein and DNA-directed RNA polymerase RNAP2, are found further downstream on the sense strand of the AM.P2 genome. Sequences encoding a putative rIIA-like protein/N4 gp33-like protein and putative rIIB-like protein/N4 gp34-like protein are also present. A homolog of the LUZ7 Drc protein is present in the genome. Drc was recently elucidated as an SSB transcription factor that interacts with RNAP2, playing a role in the transition of early- to mid-phase gene transcription (cription .(iii) Late genes. Genes involved in phage assembly are mostly found on the antisense strand. These include the major capsid protein, portal protein, and tail fiber proteins. One specific tail fiber protein contains an SGNH hydrolase domain, which suggests lipase activity. The AM.P2 genome encodes the characteristic N4-like large virion-associated RNA polymerase (vRNAP), which in N4 is coinjected with phage DNA during infection and responsible for the transcription of early genes  data from the Indian subcontinent, reports describing the incidence of resistance in community-acquired infections show undoubtedly higher rates than those in developed nations. The Indian AMR scenario is marked by a significantly high prevalence of MDR pathogens, thereby providing a potential model environment for studying the rapid rise and spread of MDR bacterial populations and uncovering possible solutions. Researchers and clinicians alike are exploring alternative and adjuvant therapies for the treatment of MDR Pseudomonas aeruginosa strains. We report here that AM.P2 efficiently lyses laboratory strain PAO1, with a 3- to 4-log decrease in number of CFU compared to that of untreated PAO1. Unfortunately, bacteria are continually evolving and adapting, and just as antibiotic resistance arises, phage resistance is also inevitable. AM.P2, like any other lytic bacteriophage, cannot be viewed as a silver bullet for the complete eradication of P. aeruginosa, as bacteriophage-insensitive mutants do arise after an extended period of phage-bacterium interaction. Proposed methods to overcome phage resistance include the implementation of polyphage cocktails or phage-antibiotic combinatorial treatments, both of which have resulted in successful outcomes in in vitro and in vivo tests . All experiments with DNA isolated from clinical strains were also approved by the TIGS Institutional Biosafety Committee (IBSC) review board (2019). Clinical strains were obtained from patients in tertiary care hospitals as part of the routine care done for patients without any additional procedures.Clinical strains were collected from multiple tertiary health care centers over a period of approximately 2 years, spanning from September 2017 to November 2019. These strains were provided on blood agar and subsequently subcultured on cetrimide agar. Single colonies were grown in Luria-Bertani (LB) broth and stored as glycerol stocks. All strains were routinely grown in LB broth aerobically (shaking at 200 rpm) at 37°C.Pseudomonas aeruginosa strain ATCC 27853 as a control. Briefly, bacteria were grown to an OD600 of 0.1 in 3 ml LB, and 300 μl of each culture was swabbed on the surface of Mueller-Hinton agar plates. Antibiotic discs (HiMedia) were placed on the swabbed culture incubated for 16 to 18 h at 37°C, following which the zones of inhibition were measured, and each strain was determined as resistant/intermediate/sensitive to each antibiotic tested according to the chart provided by the manufacturer. Strains that were resistant to three or more classes of antibiotics were considered MDR.All clinical isolates were subjected to antibiotic susceptibility testing against gentamicin, amikacin, tobramycin, ciprofloxacin, levofloxacin, ceftazidime, piperacillin, ticarcillin, aztreonam, imipenem, meropenem, and colistin by the Kirby Bauer disc diffusion method using 2HPO4) and 3 ml of overnight culture of PAO1 and enriched aerobically for phages at 37°C for 16 to 24 h. The enriched sample was centrifuged twice at 6,500 × g for 10 min, and the supernatant was treated with 1:100 chloroform and/or filtered through a 0.22-μm filter. The resulting lysate was diluted and plated by the double-agar overlay method to yield single plaques, as previously described  8000-NaCl (20% PEG-8000 and 2.5 M NaCl) to 1,200 μl of phage sample. After overnight incubation at 4°C, sample was centrifuged at 20,000 × g for 20 min, and the resultant phage pellet was resuspended in 200 μl of SM buffer.For high-titer phage stocks, AM.P2 was enriched in a plate by molten streaking on soft agar with PAO1 . After 1Virus particles were applied to a glow-discharged R1.2/1.3 holey carbon grid , blotted for 3 s with a blot force of 0, and plunge-frozen in liquid nitrogen-precooled ethane using a Vitrobot Mark-IV  at 100% humidity. Two virus samples, arising from different purification batches, were imaged on a Titan Krios 300 KV microscope equipped with a Falcon 3 direct electron detector. The first sample was imaged at ×47,000 magnification, corresponding to a pixel size of 1.78 Å. The second sample was imaged at ×59,000 magnification, which corresponds to a pixel size of 1.38 Å. Final images were processed using ImageJ .6 PFU/ml) was added to 9.9 ml of PAO1 grown in LB broth to an OD600 of 0.4. After adsorption for 5 min, 100 μl of AM.P2 plus PAO1 was added to 9.9 ml of fresh LB (A) and further diluted by transferring 1 ml to 9 ml of LB (B). At 5-min intervals over a span of 1 h, 100-μl aliquots were taken from both A and B, mixed well with 100 μl PAO1 culture and 5 ml LB soft agar (0.7%), and plated by the double-agar overlay method. Mean numbers of PFU/ml from three independent experiments were plotted along with standard errors of the means (SEM).Phage growth kinetics and latent period were determined by one-step growth curve experiments, performed as previously described, with a few modifications , 34. Bri600 of 0.2 in LB and then diluted 1:100. Twenty microliters of the diluted culture and 20 μl of 10× the desired antibiotic concentration were added to each well, and the total volume was made up to 200 μl in LB. After 24 h of static incubation at 37°C, absorbance at OD600 was measured using an Enspire Alpha multimode plate reader (PerkinElmer). MIC90 was determined as the minimum concentration of antibiotic that would inhibit bacterial growth by ≥90% compared to the untreated bacterial control.MICs were determined using the microdilution method for the antipseudomonal antibiotics colistin, gentamicin, and ciprofloxacin as described previously, with some modifications . PAO1 wa600 of 0.2 was mixed with 20 μl of 109, 108, and 107 PFU/ml AM.P2 to maintain MOIs of 10, 1, and 0.1, respectively, in a 96-well plate. For phage-antibiotic combinatorial studies, 160 μl of bacterial culture was mixed with 20 μl of 109 PFU/ml AM.P2 and 20 μl of 10× the desired concentrations of colistin, gentamicin, or ciprofloxacin. No phage controls were treated with SM buffer, and no antibiotic controls were treated with LB. Plates were incubated at 37°C for 3 h, and bacterial growth was measured by checking absorbance at OD600 every minute for PAO1 and every 5 min for all other strains/phage-antibiotic combinations using a plate reader. For 24-h incubations, plates were set up in the same manner, except the starting culture (OD600 of 0.2) was diluted 1:100 and mixed with 20 μl of 107, 106, and 105 PFU/ml AM.P2; 96-well plates were incubated at 37°C without shaking for 24 h. Final absorbance at the OD600 was measured using a plate reader. Mean triplicate values from three independent experiments were plotted along with SEM.Bacterial growth inhibition assays were done as previously described, with slight modifications . For theBactericidal activity assays were done as previously described, with slight modifications , 37. Bri600 of 0.2 and diluted 1:100 in LB. Eighteen microliters of diluted culture was combined with 20 μl of 107 PFU/ml AM.P2 (MOI of 10) in 14-ml culture tubes. Tubes were incubated with shaking at 37°C for 10 min to allow for phage adsorption. Four milliliters of LB soft agar (0.7% agar) was added to the tubes, mixed well, poured on the surface of an LB plate, and allowed to solidify before incubating at 37°C. The number of bacteriophage-insensitive mutants (BIMs) was enumerated after 24 and 48 h. Resistance frequency was calculated at these time points by dividing the number of BIMs by the number of CFU in the starting culture.AM.P2 resistance frequency was determined per the previously described protocol . PAO1 wag for 20 min, and the pellet was resuspended in 200 μl 0.05 M Tris-Cl, pH 8. The sample was incubated with 2 μl of DNase  and 2 μl of RNase  for 1 h at 37°C. An equal volume of buffered phenol  was added and tubes were mixed well, followed by collection of the aqueous layer after centrifugation at 10,000 × g for 2 min. This was followed by the addition of 200 μl of chloroform, centrifugation at 10,000 × g for 2 min, and collection of the aqueous layer. Twenty microliters of 3 M sodium acetate (pH 4.8) and 400 μl of 100% molecular-grade ethanol were added, and the precipitated DNA was pelleted at 10,000 × g for 10 min. After a 70% ethanol wash, the pellet was allowed to dry and then resuspended in 30 μl of nuclease-free water. DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher).Genomic DNA from 200 μl of high-titer AM.P2 stock was isolated using phenol-chloroform. Briefly, PEG-precipitated phage was centrifuged at 20,000 × For Illumina sequencing, phage DNA was prepared with a Nextera XT DNA library preparation kit according to the manufacturer’s instructions. Sequencing was done at the National Centre for Biological Science (NCBS), Bangalore, India, on an Illumina HiSeq 2500 platform with 100-bp paired-end reads and ∼1,000× coverage. The library for Nanopore sequencing was prepared using a one-dimensional (1D) ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies (ONT). Library preparation was a two-step process that included end-prep and adapter ligation. End-prep was performed to convert the overhangs into blunt ends and to adenylate the 3′ ends of the DNA, followed by cleanup. Adapter ligation, specific for the R9.4.1 flow cell chemistry run, was performed by adding the adapter mix and the T4 ligation kit from the 1D ligation sequencing kit to the dA-tailed DNA, followed by another cleanup. The DNA library was mixed with sequencing buffer and loading beads immediately before loading. The sample was loaded gently, in a dropwise fashion, into the SpotON sample port in the flow cell prepared for sequencing and sequenced using the MinION device. The sequencing was initiated and run for an hour with high-accuracy base calling using the MinKNOW software. Guppy was used for base calling to obtain the final sequence output in the Fastq format.https://cpt.tamu.edu/galaxy-pub) , false positives that scored less than 90, had less than a 5-bp stem without mismatch, and had fewer than four Ts following the stem-loop were filtered out manually  in the Galaxy Center for Phage Technology (CPT) server (axy-pub) , 40. Mulaxy-pub) –43, Metaxy-pub) –, and Sixxy-pub) –. Terminamanually . ARAGORNmanually . Functiomanually –50. The The AM.P2 genome was aligned to the representative RefSeq genome database to search for similar genomes using BLASTN. The top 18 hits that had >70% identity and >10% query cover were selected for further analysis. Multiple-sequence alignment of whole phage genomes was done using ClustalW, and phylogenetic trees were constructed using the neighbor-joining method in MEGA X , 51.AGAGTTTGATCCTGGCTCAG; 1492R, TACCTTGTTACGACTT)  and universal primers  . The PCR10 PFU/ml) was spotted and allowed to dry. Plates were incubated overnight at 37°C and observed for lysis.Spot tests were done by first swabbing an overnight culture of each strain on an LB plate and allowing it to dry. Two microliters of concentrated AM.P2 stock  followed by a post hoc Tukey’s test. Statistical significance was defined as a P value of  <0.05 (*).The significance of the effects of AM.P2 on PRJNA629366 and in the NCBI BioProject database under BioSample no. SAMN14794289. The Illumina and Nanopore reads were deposited as Sequence Read Archive (SRA) accession no. SRR11665926 and SRR11772201, respectively, and the AM.P2 complete genome as GenBank accession no. MT416090.All genome sequencing data have been deposited under BioProject no.


The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process. Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. Orthopneumovirus of the family Pneumoviridae and has a negative-strand RNA genome that encodes the following 11 proteins: the nonstructural proteins 1 and 2 (NS1 and NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), small hydrophobic (SH), major glycoprotein (G), fusion (F) glycoprotein, proteins M2-1 and -2, and the polymerase (L)  is the most relevant viral cause of respiratory infection in children, with extensive morbidity and nearly 200,000 deaths every year . HRSV berase (L) –5, triggase (L) –. HRSV glase (L) ––9. HRSV se (L) ––. In thisse (L) ––, 13.Proteins N and M have multiple functions during HRSV replication. The N protein directly interacts with the viral RNA, participates in the formation of virus-induced inclusion bodies, and has been shown to impair immunological synapses on the cell surface , whereas15–Although it is well established that HRSV assembles at the plasma membrane , it is p10.1128/mBio.01869-20.1FIG S1FIG S1, PDF file, 0.4 MB.HRSV inclusion bodies are surrounded by the Golgi marker giantin. (A to C) Panel C shows colocalization between HRSV N (A) and giantin (B).  Higher magnification of panels A, B, and C, respectively. Panel F shows HRSV inclusion bodies surrounded by giantin. (G to J) Panel G shows colocalization between HRSV N (H), giantin (I), and HRSM M (J), with arrowheads pointing to places where the HRSV proteins colocalize with giantin, and the arrow pointing to a cell where there is no colocalization between HRSV proteins and the Golgi at this focal plane. Approximately 73% of the infected cells containing inclusion bodies showed this pattern of localization to some extent. All images were taken at 24 hpi with a Zeiss 780 confocal microscope and are a representation of at least three different experiments. Figures shown are a single focal plane from Z-stack imaging. Magnification, ×63. Scale bar = 10 μm. Download Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the This notion was confirmed by superresolution imaging showing tight proximity between HRSV F and N proteins in giantin-labeled Golgi compartments . To test10.1128/mBio.01869-20.5SUPPLEMENTARY VIDEO 1Video S1, AVI file, 0.4 MB.Three-dimensional (3-D) video of the cropped area in Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, we investigated whether HRSV N and M proteins reach the endosomal system and the plasma membrane via the trans-Golgi network (TGN). To this end, we labeled for TGN46, a transmembrane protein that cycles between early endosomes and the TGN, where it is mostly localized at steady state . As expe10.1128/mBio.01869-20.6SUPPLEMENTARY VIDEO 2Video S2, AVI file, 3.9 MB.Three-dimensional video of the cropped area in Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To further explore the relationship between HRSV structural proteins and the Golgi complex, we treated infected cells with brefeldin A BFA), a compound that binds Arf1-GDP protein and prevents attachment of coatomer protein complex I COPI) to the Golgi. BFA treatment is known to reversibly induce Golgi stack disassembly and membrane redistribution to the ER , 24. ThePI to theA, a comp10.1128/mBio.01869-20.2FIG S2t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All the scale bars = 10 μm. Download FIG S2, PDF file, 0.3 MB.Effect of brefeldin A on the size and quantity of HRSV inclusion bodies. (A to C) IF for HRSV N protein in HRSV-infected cells not treated with brefeldin A. (D to F) IF for HRSV N protein in HRSV-infected cells treated with brefeldin A. (G to I) Quantity of HRSV aggregate/IB structures counted in HRSV-infected cells treated or not with brefeldin A, exemplified by panels H and I. All the images were taken at 24 hpi and are representative of a single focal plane from a Zeiss 780 confocal microscope. Magnification, ×63. The graph was based on the counting of at least 12 fields from two different experiments. The statistical method used was Student’s Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01869-20.3FIG S3x axes depict the time lengths of exposure to BFA. The fluorescence images were taken with a Zeiss 780 confocal microscope. Magnification, ×63.The statistical method used in panels U and V was ANOVA one-way Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All the scale bars = 10 μm. Download FIG S3, PDF file, 0.4 MB.The size of inclusion bodies is affected by the time of exposure to BFA. HEp-2 cells were infected with HRSV, and at 24 hpi they were fixed and stained for DAPI, HRSV N, and giantin. The IF images in the far-right column are merges of those in the three columns on the left. The figures are a representation of two independent experiments, and at least five fields of each experiment were used to measure the inclusion bodies. (U) Graph comparing the size of the inclusion bodies in HRSV-infected cells exposed or not to BFA during different time lengths. (V) Mander’s colocalization between HRSV N and giantin in HRSV-infected cells treated with BFA or DMSO (control). The panel U and V Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess whether HRSV N and M proteins are targeted to early endosomes, immunofluorescence IF) microscopy for sorting nexin 2 (SNX2) was performed in HEp-2 cells at different times pi. SNX2 plays an important role in the formation of the retromer coat by shaping tubular structures out of the membranes of early endosomes , and the microscoThe association of SNX2 with SNX1, Vps26, Vps29, and Vps35 forms the retromer complex, a vesicle coat involved in retrograde transport of proteins from endosomes to the TGN . In orde10.1128/mBio.01869-20.4FIG S4t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All the scale bars = 10 μm. Download FIG S4, PDF file, 0.3 MB.The early endosome marker EEA1 does not colocalize with HRSV inclusion bodies. (A to C) Noninfected HEp-2 cells with labeling for EEA1 (B) and SNX2 (C). (D) Merge of panels A, B, and C. (E to H) HRSV-infected cells at 24 hpi, labeled for HRSV N protein (E), EEA1 (F), and SNX2 (G). The arrowheads show that EEA1 marker (E) does not colocalize with HRSV N in the inclusion bodies shown in panels E, F, and H, differently from SNX2 (G), which colocalizes with HRSV N . (I) Mander’s coefficient of colocalization between HRSV N with SNX2 and EEA1. All images were taken at 24 hpi and are representative of a single plane from a Z-stack. Magnification, ×63 with a Zeiss 780 confocal microscope. The statistics were acquired with Student’s Copyright © 2020 Cardoso et al.2020Cardoso et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To verify the partitioning of SNX2 in different compartments of HRSV-infected cells, cellular fractionation and Western blotting were performed. In HRSV-infected cells, SNX2 accumulates in fractions 1 to 7, which correspond to those that contain the nonglycosylated HRSV proteins that are known to compose the inclusion bodies . This waIn keeping with previous observations that HRSV budding forms typical filamentous shapes on the plasma membrane , 29 that2 were slightly lower in cells KD for SNX2, than in those treated with the scrambled siRNA control  experiment was done using small interfering RNA (siRNA) for sorting nexins 1 and 2 in HEp-2 cells . It is w control  to H. Wel et al.  recentlyeriphery  to M, theriphery  and M. Ieriphery . In addieriphery . Consisteriphery . Nevertheriphery  and R, iIt is well known that the HRSV envelope glycoproteins G, F, and SH traffic through the Golgi apparatus, where the F protein undergoes maturation cleavages . HoweverIn the present study, HRSV M and N proteins partially colocalized with giantin, a specific marker for cis and medial Golgi, mainly at the edges of this organelle. This finding that the traffic of the M protein is associated with membranes is in agreement with results by Henderson et al. , who repThe importance of the Golgi apparatus in the HRSV protein traffic was also supported by experiments in which cell treatment with BFA resulted in smaller HRSV inclusion bodies and reduced N and F proteins in the cell surface, an effect that was reversible by BFA wash out. These observations are also in keeping with data by Céspedes et al. , who shoIt has been previously shown that the HRSV N protein reaches late endosomes, as indicated by colocalization of N with LAMP1, a late endosome marker . The preThe HRSV N and M proteins are known to interact with one another , 26, whi33–in vitro. In addition, this movement of M protein is reminiscent of what was proposed by Cifuentes-Muñoz et al.  was propagated in HEp-2 cells, and the stock was titrated by plaque assay using routine methods  with 10% fetal bovine serum (FBS) and maintained in MEM with 2% FBS in 5% CO methods .2 at 37°C. At 0, 4, 8, 12, 24, and 48 hpi the coverslips were fixed. For the immunoprecipitation and cell fractionation assays, HEp-2 cell monolayers were prepared in 75-cm2 flasks, infected at MOIs of 0.1 and 1, and harvested at appropriate times.Infection assays were done on glass coverslips inside 24-well plates, using HRSV stock diluted in phosphate-buffered saline (PBS) to reach a multiplicity of infection (MOI) of 1, with 1 hour of incubation at 4°C to synchronize virus entrance. Then, coverslips were washed three times in cold PBS, replenished with MEM with 2% FBS at 37°C, and then incubated in 5% COAt appropriate times, coverslips were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature and then washed once with PBS, permeabilized with 0.01% Triton in PBS for 20 minutes, and then blocked with PBS containing 3% bovine serum albumin (BSA) for 30 minutes. After 10 washes with PBS, coverslips were incubated with the primary antibody for one hour at 37°C and then washed 10 times with PBS and incubated with the secondary antibody for 1 hour at 37°C. Coverslips were washed 10 times in PBS and once in distilled water and then mounted on glass slides using Flouromount. Nuclei were stained with DAPI, and images were acquired on Leica SP5 or Zeiss 780 confocal microscopes or a Nikon N-SIM microscope (super resolution imaging) and were analyzed using ImageJ software Fiji.The antibodies used for immunofluorescence (IF) were rabbit polyclonal anti-TGN46 , sheep polyclonal anti-TGN46 , rabbit polyclonal anti-SNX2 and anti-VPS26  , Alexa Fg for 20 minutes at 4°C in an Eppendorf 5254R. Cell pellets were washed twice with propylene oxide, embedded overnight in EMBED 812 1:1 in propylene oxide, contrasted, and analyzed in a Jeol JEM-100 CXII transmission electron microscope.HEp-2 cells grown to 80 to 100% confluence were infected with HRSV at an MOI of 1, and at the appropriate time, the culture was fixed in a mixture containing 0.05% glutaraldehyde, 2% paraformaldehyde, and 0.025% calcium chlorate in 0.1 M cacodylate buffer pH 7.4 in a microwave oven for 7 seconds at maximum potency. Cells were then washed in 50 mM glycine in PBS for 15 minutes, incubated with 1% BSA in PBS for 45 minutes under gentle shaking, and then permeabilized with 0.05% saponin diluted in PBS with 1% BSA (PBS-BSA) for 30 minutes. Cells were then incubated with rabbit polyclonal anti-SNX2 antibody diluted in PBS with 1% BSA for 2 hours at room temperature under gentle shaking. After that, cells were washed four times for 5 minutes each with PBS containing 1% BSA, followed by incubation with anti-rabbit antibody conjugated with 6 nm colloidal gold  for 1 hour at room temperature. Cells were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 hour at room temperature, washed twice with cacodylate buffer, and soaked overnight in the same buffer. The next day, cells were washed three times for 5 minutes each with fresh cacodylate buffer, followed by three 5-minute washes with 1% BSA in PBS and three 5-minute washes in distilled water. Next, the preparations were incubated with gold enhancement mixture  for 8 minutes and then washed with distilled water. Cells were then treated for 2 hours with 1% osmium tetroxide and dehydrated in 50% to 100% alcohol series for three 5-minute changes of each dilution. Cell monolayers were detached with propylene oxide and pelleted for 10 minutes at 18,400 × g for 20 minutes at 4°C in an Eppendorf 5254R to separate the nuclear fraction. The supernatant was collected and separated in 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. Subsequently, the nitrocellulose membrane was incubated with the appropriate antibody, namely, goat anti-RSV , rabbit polyclonal anti-TGN46 , sheep polyclonal anti-TGN46 , rabbit polyclonal anti-SNX1, rabbit polyclonal anti-SNX2, rabbit polyclonal anti-GAPDH , mouse anti-beta actin , rabbit polyclonal anti-EGFR , mouse monoclonal anti-EEA1 , and rabbit polyclonal anti-LAMP1 . Except for anti-beta actin antibody, which was incubated for 1 hour, all the others were incubated overnight. This was followed by three 5-minute washes in PBS with 0.1% Tween 20, and then incubation was done with the secondary antibodies, namely, HRP-goat anti-rabbit , HRP-rabbit anti-goat , and HRP-rabbit anti-mouse , and then detected with luminol. The images were acquired in a Bio-Rad ChemiDoc.HEp-2 cells infected with HRSV were harvested at appropriate times postinfection using 0.05% EDTA, suspended in lysis buffer  for 25 minutes on ice and centrifuged at 18,400 × The protocol used was published by Sugrue et al. . Brieflyin situ) and then with primary antibodies in the following combinations: rabbit polyclonal anti-RSV N and mouse polyclonal anti-RSV M, or rabbit polyclonal anti-SNX2 and mouse polyclonal anti-RSV M. HRSV and mock-inoculated cells were incubated only with mouse anti-HRSV M as biological and technical controls, respectively. After primary antibodies, the cells were incubated with the PLA probes, and the preparation was subjected to the steps proposed by the manufacturers. Lastly, the coverslips were washed five times with the buffer solution provided by the PLA kit, mounted with Fluoromount, and analyzed in a Zeiss laser scanning 780 confocal microscope .HEp-2 cells were grown on coverslips and inoculated with HRSV at an MOI of 1 or mock, and after 24 hours, the cells were fixed with 4% PFA for 15 minutes and permeabilized with 0.01% saponin in PBS. The cells were incubated with a block solution from the PLA kit  in a proportion of 750 μl of TRIzol to 250 μl of sample. The RNA extraction was done using the TRIzol protocol proposed in the manufacturer’s protocol. Reverse transcription was carried out with the high capacity cDNA reverse transcription kit  using the manufacturer-provided protocol. Briefly, cDNA was obtained from 1 mg of total RNA denatured at 95°C for 5 minutes in the presence of 20 mM primer directed to sequences within the leader-NS1 gene of the HRSV genome. Tubes were kept on ice for 5 minutes while the reactions were assembled with 2 μl of 10 × RT buffer, 0.8 μl of 100 mM dNTP, 1 μl of MultiScribe reverse transcriptase, and water up to the volume of 20 μl. The reaction mixtures were incubated for 10 minutes at 25°C and then for 120 minutes at 37°C, and 5 minutes at 85°C. The PCR mixture contained 5 μl of SYBR green PCR master mix , 20 nM forward and reverse primers to the HRSV leader region , 1 μl of specific cDNA, and water to a final volume of 10 μl. Cycling parameters were 50°C for 2 minutes and 95°C for 10 minutes, followed by 45 cycles of 95°C for 30 seconds and 60°C for 1 minute; subsequently, one cycle was done of 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds, and specific amplification was confirmed by analyzing the melting curve. Samples were set up in triplicate on a thermocycler ABI 7500 (Applied Biosystems).HEp-2 cells were seeded in 12-well plates (containing coverslips) in 40% of confluence; the second siRNA shot (40 nM each siRNA per well) was performed and 48 hours after the first shot, and then the cells were infected with HRSV (MOI = 1). Then, 24 and 48 hours postinfection, the supernatant and cells were harvested for plaque assay, IF, real-time PCR, and Western blot analyses as described above. The sequences of each siRNA were siSNX1 (AUG AAG AAC AAG ACC AAG AGC CCA C)  and siSNX2 (AAG UCC AUC AUC UCC ACC AAG AGC CAC). The scramble sequence was acquired from Sigma-Aldrich, Brazil.HEp-2 cells were seeded on coverslips and infected with HRSV (MOI = 1), and 4 hours postinfection, fresh medium was replenished (2% FSB in MEM) containing 10 μM brefeldin A (BFA) in DMSO or DMSO alone. The media containing BFA or DMSO vehicle were maintained for 5 hours, and then BFA or vehicle was washed out, while other wells were maintained to complete 19 hours of drug treatment and 24 hours of infection. Then, after 24 hours of infection, all the cells were fixed with 4% PFA in PBS. After fixation, the cells were subjected to IF as described above. For the experiment reported in g for 5 minutes; the pellets were washed with PBS and with STE buffer  to produce an osmotic shock. Cells were passed 20 times through a 25G needle for cell lysis in 0.5 ml of STE buffer without sucrose. The cell homogenates were centrifuged at 1,000 × g for 2 minutes to remove nuclear contents, and the post-nuclear supernatants were loaded on top of a discontinuous 10 to 30% (wt/vol) glycerol gradient in STE buffer, laid on 0.5 ml of 80% sucrose cushion in ultracentrifuge tubes. After that, tubes were centrifuged at 29,000 rpm for 2 hours in a Thermo TH629 rotor. After centrifugations, fractions (1.250 ml) were carefully collected from top to bottom of the ultracentrifuge tubes. Then, trichloroacetic acid (TCA) was added to each collected fraction for protein concentration, and the pellet was suspended in sample buffer with 4% beta-mercaptoethanol. The samples were heated at 95°C, loaded in 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and analyzed by Western blotting.This protocol was based on a previous paper by Perez-Victoria , 46. HEp


Globally, the burden of papilloma virus-associated cancers is high. About 5% of all cancers worldwide are caused by the human papillomavirus (HPV). Photodynamic therapy (PDT) is considered as a useful therapeutic option to treat cancers, particularly those near the tissue surface, since it is typically well tolerated and less invasive with a lower risk of severe complications as compared to conventional treatment strategies. PDT requires the combination of a photosensitizer, light of a specific wavelength, and tissue oxygen. In the present study, we examined the effectiveness of PDT together with a curcumin (liposome)-based photosensitizer in three papilloma virus-associated cell lines. PDT with curcumin liposomes could inhibit proliferation, cell migration, and colony formation of the tested tumor cells. The results suggest that curcumin-encapsulated liposomes in conjunction with PDT could be a useful tool for the treatment of papilloma virus-associated tumors.2 (100 mA) corresponding to 1, 3 and 5 J·cm−2. After 24 h, the biological outcome of the treatment was assessed with the MTT -2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0–100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m TM, Signpath Pharma; Quakertown, PA, USA) has shown chemoprevention activity without any relevant toxicity in phase I clinical trials [1O2), which are highly reactive and toxic to cells, as well as ROS, leading to tumor destruction. Despite its numerous therapeutic effects, the clinical benefits of curcumin are limited due to poor solubility, rapid metabolism, and systemic elimination of the native compound [Papillomaviruses belong to a group of tumor viruses associated with multiple cancers including cervical, anogenital, and a major subset of head and neck squamous cell carcinomas (HNSCC), which constitute about 4.5% of all solid tumors . Cervical trials . An orall trials . Curcumil trials ,21. Curcl trials . It has l trials . As a recompound . These lcompound ,26,27. Acompound ,29. Lipocompound . The biocompound . PDT hascompound ,32. The compound ,34,35. IThe stability, safety, and efficacy of drug delivery systems depend on various physical attributes such as particle size and the polydispersive index. The mean size distribution of liposomes was assessed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS . Using this method, the particle size (hydrodynamic diameter), zeta potential, and polydispersity index (PDI) can be determined. The data were expressed as the mean ± standard deviation derived from measurements of three independent experiments .The dynamic light scattering analysis showed a PDI >0.2 suggesting the presence of a polydispersed liposome formulation . The cur−2) using a prototype light-emitting diode (LED) device with blue light (wavelength λ = 457 nm). Initial incubation of all cells for 2 h did not show any significant difference in cellular viability (data not shown), indicating the need for increasing the incubation time. Treatment of all cell lines with free curcumin without irradiation showed a dose-dependent cellular toxicity referred to as dark toxicity (50) value for each light dose was calculated by nonlinear curve fitting. A gradual reduction in half maximal inhibitory concentration of liposomes was noticed with increasing light fluence. The maximal phototoxicity was observed at a maximum radiation fluence of 5 J·cm−2. Since major effects were already seen at 3 J·cm−2, this light fluence was used for subsequent experiments. At the light fluence of 3 J·cm−2, the IC50 values of curcumin liposomes for HeLa, UD-SCC-2, and VX2 cells were 9.52 µmol/L, 7.88 µmol/L, and 20.70 µmol/L, respectively. The observed PDT effect after illumination was curcumin dose-dependent and highly significant (p < 0.001). These results are in accordance with previous reports pointing out a role of curcumin in the treatment of diverse maladies such as inflammatory diseases and cancer [MTT assays were performed to evaluate the dark toxicity (no light exposure) and phototoxicity of curcumin liposomes and free curcumin in HeLa, UD-SCC-2, and VX2 cells 24 h after exposure. This was carried out by incubation of cells with different concentrations (0–100 µmol/L) of curcurmin loaded liposomes and free curcumin dissolved in dimethyl sulfoxide (DMSO) for 4 h followed by subsequent exposure to different light fluences /PI (propidium iodide) assay was deployed to evaluate the rate of apoptosis in HeLa, UD-SCC-2, and VX2 cells after deploying various treatment modalities (mentioned in methods). Flow cytometry analysis of HeLa cells A showed A live/dead assay was performed to differentiate between live and dead cells on the basis of cell membrane integrity . HeLa FA, UD-SCCThe effects of various treatment strategies on HeLa, UD-SCC-2, and VX2 cell colony formation were evaluated by a colony formation assay . Cells wt = 24 h in HeLa  and 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) were provided by Lipoid GmbH . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich Chemie GmbH . Dimethyl sulfoxide (DMSO) was obtained from Carl Roth GmbH & Co. . Organic solvents including methanol (MeOH), ethanol (EtOH), and chloroform (CHCl3) were purchased from VWR International . Phosphate-buffered saline (PBS) pH 7.4 (with and without Ca2+/Mg2+) was freshly prepared, sterilized, and filtered using 0.2 µm polyethersulfone filters for all experiments.Curcumin was purchased from Sigma Aldrich . 1,2-Distearoyl-v/v) and curcumin (dissolved in methanol) in a round-bottom flask. Curcumin was added to the lipid mixture in a ratio of 1:30. The solvents were evaporated to obtain a thin film using a Laborota 4000 rotary evaporator  equipped with a vacuum pump . Then, 20 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution (pH 7.4) was used to rehydrate the film followed by sonication in a bath-type sonicator  at 56 °C to obtain liposomal dispersion. The liposomes were then extruded using polycarbonate membrane filters  through 200 nm using an Avanti mini-extruder® . The extruded liposomes were stored at 4 °C until further use. The hydrodynamic diameter and zeta potential of liposomes were measured by dynamic light scattering (DLS) and laser Doppler velocimetry (LDV), respectively, using a Zetasizer Nano ZS  equipped with a 10 mW HeNe laser at λ = 633 nm with scattered light detection at an angle of 173°. Prior to the measurements, liposomes were diluted (1:100) with 20 mmol/L HEPES buffer (pH = 7.4) and placed in a clear disposable folded capillary cell  for determining particle size and zeta potential. The curcumin concentrations were measured as previously described by Duse et al. [Curcumin liposomes were generated as described previously by Duse et al.  and are e et al. . The datl-glutamine , 50 μg/mL gentamicin , 100 U/mL penicillin/streptomycin , and 50 μg/mL amphotericin B . Fresh VX2 cells were derived from a VX2 carcinoma of a New Zeeland White (NZW) rabbit. The generation of VX2 tumors in NZW rabbits was approved by the regional board Giessen, Germany (V54-19c20-15 h01 MR 20/26 Nr. 83/2015) according to the German Animal Protection Law. Transiently growing VX2 cells (max. 150 passages) were then cultured in DMEM/Ham’s F-12 media containing 2 mmol/L l-glutamine  supplemented with 10% FBS, 50 μg/mL gentamicin , 100 U/mL penicillin/streptomycin , and 50 μg/mL amphotericin B . All three cell lines were cultured at 37 °C, 5% CO2 in a humidified incubator. Cells were grown as a monolayer until reaching approximately 80% confluency.The cervical cancer-derived HeLa and the HNSCC-derived UD-SCC-2  cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS , 2 mmol/L 2 irradiance at a current of 100 mA and a wavelength of 457 nm  supplied to the cells was equal to irradiance (W/cm2) multiplied by the irradiation time (s). For all PDT treatments, HeLa, UD-SCC-2, and VX2 cells were incubated with curcumin liposomes for 4 h at 37 °C, 5% CO2. Subsequently, cells were washed with PBS followed by addition of fresh media. Cells were then exposed to different light fluences and incubation was continued for 24 h. The cells were irradiated by light with a wavelength of 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3, and 5 J·cm−2, respectively, of the total light energy delivered.A low-power LED (light-emitting diode) device  was specifically designed to fit multiwell plates. The device was equipped with two different LEDs of 457 nm (blue) and 652 nm (red) wavelengths. It was supplied with the function to control settings for wavelength, current (mA) and irradiation time (s) as per the energy requirement. The device was able to deliver 220.2 W/m4 (HeLa and VX2) or ~6 × 104 (UD-SCC-2) cells per well (0.35 cm2) in a 96-well transparent microtiter plate . Twenty-four hours after seeding, cells were incubated with curcumin loaded liposomes or free curcumin (dissolved in DMSO) at different concentrations ranging from 0–100 µmol/L for 4 h. Afterwards, cells were washed twice with PBS followed by addition of fresh medium. The cells were irradiated with light fluences of 1, 3, and 5 J·cm−2. The irradiated plates were incubated for 24 h in standard conditions . Nonirradiated plates (dark) were used as a control. After 24 h, MTT solution  was added to the cells, and incubation was continued for another 4 h. Subsequently, the resulting formazan crystals were dissolved with DMSO, and the absorbance (Ab) was measured at 570 nm using the FLUOStar Optima plate reader . Cell viability was calculated using the following formula: sample represents the absorbance of cells treated with curcumin liposomes or curcumin alone, and Abcontrol refers to the absorbance of cells without any treatment. Abblank is the absorbance of wells containing media only.The MTT assay was deployed to assess the viability of cells treated with curcumin liposomes alone, PDT alone, or a combination of curcumin liposomes with PDT. Cells were seeded at ~1 × 105 cells per well. Upon reaching a confluency of 70–80%, cells were exposed to different treatments . Cells without any treatment were used as a control. Twenty-four hours after treatment, cells (1 × 105) were harvested, washed in ice-cold PBS, and resuspended in 100 μL of (1×) binding buffer , 2.5 mmol/L CaCl2). The cells were incubated with 10 μL of fluorescein-conjugated Annexin V reagent for 15 min at room temperature in the dark. Thereafter, 400 μL of (1×) binding buffer containing 5 µg/mL PI, which stains late apoptotic and necrotic cells, was added to each tube and placed on ice. The sample measurements were performed using a BD LSR II flow cytometer , and data analysis was performed with the FlowJo 10.6.2 software . Early apoptotic, late apoptotic, and necrotic cells were estimated as the percentage of the total number of cells using the BD FACSDivaTM software 7.0 . Curcumin liposomes were incubated for 4 h, and light fluence was set to 3 J·cm−2 .Flow cytometry was performed to evaluate the underlying mechanisms of cell death using the Annexin V-FITC/PI kit . Briefly HeLa, UD-SCC-2, and VX2 cells were seeded in 6 well plates overnight at a density of 5–10 × 102+/Mg2+) and stained with a solution containing 2 µmol/L SYTO9  and 4 µmol/L PI . The cells were incubated at 37 °C, 5% CO2 for 30 min, washed in PBS, and analyzed under a microscope . Curcumin liposomes were incubated for 4 h and light fluence was set to 3 J·cm−2 .The live/dead staining assay was performed on HeLa, UD-SCC-2, and VX2 cells to evaluate the toxicity of the different treatments . The cells were incubated with curcumin liposomes for 4 h followed by irradiation. After 24 h of treatment, cells were washed with PBS  and subsequently fixed with prechilled methanol for 10 min followed by another wash in PBS. Staining with 0.1% crystal violet dye was done to detect colony formation. Colonies with at least 50 cells were considered and counted. Curcumin liposomes were incubated for 4 h, and light fluence was set to 3 J·cm−2 .A colony formation assay was performed to evaluate the ability of cells to proliferate after being exposed to the different treatment modalities . HeLa, U5 cells/well and cultured until cells reached 90% confluency. After cells were subjected to different treatments , a linear scratch was made using a 10 µL sterile pipette tip followed by washing of cells with PBS and addition of fresh medium. The images were captured at t = 0 h to record the initial area of the scratch and at t = 24 h to evaluate effects of different treatments on the migratory ability of cells toward the scratch area. The images were photographed with the CKX53 microscope system . ImageJ analysis software [t= 0 h − Areat= 24 h)/(Areat= 0 h)) × 100) [−2 .The scratch (wound closure) assay was deployed to analyze the migratory ability of cells after exposure to various treatment procedures . HeLa, Usoftware  (MRI_wou) × 100) . Curcumi50 values obtained from the MTT assay. One-way ANOVA with post hoc test (Dunnett’s multiple comparison against control) was performed to identify statistically significant differences using the GraphPad Prism 5 software . A p-value <0.05 was considered as statistically significant.All experimental measurements were performed in triplicate, and the values were presented as the mean ± standard deviation. Nonlinear curve fitting functions were used to calculate the ICOn the basis of the results from this study, it can be concluded that liposomal encapsulation allows for efficient delivery of curcumin to target cells. Curcumin liposomes were capable of generating a PDT-triggered response in three papilloma virus-associated tumor cell lines, leading to major cell death. It also became evident that phototoxic effects of curcumin liposomes in combination with PDT vary from one cell line to the other. This study revealed that papillomavirus-associated tumor cell lines treated with a combination of curcumin and PDT showed enhanced cell death, inhibition of cell growth, and reduction in colony formation and cell migration. These experimental findings point to PDT in combination with curcumin-loaded liposomes as a potential useful tool for the treatment of papilloma virus-associated tumors. Prospective in vivo studies are now required to evaluate the therapeutic benefit of curcumin-loaded liposomes in combination with PDT in the treatment of solid tumors such as HNSCC and cervical cancer.