Streptococcus pyogenes and an average of 94–52% inhibition for Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive S. aureus (MSSA) and multi-drug resistant Acinetobacter baumannii (MRAB) isolates, respectively. Klebsiella pneumoniae and Enterobacter sp. isolates were however, highly resistant to 30 mM GSH. With respect to biofilm viability, all species exhibited a >50% decrease in viability with 30 mM GSH, with confocal imaging showing considerable change in the biofilm architecture of MRAB isolates. The mechanism of GSH-mediated biofilm disruption is possibly due to a concentration-dependent increase in GSH acidity that triggers cleaving of the matrix components. Enzymatic treatment of MRAB revealed that eDNA and polysaccharides are essential for biofilm stability and eDNA removal enhanced amikacin efficiency. Combination of GSH, amikacin and DNase-I showed the greatest reduction in MRAB biofilm viability. Additionally, GSH alone and in combination with amikacin fostered human fibroblast cell (HFF-1) growth and confluence while inhibiting MRAB adhesion and colonization.Bacterial antibiotic resistance has increased in recent decades, raising concerns in hospital and community settings. Novel, innovative strategies are needed to eradicate bacteria, particularly within biofilms, and diminish the likelihood of recurrence. In this study, we investigated whether glutathione (GSH) can act as a biofilm disruptor, and enhance antibiotic effectiveness against various bacterial pathogens. Biological levels (10 mM) of GSH did not have a significant effect in inhibiting growth or disrupting the biofilm in four out of six species tested. However, exposure to 30 mM GSH showed >50% decrease in growth for all bacterial species, with almost 100% inhibition of  The spread of antibiotic resistance in the hospital environment and in community settings particularly where biofilms are involved, has spurred development of new strategies to design innovative therapeutics to curtail the spread of resistance. In 2016, a review commissioned by the UK government concluded that approximately 700,000 people die each year around the globe from antibiotic-resistant infections . In AustPseudomonas aeruginosa virulence factor pyocyanin, and facilitating the growth of human lung epithelial cells  pathogens, prominent biofilm forming bacteria include S. aureus and A. baumannii. A. baumannii, a Gram-negative emerging pathogen, has garnered attention in recent years due to its inherent multidrug resistant (MDR) profile and pathogenicity, responsible for causing nosocomial infections in hospitalized patients, skin and soft tissue infections especially predominate in combat-associated wounds, morbidity, and mortality in weakened and critically ill patients  and methicillin sensitive S. aureus (MSSA), Enterobacter sp. (E. cloacae and E. aerogenes) and K. pneumoniae as well as the non-ESKAPE pathogens Streptococcus pyogenes and Escherichia coli, all clinical isolates sourced from Sydney hospital culture collections. Most of the bacterial isolates used in this study were strongly susceptible to ciprofloxacin, whereas the multi drug resistant A. baumannii (MRAB) isolates were sensitive only to amikacin. The study was also expanded in the case of A. baumannii to investigate the effect of enzymes on the efficiency of GSH plus amikacin (a triple combination therapy), in disrupting MRAB biofilms.In this study, we aimed to identify whether GSH plays a role in biofilm disruption in non-pyocyanin-expressing pathogenic species. These experiments included testing the effect of a combination treatment (CT) comprising GSH and an antibiotic of choice, on clinical strains. Strains included four bacterial species of the ESKAPE group of pathogens: S. aureus (MRSA and MSSA), S. pyogenes, A. baumannii (MRAB), Enterobacter sp., K. pneumoniae and E. coli. Strains were obtained from Royal Prince Alfred Hospital, Concord Hospital and the High Risk Foot Service clinic at Liverpool Hospital, all located in Sydney, Australia. The Tissue Act 2 did not require the study to be reviewed or approved because: all species/strains were de-identified by the hospitals concerned prior to being gifted to us, all species/strains were from their historical culture collections, and they were not collected from patients as part of this study. S. aureus MRSA and MSSA (=10 μg/ml); S. pyogenes (4 μg/ml); Enterobacter sp. (=0.5 μg/ml); E. coli (=0.5 μg/ml); K. pneumoniae (=10 μg/ml)], and the amikacin MIC was (4 μg/ml) for MRAB.Species used in this study were 2O2) were all obtained from Sigma-Aldrich . Tryptone Soy Broth (TSB) was obtained from Oxoid , Enzymes: DNase-I was obtained from Invitrogen , α-Amylase from MP Biomedicals  and Proteinase K from Sigma-Aldrich . Dulbecco’s Modified Eagle Medium (DMEM) and Fetal bovine serum (FBS) from Sigma-Aldrich .Glutathione, Glutathione disulfide (GSSG), antibiotics , resazurin dye, crystal violet solution, Phosphate buffered saline (1 × PBS) and Hydrogen peroxide (HGlutathione powder was weighed and directly dissolved into TSB (pH 7.18) and 1 × PBS  solution at room temperature under sterile conditions (bio-safety cabinet) to give 10, 20, and 30 mM of GSH. The change in pH of TSB and 1 × PBS at different GSH concentrations was determined using a pH meter  calibrated with pH standards of 4.0 and 7.0 and the pH values of TSB and 1 × PBS as a function of GSH concentration reported in g for 5 min at 10°C, followed by removal of the supernatant and resuspension of the bacterial pellet in TSB. The effect of GSH plus antibiotic on planktonically-grown isolates was determined by taking 250 μL (OD600 = 0.1 ± 0.02) of bacterial cell suspension into the wells of 96-well plates (Corning Corp. United States) and incubating for up to 48 h, at 37°C and 150 rpm. The bacterial growth media contained one of: 0, 10, 20, and 30 mM GSH, 0.25–10 μg/ml ciprofloxacin or 4–16 μg/ml amikacin (only for A. baumannii). Bacterial growth was measured at 48 h post-treatment by recording absorbance at OD600nm using a plate reader (Tecan infinite M1000 pro). The final increase in bacterial growth at 48 h was measured by subtracting the absorbance at 48 h from the absorbance at 0 h. Controls were untreated, and showing 100% growth, while tests measured percentage decrease in bacterial growth with respect to this control.All clinical isolates listed in A. baumannii  and incubated at 37°C for 60 min at 150 rpm. After 60 min, the wells were gently washed once with 1 × PBS to remove any loosely adhered bacteria. 200 μL of TSB was then added, followed by further incubation at 37°C for 48 h and 150 rpm to initiate biofilm growth. In amikacin-treated groups, biofilms were grown in the presence of 4–18 μg/ml amikacin dissolved in TSB. After 48 h of incubation, the biofilms were washed once with 1 × PBS, followed by addition of 200 μL of 1 × PBS and 15 μL of a 0.05% w/v resazurin solution. Plates were then incubated for a further 24 h at 37°C and 150 rpm. Biofilm fluorescence intensity was then determined at Ex544nm and Em590nm . Amikacin-treated groups were compared for percentage decrease in biofilm viability to 1 × PBS-treated control wells showing 100% bacterial cell viability. The percentage increase in bacterial growth at 48 h was determined as mentioned above.All 600 = 0.5 ± 0.05) was added into the wells of 96-well plates (Corning Corp. United States). Plates were incubated for 48 h, at 37°C and 150 rpm. After 48 h, the biofilm was washed once with 1 × PBS followed by treatment  as follows: Control (1 × PBS), 0.5–30 μg/mL ciprofloxacin – depending on MIC of bacterial strain, or 10 and 30 mM GSH; or a combination of ciprofloxacin + GSH. MRAB biofilms were treated with 4 μg/mL, 12 μg/mL, MBC (15–16 μg/mL) and 20 μg/mL amikacin, 10 and 20 μg/mL ciprofloxacin, 10 and 20 μg/mL Augmentin and 10 and 20 μg/mL tobramycin individually, or in combination with GSH. All antibiotic and GSH treatment solutions were prepared in 1 × PBS (pH 7.4).Bacterial isolates were grown as described above. To initiate biofilm growth, planktonic bacteria were re-suspended in TSB and 250 μL of bacterial cell suspension . Test biofilms were compared for percentage decrease in biofilm viability to 1 × PBS-treated control wells showing 100% bacterial cell viability.After 24 h, treated biofilms were washed once with 1 × PBS followed by addition of 200 μL of 1 × PBS and 15 μL of a 0.05% w/v resazurin solution. Plates were then incubated for a further 24 h at 37°C and 150 rpm. After incubation, the fluorescence intensity of the biofilm was determined at ExA. baumannii biofilms were grown for 48 h in 96-well plates as described above. After 48 h, biofilms were washed once with 1 × PBS followed by incubation for 24 h, at 37°C, 150 rpm with: 4, 12, and 20 μg/mL amikacin or 10, 15, and 30 mM GSH or a combination of 30 mM GSH + 4 μg/mL amikacin whereas, controls were treated with 1 × PBS. All test solutions were prepared in 1 × PBS (pH 7.4). After 24 h, treated biofilms were washed once with 1 × PBS. The biofilm biomass attached to the wells was then stained with 200 μL 0.05% (w/v) CV and incubated a further 1 h at 37°C, 150 rpm. After incubation, cells were washed three times with 1 × PBS to remove excess CV stain. The pre-stained biofilm was then allowed to dry for 15 min at 37°C, after which it was dissolved using 80% v/v ethanol and transferred into new 96-well plate for biomass quantification at OD550nm using a Tecan plate reader (Infinite M1000 pro). Controls were treated with 1 × PBS and showed 100% growth, while tests measured the percentage decrease in biofilm biomass with respect to this control.Multi-drug resistant A. baumannii biofilms were grown for 48 h in 96-well plates as described above. After 48 h, biofilms were washed once with 1 × PBS followed by treatment with different enzyme solutions prepared in 1 × PBS, pH 7.4, including: 5, 10, 20, and 40U DNase-I, 100, 200, 500 and 1000 μg/ml amylase and 50, 100, 200 and 500 μg/ml Proteinase K, for 24 h, at 37°C and 150 rpm, the control was treated with 1 × PBS alone. After 24 h, treated biofilms supernatant washed once with 1 × PBS. The biofilm biomass attached to the wells was then stained with 0.05% (w/v) CV and biofilm biomass quantified using the above protocol. Control group normalized as 100% biofilm biomass and enzyme-treated groups were compared with the control to measure percentage decrease in biomass.Multi-drug resistant 600 = 0.5 ± 0.05) in TSB was added to microscope glass slides and incubated at 37°C in a static incubator for 48 h. After 48 h, biofilms were washed once with 1 × PBS and treated using one of the following: 30 mM GSH or 4 μg/mL amikacin individually or a combination of 30 mM GSH + 4 μg/mL amikacin. Enzymatic treatment included 40U DNase-I, 1000 μg/ml Amylase, 500 μg/ml Proteinase K and a combination of 40U DNase-I + 4 μg/mL amikacin or 40U DNase-I + 30 mM GSH. Finally, MRAB-3 biofilms were also subjected to a three-part combination treatment: 30 mM GSH + 40U DNase-I + 4 μg/mL amikacin. For control, MRAB-3 biofilms were treated with 1 × PBS. All treated biofilms were then incubated for 24 h at 37°C in a static incubator. After 24 h incubation, control and all treated biofilms were washed three times with 1 × PBS to remove any planktonic/loosely bound bacterial cells. Biofilms were then stained with a live/dead stain  for 30 min in the dark and cells were then visualized by CLSM  with Ex473 and 559nm and Em500 and 637nm, for Syto-9 (green-live) and propidium iodide (red-dead) staining, respectively. ImageJ software was used to generate images and quantify percentage of live and dead biofilm cells for biovolume.To initiate biofilm growth, 500 μL MRAB-3 (ODA Circular Dichroism (CD) Spectropolarimeter  was used to investigate DNA-GSH reactions in a 1-mm path length quartz cuvette. dsDNA  and GSH stock solutions were prepared in sterile MilliQ water. To study the interaction, 200 ng/μl dsDNA incubated for 24 h at 37°C, 100 rpm, in either absence or presence of 1 mM GSH at intrinsic pH or 1 mM GSH at neutral pH (7.2). 300 μl aliquots pipetted into cuvettes and scanned by CD at 200–320 nm wavelength in a static condition at 25°C.A total of 200 ng/ μl dsDNA was incubated for 24 h at 37°C, 100 rpm, in either presence or absence of 1 mM GSH at intrinsic pH and in 1 mM GSH at neutral pH (7.2). After 24 h, the dsDNA concentration was quantified using a fluorescent dye assay , and monitored with a Qubit 3.0 Fluorometer , yielding a dsDNA concentration in μg/mL.2O2 production by 10 and 30 mM GSH at its intrinsic pH  and at pH 7 ± 0.2 (buffered using NaOH) in 1 × PBS was analyzed using a Hydrogen Peroxide Assay Kit – (Fluorometric-Near Infrared)  complemented with a fluorescence plate reader (Tecan infinite M1000 pro). The protocol used to measure H2O2 in this study was as published by the manufacturer (Abcam – Hydrogen Peroxide Assay Kit).Quantification of HA. baumannii biofilms were grown for 48 h in 96-well plates as described above. After 48 h, biofilms were washed once with 1 × PBS followed by treatment with different concentrations of 0, 5, 10, 25, and 50 μM H2O2  H2O2 in 1 × PBS) for 24 h, at 37°C and shaking at 150 rpm. After 24 h, treated biofilm supernatant was washed once with 1 × PBS followed by addition of 200 μL of 1 × PBS plus 15 μL resazurin 0.05% w/v solution, and incubated for a further 24 h, at 37°C, 150 rpm. After 24 h, the fluorescent intensity of the biofilm was determined at Ex544nm and Em590nm (Tecan infinite M1000 pro). The control comprised a 1 × PBS treated biofilm showing 100% viability and percentage decrease in biofilm viability in the treated samples was calculated with respect to this control.Multi-drug resistant A. baumannii biofilms were grown for 48 h in 96-well plates as described above. After 48 h, biofilms were washed once with 1 × PBS followed by treatment with 30 mM GSH (pH 7 ± 0.2) and 30 mM GSH (pH 7 ± 0.2) + 4 μg/mL amikacin and also with 30 mM GSSG and 30 mM GSSG + 4 μg/mL amikacin for 24 h, at 37°C and 150 rpm. After 24 h, treated biofilms were analyzed for viability using the resazurin assay as described above.Multi-drug resistant Biofilms were treated with either GSH, antibiotics, or a combination of both, for 24 h as described above. Biofilms were then washed once with 1 × PBS and thoroughly homogenized by pipetting, with 200 μL of 1 × PBS. To establish a CFU count, 100 μl of homogenized suspension from each well was added to 900 μl of 1 × PBS . The biofilm suspension was then serially diluted in 1 × PBS and 100 ml aliquots were spread on TSB plates and incubated for 24 h at 37°C. After 24 h the colonies on the plates were counted and numbers expressed as CFU/ml.2 atmosphere and harvested at 90% confluence using 0.12% trypsin-EDTA. Cells were collected by first quenching Trypsin 1:1 (v/v) with supplemented media and transferred to 15 ml Falcon tubes, followed by centrifugation . The supernatant was aspirated and the cell pellet was suspended in supplemented DMEM media for further experiments.The HFF-1 cell line (ATCC-SCRC-1041) was cultured in DMEM, supplemented with 12% (v/v) FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. HFF-1 cells maintained in a T-25 cell culture flask  at 37°C in a 5% (v/v) CO5 cells/mL in six-well plates (Corning) and allowed to incubate for 72 h at 37°C in a 5% (v/v) CO2 atmosphere to a confluence of 90%. 100 μL of MRAB -3 (OD600nm = 0.1 ± 0.02) suspended in 1 × PBS was then introduced into the confluent HFF-1-containing media and the plates allowed to incubate for a further 24 h. Where indicated, HFF-1-containing media were also incubated with a different treatment of either 10 or 30 mM GSH, or 4 μg/mL amikacin alone, or a combination of 10 mM GSH + 4 μg/mL amikacin, either in the presence or absence of bacteria. After 24 h, the HFF-1 cells in the well plates were imaged using phase contrast microscopy  for growth appearance, adherence and confluence.To study the effect of MRAB-3 on pre-confluence of HFF-1 cells, HFF-1 cells were cultured and harvested as above. After harvesting, cells were plated to a density of 6 ± 0.5 × 10600nm = 0.1 ± 0.02) was re-suspended in TSB and added to six-well plates (Corning Corp. United States) for incubation  to initiate bacterial adhesion and biofilm growth. Where indicated, biofilm growth was also initiated in both presence and absence of 5, 10, and 30 mM GSH. After 24 h, the biofilm was washed once with 1 × PBS and imaged using phase contrast microscopy , for bacterial adhesion and colonization. Bacterial cells adherent to the surface of six-well plates were enumerated using ImageJ, where number of maxima present were quantified as explained on the ImageJ process menu, on its website3. The maxima were enumerated as this provided a more accurate presentation of individual cells clumped together, in comparison to simple thresholding.MRAB-3 was cultured and harvested as above and after harvesting at a bacterial density  at 30 mM GSH. Gram-negative species: For A. baumannii isolates, growth varied between 41 and 54%, Enterobacter sp. (77–91%), E. coli (4–8%) and K. pneumoniae (52–100%). At lower concentrations (10 and 20 mM GSH), only MRSA, S. pyogenes and E. coli showed growth ≤50%. These decreases in bacterial growth specifically at 30 mM GSH were statistically significant (P < 0.05) when compared to the control and 10 mM GSH-treated condition.P < 0.05) decreases in biofilm viability at all concentrations of amikacin, except for MRAB-4 (catheter isolate), which showed a significant decrease starting at 10 μg/ml amikacin  showed significant decreases in biofilm viability  in comparison to untreated controls. An increase in ciprofloxacin concentration: to 30 μg/ml (S. aureus) and 12 μg/ml (S. pyogenes) showed further decreases in biofilm viability for few isolates: an average of 25% for strains MRSA-1 MRSA-2 and MSSA-3, and 6% for S. pyogenes (SP-1). Treatment with GSH at biological concentration (10 mM) did not show any significant decrease in biofilm viability in any isolates of MRSA and MSSA. However, S. pyogenes (SP-1) (56%) and SP-2 (68%) were more sensitive to GSH and showed a significant decrease in biofilm viability (P < 0.05). At a GSH, concentration of 30 mM an increase in biofilm disruption amongst all Gram-positive isolates was observed. For MRSA and MSSA, biofilm viability at 30 mM GSH ranged between 1–33% and 1–20%, respectively, and for S. pyogenes isolates, between 4 and 22%. Combination treatment comprising 30 mM GSH and 10 μg/ml ciprofloxacin showed further significant (P < 0.05) decreases in biofilm viability in all MRSA strains (0–22%). For MSSA (1–10%), there was a significant decrease only for strains MSSA-2 and MSSA-3, and significant decreases in for all S. pyogenes strains (0–12%).E. coli were found to have the lowest ciprofloxacin MIC (0.5 μg/ml) while for K. pneumoniae, the ciprofloxacin MIC was 10 μg/ml. At their respective MIC, biofilm viability of all isolates of Enterobacter sp. (15–64%), E. coli (30–58%), and K. pneumoniae (29–55%) showed significant decrease in viability compared to the control. However, further increases in ciprofloxacin concentration (to 2 μg/ml) did not result in significant decreases in viability for Enterobacter sp., and E. coli. On the other hand, all K. pneumoniae strains showed a large decrease in biofilm viability (21–25%) at 30 μg/ml ciprofloxacin, with statistical significance (P < 0.05) for K. pneumoniae (KP-3) neck wound isolate. In the case of amikacin sensitive A. baumannii isolates, treatment at the MIC (4 μg/ml) of amikacin showed a significant (P < 0.05) decrease in biofilm viability (36–67%). A further significant (P < 0.05) decrease in biofilm viability (8–42%) observed when amikacin concentration increased to 20 μg/ml.Amongst the Gram-negative bacterial isolates, Enterobacter sp., and P < 0.05) decrease in biofilm viability for all clinical isolates including A. baumannii (16–38%), Enterobacter sp. (0–20%), E. coli (1–12%) and K. pneumoniae (9–17%). Combined treatment with 30 mM GSH the + MIC concentration of antibiotics showed significant decreases (P < 0.05) in biofilm viability for most of the isolates: A. baumannii (4–10%), Enterobacter sp. (0–9%), E. coli (1–6%) and K. pneumoniae (2–10%) when compared to treatment with MIC concentration antibiotic or 30 mM GSH alone.A biological GSH concentration (10 mM) did not show any significant decrease in biofilm viability in most of the Gram-negative isolates. This is in contrast to use of the highest GSH concentration (30 mM), which showed a drastic . Interestingly, a combination of 30 mM GSH + 4 μg/ml amikacin showed statistically significant decreases in biomass (40–45%) for all isolates in comparison to 4 μg/ml amikacin alone.Crystal violet assays measured biofilm biomass of MRAB isolates and showed a statistically significant decrease in biomass when treated with 4–20 μg/ml amikacin or 30 mM GSH alone or in combination, compared to untreated biofilm . With MIWe also investigated the effect of DNase-I, amylase and Proteinase K on all MRAB biofilm biomass using CV assays . In compThe biofilm architecture of MRAB-3 changed significantly when subjected to different treatments Circular Dichroism peaks clearly indicated that GSH at its intrinsic pH drastically modulates the DNA sugar phosphate backbone (peak 247 nm) whereas GSH at neutral pH did not alter this peak . GSH by P < 0.05) when compare to both control and 30 mM GSH (pH 7) alone treatment.MRAB biofilms subjected to treatment with 30 mM GSSG at intrinsic pH showed statistically significant decreases in biofilm viability (11–14%) when compared to control biofilms. Further significant decreases of 5–6% in MRAB biofilm viability compared to GSSG alone treatment were observed when biofilms were exposed to 30 mM GSSG + 4 μg/ml amikacin .2O2 concentration, as measured using a H2O2 assay kit. Standard H2O2 data were used to determine H2O2 production by GSH at intrinsic pH i.e., 10 mM = pH 5.51 and 30 mM = pH 3.2, and at buffered neutral pH 7. In general, at intrinsic pH GSH produced a significantly higher concentration H2O2 (higher fluorescent intensity) than at its corresponding neutral pH (P < 0.05). 30 mM GSH (intrinsic pH) showed 3 and 4 fold increases in H2O2 production in comparison to 30 mM GSH (pH 7) and 10 mM GSH (at intrinsic pH), respectively,  showed a statistically significant decrease in biofilm viability, to 53 and 58%, respectively, at 50 μM H2O2 (P < 0.05). However, for the catheter isolates (MRAB-2 and MRAB-4), biofilm viability was similar to control at all H2O2 concentrations (Multi-drug resistant trations .10 of 9-10.2. The CFU/ml of amikacin (4–80 μg/ml) and GSH (30 mM)-treated biofilms were log10 5.1-8.2 and 5.1-6.5, respectively. Double combination treatments comprising: DNase-I (40U) + amikacin (4 μg/ml), GSH (30 mM) + amikacin (4–80 μg/ml) and GSH (30 mM) + DNase-I (40U) resulted in decreased CFUs/ml of log10 4.9–6.2, 3.7–5.3 and 4.6–5.9, respectively. GSH (30 mM) + amikacin (MBC) showed the most effective decrease in CFU/ml (1og10 3.7-4.1). Whereas, triple combination treatment (30 mM GSH + 40U DNase-I + 4 μg/ml amikacin) resulted in a CFU/ml between log10 4.3–4.9. For all treatment conditions  the decreases in CFU/ml were statistically significant when compared to 1 × PBS treated control (P < 0.05). In addition, when treatment with amikacin alone was compared with GSH + amikacin, the reduction in CFU/ml in combination treatment was significant, (P < 0.05) especially at higher amikacin concentrations (Control/untreated biofilms and DNase-I-treated biofilms had a CFU/ml at logtrations .4 bacteria per 0.25 × 0.325 mm2 (area) for the 1 × PBS treated control whereas, in the presence of GSH bacterial adhesion decreased significantly (>5 × 103 bacteria) especially at 10 and 30 mM GSH  has up to 10 mM of GSH whereas the plasma GSH concentration is very low (micromolar range) . In cystus study , which a0 mM GSH . This co density .P > 0.05, for all isolates). Reduction in biofilm viability achieved by treatment with 30 mM GSH alone is either analogous to or improved in comparison to maximum antibiotic concentrations  used in this study. Most remarkably, for almost all bacterial isolates, 30 mM GSH was shown to enhance antibiotic efficiency, indicating the practicality of GSH in treatment of biofilm-associated infections  (E. coli) with antioxidants (GSH and Ascorbic acid), reduces antibiotic-mediated reactive oxygen species production and consequently limits antibiotic lethality.Biofilm disruption by GSH acidity is triggered through cleaving and destabilization of biopolymers such as extracellular DNA (eDNA), polysaccharides and proteins. By using CD and Qubit fluorometry, we confirmed that acidic GSH cleaves dsDNA and destabilizes its double helix structure . A signine (G-C) . Similarne (G-C) . Bacterine (G-C) . Rao et A. baumannii biofilm formation and stability -triggered modulation of bacterial cell ATP synthesis (The efficacy of GSH is enhanced by its intrinsic acidity (pH 3.21 in 1 × PBS and 4.77 in TSB) at 30 mM GSH. Buffering of 30 mM GSH (in TSB and 1 × PBS), with pH to neutral (7) had no impact on bacterial growth (data not shown) and very minimal impact on biofilm viability . On the ynthesis .N-acetylcysteine (NAC), at its intrinsic pH, showed that it destabilizes the biofilm matrix by chelating divalent cations , inhibition of bacterial extracellular polysaccharide production, modulation of bacterial cell physiology and the impact on its metabolism  tested in this study (Our investigation of H results . Previoults (2O2 . In this H+ ions . GSH-medductions . MRAB aris study , indicatis study . An addiis study . Howeveris study .The thiol-based antioxidant NAC has undergone clinical trials for use in respiratory related infections . HoweverIn this study, we demonstrated that GSH acidity plays a key role in biofilm disruption of many MDR bacterial isolates and enhances antibiotic efficacy. With MRAB, biofilm integration is primarily dependent on eDNA and polysaccharides and the cleaving of these biopolymers enhances the antibiotic’s efficiency in killing the bacteria.The datasets generated for this study are available on request to the corresponding author.TD designed and conducted the experiments and wrote the manuscript. DP designed and conducted the experiments. AM and JF conducted the experiments. GW, FK, TG, and JM designed the experiments and wrote the manuscript.GW, FK, and TG are employed by Whiteley Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phospho-proteome profiling array displayed that GDF11 significantly increased the phosphorylation of 13 serine/threonine kinases (p < 0.01), including p-p38, p-ERK and p-Akt, in C17.2 cells, which implied the activation of MAPK pathway. Western blot validated that the results of phospho-proteome profiling array were reliable. Based on functional analysis, we demonstrated that the differentially expressed proteins were mainly involved in signal transduction which was implicated in cellular behavior. Collectively, our findings suggest that, for neurogenesis, GDF11 might not be the desired rejuvenation factor, but a potential target for pharmacological blockade.GDF11, a member of TGF-β superfamily, has recently received widespread attention as a novel anti-ageing/rejuvenation factor to reverse age-related dysfunctions in heart and skeletal muscle, and to induce angiogenesis and neurogenesis. However, these positive effects of GDF11 were challenged by several other studies. Furthermore, the mechanism is still not well understood. In the present study, we evaluated the effects of GDF11 on C17.2 neural stem cells. GDF11 induced differentiation and apoptosis, and suppressed migration of C17.2 neural stem cells. In addition, GDF11 slightly increased cell viability after 24 h treatment, showed no effects on proliferation for about 10 days of cultivation, and slightly decreased cumulative population doubling for long-term treatment ( Growth differentiation factor 11 (GDF11), also known as bone morphogenetic protein 11 (BMP11), is a secreted glycoprotein belonging to the transforming growth factor β (TGF-β) superfamily . GDF11 pRecently, In the present study, we focused on the effects of GDF11 on the cellular behavior of C17.2 neural stem cells , the changes in the phospho-proteome and the corresponding signaling pathways. We herein showed that GDF11 promoted differentiation and apoptosis, and suppressed migration of C17.2 cells. In addition, GDF11 stimulated cellular proliferation in a short time (within 24 h), whereas high concentrations of GDF11 inhibited proliferation in a long-term cultivation (∼20 days). Pathway-oriented proteome profiling revealed that GDF11 stimulation significantly activated phosphorylation of 15 proteins, including Smad2/3, Erk1/2, Akt1/2/3, p38, p70S6k, GSK-3α/3β and HSP27, which were mainly involved in MAPK signaling pathway. These data demonstrated the effects of GDF11 on neural stem cell  through the MAPK pathway.C17.2 neural stem cell line was purchased from zqxzbio . Dulbecco’s modified Eagle’s medium , Penicillin-Streptomycin Solution  and Trypsin  were obtained from Hyclone . Fetal bovine serum  and horse serum  were purchased from Biological Industries . Recombinant human/mouse/rat GDF-11/BMP-11  and Phospho-MAPK proteome profiler array kit  were obtained from R&D systems . Dimethyl sulfoxide  were purchased from Sigma-Aldrich . NuPAGE LDS loading buffer  and NuPAGE 4–12% Bis-Tris gel  were obtained from Invitrogen . Phosphatase inhibitor cocktail  and protease inhibitor cocktail  were from MedChem Express . RIPA buffer  was from Pierce . Rabbit anti-Smad2/3 , rabbit anti-p-smad2/Smad3  , rabbit anti-CREB , rabbit anti-p-CREB , rabbit anti-ERK , rabbit anti-p-ERK , rabbit anti-p38 , rabbit anti-p-p38 , mouse anti-nestin , rabbit anti-βIII-tubulin , rabbit anti-GFAP , rabbit anti-β-actin , rabbit anti-GAPDH  and goat anti-rabbit IgG  were obtained from Cell Signal Technology . Enhanced Cell Counting Kit-8  and BCA Protein Assay Kit  were obtained from Beyotime Biotechnology . Enhanced chemiluminescence  was form Pierce .2 culture flasks in complete medium (DMEM supplemented with 10% (v/v) FBS, 5% (v/v) HS, 100 U/ml penicillin and 100 µg/ml streptomycin) at 37 °C, 5% CO2 in a humidified atmosphere. Media were changed every 2–3 days. After reaching 70–80% confluence, C17.2 cells were trypsinized and re-seeded at a density of 4*104 cells/mL in complete medium which was changed to starved medium (DMEM supplemented with 0.5% HS and 1% FBS) one day post seeding. After 6 h of serum starvation, different concentrations of GDF11  were added, respectively.C17.2 cells were cultured on 25-cm4 cells/well in 0.1 mL complete medium. After adherence, complete medium was replaced with starved medium for 6 h and then, various concentrations of GDF11 were introduced when appropriate. GDF11-untreated cells were served as control.C17.2 cells were seeded onto the 24-well plates at a density of 4*10® viability/cytotoxicity kit  for mammalian cells  according to the manufacturer’s instructions under inverted fluorescence microscope . The live cells were stained with calcein AM in green, and the dead cells were stained with ethidium homodimer-1(EthD-1) in red.The cell morphology and viability were examined using LIVE/DEADe . Then, by comparing the absorbance of GDF11-treated and untreated cells, percentage viability was calculated.Cell viability was assessed by CCK-8 assay. Briefly, 10 µL of CCK-8 agent was added to each well 2 h before the termination of the experiment. The optical density (OD) values at 450 nm were determined using SpectraMax M24/mL cells were seeded in 12-well plates in triplicates. When the cell cultured to ∼80% confluence , cells were trypsinized and manually counted using a haemocytometer. Cell population doubling (PD) was calculated using the following formulae: N0 represents the number of cells seeded at the initial passage, N is the final number of cells.For proliferation assay, 1*105 cells were analyzed in each experimental group. The cell populations were distinguished according to their positioning of quadrants: live cells (Annexin V−/PI−), early/primary apoptotic cells (Annexin V+/PI−), late/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells (Annexin V−/PI+).To investigate the apoptosis-inducing effect of GDF11, we identified apoptotic and necrotic cells by Annexin V-FITC and propidium iodide (PI) dual staining using FACScan flow cytometry . Approximately 1*104 cells/well. After reaching ∼80% confluence, a single uniform scratch was made by using a 200 µL pipette tip along the center of each monolayer. The scratch was lightly washed with PBS twice to remove the detached cells, and then starved medium supplemented with various concentrations of GDF11 was added . The scratches were monitored at 0 h, 12 h and 36 h after scratching by taking photos with inverted microscope to measure the wound closure. The wound closures of various treatments at different time points were calculated with Image J software.C17.2 cells were cultured with complete medium in a 48-well plate at a density of 5 × 104 cells per well under standard conditions. Upon reaching 80% confluence, the complete medium was changed to starved medium. After 6 h of serum starvation, plates were treated with either indicated concentrations of GDF11  or vehicle in starved medium for 4 h. Total RNA was extracted from the cultured cells using TRIZOL reagent according to the standard procedure. Total RNA (1 µg) was reverse transcribed in a final volume of 20 µL in a reaction containing random primers, using iScriptTM cDNA Synthesis kit . qRT-PCR was done using the Quantitect SYBR Green PCR kit  with a ABI StepOnePlus Real-time PCR system . Relative expression was calculated using the 2−ΔΔCt method by normalizing with GAPDH housekeeping gene expression and presented as fold changes relative to control. The primers for qRT-PCR were synthesized by Beijing Genomics Institute  and the details of primer sequences are shown in C17.2 cells were cultured on 12-well plates at a density of 4*107 cells/mL. After rocking gently at 2–8 °C for 30 min, the lysates were centrifuged at 14,000 g for 5 min, and the supernatant was collected and detected the protein contents using BCA protein assay. The arrays were blocked by Buffer 5 for 1 h on a rocking platform shaker. Afterwards, the mixture of sample and detection antibody cocktail were introduced and incubated overnight at 2–8 °C on a rocking platform shaker. The following day, the membranes were washed three times, and then were incubated in streptavidin-HRP for 30 min followed by three washes. The protein blots were developed by ECL reagents. Densitometry analysis was measured with the Quantity One software, and the averaged intensity was calculated by subtracting the averaged background signal. The fold change was obtained by comparing GDF11-treated samples with the untreated control : Human phospho-MAPK array kit was used to determine the relative levels of phosphorylation of mitogen-activated protein kinases (MAPKs) and other serine/threonine kinases with or without GDF11 treatment. Briefly, C17.2 cells were rinsed with PBS and solubilized with Lysis Buffer 6 (provided in Human Phospho-MAPK Array Kit) at 1*10C17.2 cells were cultured in six-well dishes in starved medium with or without GDF11 for 24 h. Then, the cells were lysed in RIPA buffer containing 1 × phosphatase inhibitor cocktail and 1 × protease inhibitor cocktail on ice for 30 min, and centrifuged at 14,000 g for 5 min at 4 °C. The supernatants were collected, and the protein concentration was determined by BCA protein assay kit. The samples were mixed with 4 × NuPAGE LDS loading buffer, separated on NuPAGE 4–12% Bis-Tris gels, and subsequently transferred to PVDF membranes by a wet transfer apparatus . Following blocking with superblock at room temperature for 2 h, the membranes were incubated with rabbit anti-β-actin , anti-Smad2/3 , anti-p-Smad2/3 , anti-CREB , anti-p-CREB , anti-ERK , anti-p-ERK , anti-p38 , anti-p-p38 , anti-nestin , anti-βIII-tubulin , anti-GFAP  and anti-GAPDH , respectively, at 4 °C overnight. After washing with PBST (PBS supplemented with 0.1% Tween 20), membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  at room temperature for 2 h, and were visualized by ECL reagents. ImageJ software  was usedTo further understand the functions and features of the identified and quantified proteins, we annotated functions and features of proteins from several different categories, including subcellular localization, domain, Gene Ontology (GO) and pathway.http://www.jci-bioinfo.cn/iLoc-Animal) were used to predict subcellular localization of all identified differentially expressed proteins.WoLF PSORT  and iLoc-Animal (http://www.ebi.ac.uk/interpro/).The domain functional description of the differentially expressed proteins were annotated by InterProScan  based on protein sequence alignment method, and the InterPro domain database was used (http://www.ebi.ac.uk/GOA/), and the differentially expressed proteins were classified by GO annotation based on the three categories (GO term level 1): biological process, cellular component and molecular function. According to GO annotation information of the identified proteins, we summed up the amount of the differentially expressed proteins in each GO term of level 2.GO annotation was derived from the UniProt-GOA database (The protein–protein interaction networks and pathways were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) database.t –tests when appropriate. In addition, Statistical analyses were conducted using SPSS statistics software,version 17.0 , and p < 0.05 was considered statistically significant.The results were presented as the mean ± standard error (SE). Multi-group comparisons were performed by one-way ANOVA followed by Tukey’s post hoc test. Paired analysis of control and treatment was accomplished using two-tailed unpaired or unpaired Student’s When compared with the counts of C17.2 cells initially seeded, both GDF11- and vehicle-treated cells significantly proliferated after 72 h of cultivation . Imagingp < 0.05) cell viability after 24 h treatment, whereas it did not affect the cell viability after 72 h treatment  for a 72 h period, followed by CCK-8 assays. GDF11 slightly increased  and the exposure of C17.2 cells to 100 ng/mL GDF11 resulted in the lowest cumulative population doubling level during the 6 passages of cultivation amongst the five groups, which was approximately 17% lower than control (p < 0.05).As displayed in p > 0.05). These provide a potential molecular basis for the effects of GDF11 on C17.2 cell viability and proliferation.Next, we detected the mRNA expression of cyclin D1 and cyclin D2, the cell cycle-related proteins. GDF11 slightly but not significantly attenuated the expression of cyclin D1 and cyclin D2 in the mRNA levels  cultivation and showed no effect on cell viability from 1st to 4th passage of cultivation (approximately 10 days), whereas high concentrations of GDF11 significantly suppressed cumulative population doubling for a long-term treatment.p < 0.01). By contrast, the GDF11-treated groups showed significant increase in βIII-tubulin  and GFAP (astrocytic biomarker) mRNA expression as compared to the control (p < 0.05). These all indicated the maturation and differentiation of C17.2 neural stem cells. The differences in nestin mRNA expression among the groups of GDF11 treatment were, however, not significant, similar to βIII-tubulin and GFAP. Concomitantly with the mRNA expression, the protein levels of nestin, βIII-tubulin and GFAP confirmed the similar results by western blot . Meanwhile, the amount of necrotic cells showed a slight but significant increase when treated with GDF11.The results of Annexin V-FITC/PI dual staining revealed that GDF11 substantially induced apoptosis of C17.2 cells. As shown in The migration of C17.2 cells was performed by a “scratch wound healing” assay. The wound closure data are shown in p < 0.05). However, no dose-dependent effect of GDF11 on Smad2/3 phosphorylation was observed. Moreover, we investigated the mRNA levels of receptors of TGF-β superfamily, activin type IIB receptor (ActRIIB) and the type I receptors, activin receptor-like kinase 5 (ALK5). The results of qRT-PCR revealed that GDF11 didn’t alter mRNA expression of ActRIIB and ALK  in C17.2 neural stem cells. GDF11 showed no effect on both the mRNA and protein levels of Smad3  and 4A.  and ALK .α/β , p38 α/β (3.21 and 1.73 times increased), Erk1 (1.57), MKK3/6  and p70s6k (1.93 times increased) (p < 0.05). These indicated that GDF11 activated the MAPK/Erk and p38 MAPK pathways but not JNK pathway in C17.2 neural stem cells. Remarkably, many of these differentially expressed proteins are involved in signal transductions of cell survival and apoptosis.In order to further research the signal pathways affected by GDF11, we compared the phosphorylation levels of MAPKs in C17.2 cells treated with vehicle or GDF11 using a phospho-MAPK array kit. The fold changes were calculated from the ratio of intensity of the MAPK array from the GDF11 treated C17.2 cells to the control (untreated cells). Cut-off values were set 1.5-fold for up-regulated expression and 0.67-fold for down-regulated expression of a protein. We observed significant increases in the phosphorylation levels of several proteins in GDF11-treated cells compared with the untreated cells . Overalln = 8), nuclear (n = 7) and mitochondrial (n = 1) proteins.As shown in For an overview of the differentially expressed proteins, GO annotation was carried out to identify the significantly enriched GO functions. According to the analysis, the 15 differentially expressed proteins between GDF11-treated cells and control were mainly clustered into 38 functional groups, including 18 biological processes, 12 cellular components, and eight molecular functions .The biological process category according to GO annotations indicated that all of the 15 differentially expressed proteins were involved in the metabolic process. Other significant function groups included cellular process (13/15), response to stimulus (13/15), signaling (13/15) and localization (12/15), etc .In the category of cellular components, the differentially expressed proteins were mainly involved in the cell (13/15), organelle (14/15), cytoplasm (12/15) and nucleus (13/15), indicating the similar subcellular localization that was obtained from WoLF PSORT . Not onlThe most representative molecular function category was “binding”, which accounted for all the 15 differentially expressed proteins, and most of the differentially expressed proteins were also involved in catalytic activity (11/15), transferase activity (11/15) and kinase activity (11/15). These results also elucidated that the related signal pathways activated by GDF11.To explore the potential mechanisms for GDF11-mediated cell behavior  in C17.2 neural stem cells, we used the KEGG database to determine the protein-protein interaction networks and pathways involved in the up-regulated phosphoproteins. The 15 differentially expressed proteins were mainly mapped to 51 pathways according to the KEGG database, which were mainly associated with environmental information processing , organismal systems , cellular processes  and human diseases  . These aα/β . These results provided a possible resource for future studies of the proteins involved in GDF11-treated C17.2 cells.A major overlapping network was enriched in this analysis . Three cp < 0.05). These western blot results were generally consistent with the results of the phospho-MAPK array.To confirm the results of phospho-MAPK array, three differentially expressed protein candidates  were selected for further validation using western blot. Total protein lysates from C17.2 cells cultured with indicated concentrations of GDF11  were prepared and the phosphorylation levels were determined by their respective phosphorylated antibodies. When compared with control, no detectable changes in total Creb, p38 or Erk protein expression were observed in GDF11-treated C17.2 cells. Nevertheless, GDF11-treatment significantly increased the phosphorylation levels of Creb, p38 and Erk . To the best of our knowledge, we are unaware of any similar published results. Strikingly, similar to our results, it was found that GDF11 slightly increased cell viability after short-term treatment and slightly decreased cell viability after long-term treatment in human umbilical vein endothelial cells  and showed no effects on cellular viability for medium-term cultivation . For long-term cultivation (>4 passages), high concentrations of GDF11 significantly inhibited the proliferation of C17.2 cells  were activated, but not JNK pathway. Similarly, it also reported that GDF11 activated TGF-β/Smad2/3 but suppressed JNK signaling pathways in apolipoprotein E-null mice (Proteins, not genes, are the specific practitioners of cellular life organisms. Although genome and transcriptome analyses are very useful to reveal the mechanism of GDF11 stimulation, proteomic profiles may not be accurately predicted by transcriptome profiling due to several factors, such as post-translational modifications. Therefore, research on proteomics is helpful to provide new information concerning the C17.2 cells response to GDF11 stimulation. Based on phospho-proteome profiling array and bioinformatic analysis  and 6, wull mice . The funull mice . Variousull mice . Althougull mice , it was ull mice . MAPK/Erull mice , howeverull mice . All of Neuronal migration is a complex and key process in physiological and pathological conditions. Increasing the quantity of nerve cells and the migration of neurons to the final position are critical to reverse age-related dysfunction in brain . It shouIn the present study, we found that GDF11 was an important regulator of neural stem cell. In C17.2 neural stem cells, GDF11 showed a positive effect on cell viability after 24 h treatment but displayed a tendency of a negative effect for long-term cultivation. In addition, GDF11 significantly induced differentiation and apoptosis, and suppressed migration of C17.2 neural stem cells. Further analysis of MAPK signaling pathway, which was activated by GDF11, preliminary illustrated the potential mechanism of action by which the cellular behavior was induced. Taken together, our current findings implied that GDF11 might be a potential target for pharmacologic blockade instead of a rejuvenated factor for neural stem cells.10.7717/peerj.5524/supp-1Table S1Click here for additional data file.10.7717/peerj.5524/supp-2Table S2Click here for additional data file.10.7717/peerj.5524/supp-3Table S3Click here for additional data file.10.7717/peerj.5524/supp-4Figure S1Images were obtainedat 50Xmagnification by inverted fluorescence microscope. The live cells were stained with calcein AM in green, and the dead cells were stained with EthD-1 in red.Click here for additional data file.10.7717/peerj.5524/supp-5Figure S2Click here for additional data file.10.7717/peerj.5524/supp-6Data S1Click here for additional data file.10.7717/peerj.5524/supp-7Data S2Click here for additional data file.


Liquid solder laser-assisted vascular welding using biocompatible polymeric scaffolds (ssLAVW) is a novel technique for vascular anastomoses. Although ssLAVW has pronounced advantages over conventional suturing, drawbacks include low welding strength and extensive thermal damage.To determine optimal ssLAVW parameters for maximum welding strength and minimal thermal damage.Substudy 1 compared breaking strength (BS) of aortic strips welded with electrospun poly(ε-caprolactone) (PCL) or poly(lactic-co-glycolic acid) (PLGA) scaffold, 670-nm laser, 50-s single-spot continuous lasing (SSCL), and semi-solid solder /0.5% methylene blue (MB)/3% hydroxypropylmethylcellulose (HPMC)). Substudy 2 compared the semi-solid solder to 48% BSA/0.5% MB/0.38% genipin and 48% BSA/0.5% MB/3% HPMC/0.38% genipin solder. Substudy 3 compared SSCL to single-spot pulsed lasing (SSPL).2 and remained stable up to 7d of hydration. PLGA-ssLAVW obtained higher acute BS (408.6 ± 78.8 N/cm2) but revealed structural defects and a BS of 109.4 ± 42.6 N/cm2 after 14 d of hydration. The addition of HPMC and genipin improved the 14-d BS of PLGA-sLAVW (223.9 ± 19.1 N/cm2). Thermal damage was reduced with SSPL compared with SSCL.PCL-ssLAVW yielded an acute BS of 248.0 ± 54.0 N/cmPCL-ssLAVW yielded lower but more stable welds than PLGA-ssLAVW. The addition of HPMC and genipin to the solder increased the post-hydration BS of PLGA-ssLAVW. SSPL regimen reduced thermal damage. PLGA-ssLAVW using 48% BSA/0.5% MB/3% HPMC/0.38% genipin solder and SSPL constitutes the most optimal welding modality.Surgical patients requiring vascular anastomoses may benefit from the advantages that ssLAVW potentially offers over conventional sutures (gold standard). These include no needle trauma and remnant suture materials in the patient, reduction of foreign body reaction, immediate liquid-tight sealing, and the possibility of a faster and easier procedure for minimally invasive and endoscopic anastomotic techniques. The use of sutures, which currently comprises the gold standard approach, is associated with several drawbacks, including needle trauma, the introduction of exogenous materials (suture threads), induction of foreign body reactions, and leakage through the anastomosis -4. AlterModern approaches to LAVW are based on the thermal coagulation of a chromophore-containing protein solder that is placed over the coapted vascular segments, referred to as solder-mediated LAVW (sLAVW) -13. The 2). Welding strength depends on the adhesive strength (the strength of the solder-tissue bond) and the cohesive strength (the strength of the tissue-tissue bond and inter-solder bonding). sLAVW produces welding strengths that exceed > 250 mmHg [The ultimate goal of LAVW is to achieve welding strengths well above malignant hypertension pressure (> 250 mmHg), measured by the bursting pressure (mmHg) or breaking strength . In this approach, a semi-porous scaffold composed of biocompatible polymeric material is drenched in chromophore-containing solder, placed over the coaptation, and irradiated. The scaffold provides an intertwining fiber network that, after laser irradiation, solidifies/fortifies the thermally coagulated solder. ssLAVW produces greater welding strengths than sLAVW due to a greatly enhanced cohesive strength -23. To dPLGA has a higher melting point than PCL. Depending on the glycolic:lactic acid ratio in the copolymer, the melting point of PLGA varies from 173 °C (ratio of 5:95) to 201 °C (ratio of 90:10) . BecauseA persistent challenge in (ss)LAVW has been to improve adhesive bonding while minimizing thermal damage to the vascular wall. It was shown that the addition of a protein cross-linker, genipin, to solid albumin solder yields a significant increase in welding strength due to cross-linking of albumin to tissue collagen , 27. AccIn previous work  it was d2.The concentrations listed throughout the manuscript refer to final concentrations. Supplemental figures are indicated with a prefix ‘S.’2.1.Fresh porcine aortas (n = 40) were harvested at the slaugh-terhouse. Aorta strips were stored in PBS at 4 °C and were used within 3 d. Perivascular tissue was trimmed and the aortas were cut along the longitudinal axis. Subsequently, 30 × 5-mm longitudinal strips (n = 530) were punched from the unfolded slabs of vascular tissue.2.2.w = 120,000, CAPA 6800, Perstorp UK, Cheshire, UK) was dissolved in chloroform ata 17% (w/w) concentration. The polymer solution was electrospun for 25 min at 23 °C and 50% relative humidity with a flow rate of 60 μL/ min and 15 kV to produce a mesh consisting of fibers with a diameter range of 12-14 μm.All scaffolds were electrospun in a climate-controlled electrospinning cabinet  equipped with a 14-G capillary and a distance of 15 cm between capillary and the target drum (25 × 120 mm). PCL scaffolds were prepared as described previously -21. BriePLGA  scaffolds with different fiber diameter were produced as follows. A 15% (w/w) PLGA in chloroform solution was electrospun at 23 °C and 50% relative humidity at 20 kV using a flow rate of 50 μL/min for 26-μm fibers and 35 μL/min for 19-µm fibers. Meshes with 10-µm diameter fibers were electrospun from a 13% (w/w) chloroform solution at 23 °C and 50% relative humidity with a flow rate of 35 μL/min and 20 kV. The same 13% (w/w) solution was electrospun for 60 min at 23 °C and 40% relative humidity with 16 kV and 20 μL/min to produce scaffolds with a 14-µm fiber diameter.Fiber diameter was confirmed by scanning electron microscopy  using analytical software . For mechanical testing and ssLAVW, scaffolds were punched out of the mesh in 20 × 5-mm and 5 × 5-mm arrays, respectively. Scaffold thickness was measured with a digital micrometer between two glass slides.2.3.Semi-solid albumin solder consisted of 48% (w/v) bovine serum albumin , 0.5% (w/v) methylene blue , and 3% hydroxypropylmethylcellulose  in MilliQ water , 28. TheFor preparation of the BSA-genipin and BSA-HPMC-genipin solder, genipin was dissolved in ice-cold MilliQ (1% (w/v) final concentration) for 24 h under continuous stirring and at 4 °C until a homogenous suspension was obtained. Next, the genipin solution was placed in a water bath at 37 °C for 30 min to maximally dissolve the genipin , 29. The2.4.Aorta strips (n = 530) were pinned to a slab of silicone rubber  and placed in a petri dish. The aorta strips were cut in half along the longitudinal axis and realigned, after which 50 μL of solder and scaffold was applied over the adventitial surface of the incised strip.2, accounting for an irradiance of 5.8 W/cm2. The scan speed was dictated by MB transiting into its leuco-form, i.e., irradiation of the subsequent scaffold volume was performed only after the scaffold area had turned white. This lasing regimen was standardly applied twice per scaffold.A 670-nm diode laser  was used in continuous wave mode for ssLAVW with a low-power red HeNe laser aiming beam. For mechanical testing and structural analysis only  was used for ssLAVW in all other experiments. The lasing modality was performed by positioning the laser probe at a 3.0-cm distance perpendicular to the scaffold surface to generate a spot diameter of 1.3 cm. The laser power was set to 1.6 W and irradiation was performed for 50 s, yielding an irradiance of 1.2 W/cme ssLAVW .Modifications to the single spot lasing regimen to reduce thermal damage are explained in section 2.5.3.2.5.2.5.1.2.5.1.1.2 (Fifty-six scaffolds (n = 7/treatment/scaffold) were prepared for the determination of the stress–strain relation. Native electrospun PCL and PLGA scaffolds were tested without any modification as control. Solder-soaked scaffolds were prepared by soaking scaffolds in 1 mL of BSA-HPMC solder dispersed over a small area in a petri dish. Scaffolds were gently dabbed to enhance solder penetration. To investigate the effect of irradiation on the solder-soaked scaffold, PCL and PLGA scaffolds underwent a similar soaking procedure, after which they were subjected to dual pass irradiation at 5.8 W/cm2 . Lastly,Mechanical tests were performed on scaffolds using a tensiometer (n = 7/group). The force of the tensiometer was zeroed prior to mounting the scaffolds into the metal clamps. The grip-to-grip separation was set at 5 mm and the test was performed at a test speed of 5 mm/min. Stress-strain curves during mechanical testing were made to compare mechanical behavior between PCL and PLGA scaffolds, while E-modulus, yield strength, and yield stress were calculated in MatLab  to determine ssLAVW- induced alterations of the mechanical properties.2.5.1.2.nd heating was performed to determine the melting point.The melting points of PCL and PLGA scaffolds were measured using a differential scanning calorimetry (DSC) . DSC scans were performed at 10 °C/min from 0 to 120 °C for PCL scaffolds and from 0-200 °C for PLGA scaffolds. The 2Scaffolds (n = 3/treatment/scaffold) were prepared and irradiated as described in 2.5.1.3.To define the optimal scaffold properties of PLGA scaffolds, PLGA was spun at different electrospinning settings to produce scaffolds with a fiber diameter of 10, 14, 19, or 26 μm and a thickness between 120-200 μm . Scaffol2.5.1.4.After determination of the optimal scaffold properties of PCL  and PLGA2.5.2.PCL and PLGA ssLAVW was performed by SSCL  with dif). Histology samples were prepared to investigate thermal damage to the vessel  was determined in a separate set of experiments. To avoid the melting of PCL fibers to the thermocouple , this paThe denaturation/melting temperatures of BSA, MB, and all three solder compositions were measured by DSC. A sealed pan was used to reduce the background of water evaporation. DSC scans were performed at 10 °C/min from 30-95 °C. A difference measurement was obtained by completing two sequential scans for a single sample. The first scan was performed to denature solders, whereas the second scan was performed to obtain a background thermal profile for the remaining denatured sample. The thermal difference between the first and the second scan was defined as irreversible denaturation of the solder [2.5.3.In order to reduce thermal damage, single spot ssLAVW was performed with BSA-HPMC-genipin solder, PLGA scaffold, and with either 50-s SSCL  or with 2.6.2) of the scaffold and solder. The cross-sectional area of the solder was measured right before the breaking strength (BS) measurement was performed (after welding and after hydration period).BS tests were performed after every 5 consecutive ssLAVW procedures, with the exception of the hydration experiments, where BS testing was performed directly on all hydrated samples. The ssLAVWed aorta strips were kept moist in PBS-soaked gauzes between the ssLAVW procedure and BS testing. The strips were mounted in the metal clamps of a tensiometer . The distance between the clamps was 10 mm. BS analysis was performed at a test speed of 10 mm/min. BS was defined as the force (N) required to completely tear two halves of the strip, divided by the cross-sectional area . When differences were found in the multiple comparison tests, a Bonferron (ANOVA) or Dunn’s  post-hoc test was performed. When comparing various treatment regimens to a control group  a Dunnet’s post hoc multiple comparison test was employed. The choice for parametric or nonparametric testing was based on the D’Agostino Pearson omnibus test of normality, which was performed on each data set. The number of symbols designates the strength of the p-value, i.e., (*), (**), and (***) designate a p-value of ≤ 0.05, ≤ 0.01, and ≤ 0.001, respectively, throughout the text.3.3.1.3.1.1.The effect of heating on scaffold properties was investigated in the MB-soaked scaffold group. In the absence of a solder, the irradiated PLGA scaffold remained stiffer and stronger than PCL. The E-modulus of MB-soaked PLGA and PCL scaffolds was 1.3 ± 0.5 MPa and 0.7 ± 0.2 MPa †, , respect3.1.2.m = 62 °C, m = 148 °C, To determine the effect of laser-induced heating on the scaffold material, PCL and PLGA scaffolds were soaked in solder or MB, irradiated, and investigated macroscopically and by SEM. Macroscopic images of irradiated solder-soaked and MB-soaked PCL vs. PLGA scaffolds are presented in The complete set of SEM images of native, solder-soaked, and irradiated solder-soaked PCL and PLGA scaffolds is presented in Figure S1.3.1.3.2), whereby the BS decreased with increasing scaffold thickness. For scaffolds with a fiber diameter of 26 μm, an increased scaffold thickness had no impact on welding strength. The 19-μm fiber diameter group yielded comparable welding strength as the 26-μm fiber diameter group. The combination of 14-μm fiber diameter and a scaffold thickness of 150-180 μm produced the highest acute BS, and was therefore used in the subsequent substudies.Next, the ssLAVW modality was optimized for PLGA scaffolds using single spot irradiation, as was performed for PCL previously . Figure 3.1.4.2 and 248.0 ± 54.0 N/cm2, respectively. SEM imaging revealed seamless adhesive bonds in both PCL and PLGA ssLAVWed aortas , genipin (protein cross-linker), or both. HMPC and genipin were shown to improve welding strengths in intestinal welding , 29.Irradiation of MB- and genipin-containing solder caused the solder to turn dark purple rather than white (leuco-form of MB), as was the case with MB-containing solder. 2 vs. 408.6 ± 78.8 N/cm2, respectively, 2 versus 408.6 ± 78.8 N/cm2 (ns), respectively. The welding strengths during 14 d of hydration were higher in the PLGA + BSA-HPMC-genipin group compared to the PLGA + BSA-HPMC group, comprising 258.7 ± 43.9 N/cm2, 209.0 ± 24.4 N/cm2, and 223.9 ± 19.1 N/cm2 for 1-, 7-, and 14 d of hydration, respectively. These welding strength were similar to those achieved in the PCL scaffold group using BSA-HMPC solder, which produced welding strengths of 209.0 ± 24.4 N/cm2 (§§), 207.0 ± 35.1 N/cm2 (ns), and 202.9 ± 39.6 N/cm2 (ns), respectively.In the PLGA ssLAVW group, genipin-enriched BSA solder produced weaker welds than the BSA-HPMC solder . Upon irradiation, a dark purple color change occurred at the center of the laser spot and expanded laterally. After this color change, from 15-25 s the temperature increased gradually at a rate of 0.91 °C/s, 0.89 °C/s, and 0.7 °C/s for BSA-genipin, BSA-HPMC, and BSA-HPMC-genipin solders, respectively. In the last 25 s, the temperature increased at a much slower rate (0.2 °C/s) in all solders and the color change had extended over the entire scaffold area. At the end of the 50-s SSCL, the highest temperature at the s-t interface was achieved in the BSA-genipin solder group, i.e., 93.3 ± 4.2 °C (* vs. 79.9 ± 4.9 °C for BSA-HPMC and * vs. 84.7 ± 4.9 °C for BSA-HPMC-genipin). The aortas welded with BSA- genipin solder exhibited the most extensive thermal damage , which corresponds to the temperature profile . Thermal profiles of BSA, MB, BSA-HPMC, BSA-genipin, and BSA-HPMC-genipin are presented in Figure S3.3.3.2  on the basis of the observed color change. The mean ± SD s-t temperature after the first pulse was 80.7 ± 6.8 °C in all multiple pulse regimens. After a 5-, 10-, or 15-s cooling interval, the administration of a second pulsed raised the s-t temperature to 92.5 ± 4.9 °C, 85.7 ± 7.1 °C, and 82.9 ± 5.1 °C, respectively. The two-pulse regimens inflicted thermal damage up to 1/3 of the aortic wall . A third2 and 323.4 ± 25.2 N/cm2, respectively  and in which the least thermal damage was found . After 1-d of hydration, the BS in the control, 25-|10|-15 s, 25-|15|-15 s, and 25-|10|-15-|10|-10 s groups had decreased by 27%, 43%, 32%, and 21%, respectively . Followi4.The aim of this research was to optimize the previously described ssLAVW modality . The stu2, which was significantly lower than the 408.6 ± 78.8 N/cm2 produced by PLGA ssLAVW. This corresponds to a breaking force of 1.4 ± 0.3 N for PCL ssLAVW\and 2.2 ± 0.4 N for PLGA ssLAVW. Earlier studies on ssLAVW with solvent casted particulate leached PLGA scaffolds reported breaking forces of 1.31 N [The thermo-mechanical properties of a biomaterial in part dictate the suitability of the biomaterial for a given application, particularly if the application is associated with the generation of high temperatures such as during (ss)LAVW. Due to the relatively low melting point of PCL (62 °C) versus PLGA (148 °C), it was expected that thermal denaturation of PCL fibers would compromise post-LAVW welding strength. Indeed, mechanical tests on PCL and PLGA scaffolds confirmed that PLGA was stronger than PCL after irradiation, both as free solder-impregnated scaffold and as a scaffold imposed on a vascular segment during ssLAVW. With respect to the latter, ssLAVW with PCL yielded an acute BS of 248.0 ± 54.0 N/cmf 1.31 N , 1.5 ± 0f 1.31 N , and 1.1f 1.31 N , which wf 1.31 N  but lowef 1.31 N .Despite the higher acute BS, aortas welded with PLGA + BSA-HPMC solder exhibited deterioration of welding strength under quasi-physiological conditions. Similar results have been reported by Sorg and Welch . AlthougThe improvement in post-hydration welding strength in PLGA ssLAVW and the corollary enhancement in adhesive bonding by the addition of genipin reflect the important role of genipin in promoting cross-linking between albumin and tissue collagen . However2 and 292.3± 59.3 N/cm2, respectively), were considerably higher than the breaking force reported by Alfieri et al. for the 15-|15|-15 s regimen, namely 0.6 ± 0.04 N [To determine the relationship between heat build-up at the s-t interface and welding strength as well as thermal damage, the temperature profiles during ssLAVW were determined for different welding regimens. The s-t temperature during a 50-s single spot laser pulse, namely 80-85 °C, was similar to values reported by Sorg and Welch . This re± 0.04 N .5.In the final analysis, the previously described ssLAVW modality was optimized, but neither of the modalities proved to be superior over the other regarding post-hydration BS, the most important parameter for in vivo LAVW. Whereas PLGA ssLAVW yielded better acute cohesive bonding, PCL ssLAVW produced more stable welds. The quality of each modality should therefore be tested in ex vivo or in vivo models for further evaluation. Nevertheless, it is compelling to argue that a dual-layer scaffold composed of an inner PCL layer and an outer PLGA layer may provide more stable and stronger welds than ssLAVW with either biomaterial alone. Electrospinning allows the fabrication of a double-layered scaffold . Also, g


The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells.3-Iodothyronamine (3-T Clas1AM . Its act1AM . Togethe1AM , but als1AM  and neur1AM , 29.1AM raises the question of what could be the 3-T1AM-induced signalosome and whether there is a link between stimulatory effects of 3-T1AM in tissues that pertain to metabolic- and/or thermo-regulation and TRPs.Different studies demonstrated that TRPs are the major downstream effectors of GPCRs and the signaling cascades that emanate from the activation of GPCR evoke TRP channel activity , 31. The1AM in murine hypothalamic nuclei and explored the underlying mechanism behind this effect in murine hypothalamic cell lines. The results of this study show a stimulatory effect of 3-T1AM on Ca2+ mobilization and whole-cell currents in murine hypothalamic cells and that this effect is associated with TRPM8 activation.Here, we identified the stimulatory effect of 3-T1AM solved in 60% DMSO and 40% PBS, control mice with 60% DMSO and 40% PBS (volume of injection was 5 μl/g body weight). After 60 min, animals were transcardially perfused with PBS and 10% formalin (European Community Council Directives (86/609/EEC) and approved by Stockholm's Norra Djurförsöksetiska Nämnd). Fixated murine brains were successively incubated in 10, 20, and 30% sucrose solution over several days. Brains were cut at a cryotom into 30 μm slices and placed in a 48 well plate filled with PBS. Slices were blocked with a blocking buffer  for 2 h, subsequently incubated with a c-FOS antibody, rabbit anti mouse  over night at 4°C and finally with an Alexa Fluor 549 antibody, goat anti rabbit  for 2 h at room temperature. Between each step, the slides were washed 3 × 1 min with TBS . The brain slices were placed on glass slides and mounted with VectaShield containing DAPI . Pictures were taken with a Keyence BZ-9000 microscope (20 × magnification) and optimized with the ImageJ software. C-FOS positive cells were counted with the ImageJ software using two brain slides of each animal for each brain loci.In collaboration with the Karolinska Institute, Sweden, C57BL/6J mice (4 in each group) were i.p. injected with 50 mg/kg body weight 3-Thttps://www.cedarlanelabs.com/Products/Listing/Hypothalamic). The cells were cultured in Dulbecco's modified Eagle's medium , supplemented with 10% fetal calf serum (FCS) and 1% penicillium/streptavidin at 37°C in humidified air containing 5% CO2.GT1-7  were purchased from MERK, . mHypoE-Cells were seeded in T75 flask and grown to 80% confluence. Cells were harvested in three different passages, spanning from passage three to passage six. Total mRNA was isolated by chloroform/phenol extraction, a DNase digestion was performed and samples were stored at −80°C.Pgk1 was chosen as the reference gene, as previously recommended (−1/slope). Each of the three passages of every cell line was measured in duplicates together with a sample without reverse transcription to exclude genomic DNA contamination.A quantitative PCR (qPCR) was performed to determine the expression level of several GPCRs and TRPs. QPCR primers including their efficiency are listed in Supplemental Table ommended . First, s and Gi/o signaling were determined by measuring cAMP accumulation using the AlphaScreen technology  as previously described (−4 cells / well). Seventy-two hours after seeding, stimulation was performed by means of using a stimulation buffer . For Gαs signaling, cells were incubated for 45 min with either 3-T1AM , serotonin , norepinephrine , isoproterenol , or phenethylamine  in a concentration of 10 μM or only stimulation buffer to monitor the basal cAMP content. 3-T1AM was diluted from a 10 mM stock solution using DMSO as solvent. H2O was used as solvent for serotonin and norepinephrin and PBS with 0.1% BSA was used as the solvent for isoproterenol and phenethylamine. For Gi/o pathway examination, cells were additionally stimulated with 50 μM forskolin  to activate the adenylyl cyclase for a total of 45 min. Afterwards, cells were lysed at 4°C on a shaking platform. Intracellular cAMP accumulation was determined by a competitive immunoassay based on the AlphaScreen assay kit according to the manufacturer's instructions and measured using a Berthold Microplate Reader . Cyclic AMP concentrations were normalized to protein contents, which was measured with the Pierce BCA Protein Assay Kit .Gαescribed . Cells w2+ levels ([Ca2+]i) in single-cells, cells were pre-incubated with culture medium containing fura-2/AM (2 μM) for ~30 min at 37°C. Loading was stopped with a Ringer-like (control) solution containing: 150 mM NaCl, 6 mM CsCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, and 1.5 mM CaCl2 at pH 7.4. Where a blocker was used, pre-incubation was performed 30 min before the measurement. Fluorescence measurements were performed on the stage of an invert microscope (Olympus BW50WI) and a camera (Olympus XM-10) in connection with a LED-Hub . Fura-2/AM fluorescence was excited at 340 and 380 nm alternatingly and emission was detected from small cell clusters every 4 s at 510 nm. Results are shown as mean traces of f340nm/f380nm ± SEM. Drugs were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution and diluted in Ringer-like solution to obtain a working concentration which did not exceed 0.1%. For image acquisition and data evaluation, the life science imaging software cellSens was used .To monitor time-dependent changes in intracellular free Ca2, 60 mM CsF, 20 mM EGTA, and 10 mM HEPES, pH 7.2 and osmolarity 288 mOsM. Cs in the internal solution blocks potassium channel activity. A single cell suspension was added to an external solution of the following composition: 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2F, 5 mM D-glucose monohydrate, and 10 mM HEPES, pH 7.4 and osmolarity 298 mOsM. Whole-cell currents were recorded using a ramp protocol ranging between −60 to +130 mV for 500 milliseconds. The mean membrane capacitance of N41 cells was 9 pF ± 1 pF (n = 10). Mean access resistance was 15 ± 1 MΩ (n = 10). The holding potential (HP) was set to 0 mV in order to eliminate any possible contribution of VDCCs or sodium channels. All plots were generated with SigmaPlot software version 12.5 .For electrophysiological recordings, the semi-automated planar patch-clamp technique was used as previously described . Whole-c2+ imaging, statistical significance was determined by an unpaired t-test with Welch's correction. In figure legends, the number of experiments and the type of comparison are given. Statistical significance was defined as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.GraphPad Prism 6.0  was chosen for visualization and data analysis. Data are shown as means ± SEM of independent experiments. Statistical analysis was carried out using one-way and two-way ANOVA, followed by Sidak correction. For Ca1AM in selected brain nuclei such as locus coeruleus and PVN of the hypothalamus , while DMSO/PBS treated mice showed only few c-FOS positive neurons (16 ± 4 c-FOS positive cells per brain slide) , the supraoptic nucleus (SON), the dorsolmedial nucleus of the hypothalamus, the periaqueductal gray (PAG) and the ventral tegmental segment (VTA) , and mHypoE-N41 (N41). These cell lines are established models to study neuroendocrine mechanisms and known to express PVN-like markers . In all cell lines, 3-T1AM stimulation (10 μM) did not increase cAMP concentration compared to the basal cAMP content , N41 (~1.8 fold for NorEpi and ~2.1 fold for ISOP), and N39 (~3.5 fold for NorEpi and ~5.9 fold for ISOP) cells . The specific ligands for TAAR1, 5-HT1b, ADRA2A, ADRB1, and ADRB2 did not activate Gi/o signaling  and Trpv1 (ratio to Pgk1 514 ± 226) were the highest expressed genes, followed by Trpm7 (ratio to Pgk1 29 ± 23) and Trpm8 (24 ±18). Trpm2 and Trpm1 were lower expressed with ratios of 0.029 ± 0.025 and 0.0014 ± 0.0011 compared to the reference gene. Trpm3 and Trpm6 were least expressed in the transcriptome of N41 cells with ratios to Pgk1 of 0.000089 ± 0.000076 and 0.0000022 ± 0.0000013  in single-cells. 3-T1AM (10 μM) increased the f340nm/f380nm ratio from 0.70 ± 0.01 to 0.77 ± 0.05; (n = 10) in GT1-7, from 0.70 ± 0.008 to 0.84 ± 0.03 (n = 15) in N39 cells and from 0.70 ± 0.009 to 1.88 ± 0.03;  in N41 cells , 0.70 ± 0.009 in N39 cells (n = 10) and 0.70 ± 0.01 in N41 cells after the same period (n = 15)  which also has the highest expression level of adrenergic receptors and TRPs.To investigate the involvement of TRPs in the 3-T1AM effects on whole-cell currents of N41 cells to determine if increases in their magnitude underlie rises in plasma membrane Ca2+ influx in this cell line. At−60 mV, 10 μM 3-T1AM increased inward currents from −14.38 pA/pF to −60.78, which are attributable to Ca2+ influx because of the internal Ca2+ free solution. At +130 mV, outward rectifying currents strongly increased from 83.71 to 177.38 pA/pF in the presence of 3-T1AM.In the next step, we evaluated 3-T1AM affects TRPM8 activation at a constant temperature in different cell types  (p ≤ 0.001)  as a specific TRPV1 blocker. In the presence of 10 μM AMTB, the f340nm/f380nm ratio decreased from 1.83 ± 0.08 to 1.12 ± 0.05  which is involved in the mitochondrial function  could strongly inhibit the 3-T1AM-induced Ca2+ influx and whole-cell current.Previous studies demonstrated the co-expression of TRPV1 with TRPM8 in different cell types including rat hippocampal neurons, intralobar pulmonary arteries, aorta, neuroendocrine tumor cells, retinoblastoma cells, uveal melanoma cells, and corneal cells , 73–76. 2+ signal transduction pathways induced by 3-T1AM and provided evidence of TRP channel modulation via this TH derivative in hypothalamic cell lines. Characterization of the intracellular signaling cascade induced by 3-T1AM might explain the underlying mechanism behind the profound physiological effects of this metabolite.Collectively, the results of this study demonstrated the CaHB, NK, and JB designed the study. JB, SJ, and NK performed the experiments and analyzed the data. SJ, CH, and JM performed the mouse studies. NK, JB, SM, and HB wrote and edited the manuscript. CH, JM, and MR discussed data and edited the manuscript. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure containing both inner and outer membranes. While we generally think of cellular membranes being composed largely of phospholipids, cellular elongation occurs when environmental phosphate, and therefore phospholipid synthesis, is limited. In order to adapt to these environmental constraints, C. crescentus synthesizes several glycolipid species, including a novel glycosphingolipid. This finding is significant because glycosphingolipids, while ubiquitous in eukaryotes, are extremely rare in bacteria. In this paper, we identify three proteins required for GSL-2 synthesis and demonstrate that they contribute to phage resistance. These findings suggest that bacteria may synthesize a wider variety of lipids in response to stresses than previously observed.Bacteria adapt to environmental changes in a variety of ways, including altering their cell shape.  Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure. The stalk is an extension of the Gram-negative envelope containing inner and outer membranes as well as a peptidoglycan cell wall. Cellular elongation requires a 6- to 7-fold increase in membrane synthesis, yet phosphate limitation would preclude the incorporation of additional phospholipids. In the place of phospholipids, C. crescentus can synthesize several glycolipid species, including a novel glycosphingolipid (GSL-2). While glycosphingolipids are ubiquitous in eukaryotes, the presence of GSL-2 in C. crescentus is surprising since GSLs had previously been found only in Sphingomonas species, in which they play a role in outer membrane integrity. In this paper, we identify three proteins required for GSL-2 synthesis: CcbF catalyzes the first step in ceramide synthesis, while Sgt1 and Sgt2 sequentially glycosylate ceramides to produce GSL-2. Unlike in Sphingomonas, GSLs are nonessential in C. crescentus; however, the presence of ceramides does contribute to phage resistance and susceptibility to the cationic antimicrobial peptide polymyxin B. The identification of a novel lipid species specifically produced upon phosphate starvation suggests that bacteria may be able to synthesize a wider variety of lipids in response to stresses than previously observed. Uncovering these lipids and their functional relevance will provide greater insight into microbial physiology and environmental adaptation. Escherichia coli, cells incorporate an increasing proportion of unsaturated fatty acids as temperatures decrease , glucuronosyl diacylglycerol, and diacylglycerol trimethylhomoserine (DGTS)  (C. crescentus either (i) increases the proportion of existing glycolipids or (ii) synthesizes novel lipid species to replace phospholipids.Several alphaproteobacteria adapt to phosphate limitation by increasing the production of glyceroglycolipids and ornithine lipids. For example, e (DGTS) , 11, whine lipid . Glycoli to 62%) , but phoC. crescentus increases the amount of monohexuronosyl DAG (MHDAG) and synthesizes a novel hexosyl-hexuronosyl-ceramide glycosphingolipid (HexHexA-Cer). This glycosphingolipid (GSL) represents a novel bacterial lipid species. In this report we characterize this GSL, identify the enzymes responsible for initiating ceramide synthesis and its sequential glycosylation, and address the physiological importance of ceramide-based lipids.Analysis of total membrane composition following phosphate limitation revealed that both hypotheses were correct. C. crescentus  and phosphate-starved (1 µM phosphate) conditions by using normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS), we saw a dramatic decrease in phosphatidylglycerol and a corresponding increase in several species of glycolipids (16 ceramide [d18:1/16:0])   . These lglycerol . Non-phode (LPS) . Structuar units  14, 15), 15C. cred GSL-2 .C. crescentus encodes three putative oxoamine synthases: CCNA_01220 (BioF), CCNA_01417 (HemA), and CCNA_01647 (BioF).Sphingolipid synthesis begins with the production of a ceramide molecule which is then modified with various polar groups. In eukaryotes, ceramide synthesis is a four-step process that begins with the condensation of serine and a fatty acyl coenzyme A (acyl-CoA) to produce 3-oxo-sphinganine in a reaction catalyzed by oxoamine synthase . While eccna_01220, and Δccna_01647 cells. We were unable to obtain a deletion of ccna_01417 since this is an essential gene  nosferases , and (iina_00793 . Both cana_00793  22). De. DeC. crceramide , whereas species . Complem species B and C.  species . Sgt1 fu species  and D. Td lipids A and B.10.1128/mBio.00107-19.5TABLE S4Table S4, DOCX file, 0.01 MB.Selection criteria for assessing candidate glycosyltransferases. Download Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00107-19.7FIG S2E. coli. Download FIG S2, PDF file, 0.8 MB.Expression of Sgt1 and -2 in Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ccbF mRNA levels are reduced during phosphate starvation, despite higher levels of ceramide production  or low-phosphate (1 µM) minimal medium  (ccbF deletion strains infected with ΦCr30 in peptone-yeast extract (PYE) demonstrated that ceramides are important for increasing phage resistance –1 µM phosphate. Both of the mutant strains had an enhanced rate of phage adsorption which was restored to normal upon complementation  . Growth sistance ; this insistance . The incentation , suggestentation ; howeverse cells .C. crescentus by binding to the cell envelope S-layer, a crystalline lattice composed of the protein RsaA (C. crescentus strains were grown in HIGG–1 µM phosphate and incubated with fluorescein isothiocyanate (FITC)-labeled dextran  medium (C. crescentus was grown in Hutner base-imidazole-glucose-glutamate media (HIGG) with variable amounts of phosphate   for E. coli and 5:25 for C. crescentus; ampicillin, 50:100 for E. coli; tetracycline, 12:12 for E. coli and 1:2 for C. crescentus; gentamicin, 15:20 for E. coli and 0.5:5 for C. crescentus; and spectinomycin, 50:50 for E. coli and 25:100 for C. crescentus. Gene expression was induced in C. crescentus with either 0.3% (wt/vol) xylose or 0.5 mM vanillate. E. coli gene expression was induced with isopropyl-β-d-1-thiogalactopyranoside . Phage titering was performed by adding 1 to 10 µl of ΦCr30 to 100 µl of an overnight culture of NA1000 in PYE. This mixture was added to 4 ml of soft agar (0.3% [wt/vol] agar in PYE) and overlaid on a PYE-agar plate. After solidifying, the plate was incubated overnight at 30°C and plaques were counted.The strains, plasmids, and primers used in this study are described in ) medium  for rout,000 µM) . E. coli10.1128/mBio.00107-19.2TABLE S1Table S1, DOCX file, 0.02 MB.List of strains used in this study. Download Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00107-19.3TABLE S2Table S2, DOCX file, 0.02 MB.List of plasmids used in this study. Download Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00107-19.4TABLE S3Table S3, DOCX file, 0.02 MB.List of primers used in this study. Download Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Morphometrics , and the pellet was resuspended in 30 µl of 0.5× phosphate-buffered saline (PBS). Ten microliters of the cell suspension was mixed with 5 µl of FITC-dextran  and 1 µl of SlowFade Diamond mountant (Thermo Scientific). Two microliters of this mixture was spotted onto a glass slide, coverslipped, and sealed with vaseline-lanolin-paraffin  for imaging.Cells were spotted onto 1% agarose pads made in the corresponding growth medium. Phase microscopy was performed on a Nikon TiE inverted microscope equipped with a Prior Lumen 220PRO illumination system, Zyla sCMOS 5.5-megapixel camera, CFI Plan Apochromat 100× oil immersion objective , and NIS Elements software for image acquisition. Cell and stalk dimensions were measured using ometrics  and Imagometrics . BrieflyTC) method. rpoD expression was used as the loading control.RNA was extracted from bacterial cultures using the Qiagen RNeasy kit. Following DNase digestion, RNA (5 ng/µl) was reverse transcribed using a high-capacity cDNA reverse transcription (RT) kit (Applied Biosystems). One microliter of cDNA was used as a template in a 10-µl quantitative RT-PCR (qRT-PCR) performed with Power SYBR reagent (Applied Biosystems). qRT-PCR was performed on an ABI QuantStudio 6 using the threshold cycle  chloroform-methanol was added, and the samples were mixed by vortexing. Chloroform (1.25 volumes) and water (1.25 volumes) were added sequentially with vortexing to create a two-phase system, and the samples were centrifuged at 200 × g for 5 min at room temperature. The bottom, organic phase was transferred to a clean tube with a Pasteur pipette and washed twice in “authentic” upper phase. Subsequently, the residual organic phase with the lipids was collected and dried under argon.and Dyer . Cells w2; GSI, 20 lb/in2; DP, −55 V; and FP, −150 V. The MS/MS analysis used nitrogen as the collision gas. Data analysis was performed using Analyst TF1.5 software (Applied Biosystems).Methods for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) have been described previously , 44. BriC. crescentus cells were diluted to an optical density at 660 nm (OD660) of 0.05 in HIGG with 1 mM or 1 µM phosphate, treated with 30 µg/ml of polymyxin B, and incubated with shaking (250 rpm and 30°C). Complementation gene expression was induced with 0.3% xylose. Samples were taken at the desired times for absorbance measurements (OD660). For phage sensitivity assays, cells were grown in PYE and diluted to an OD660 of 0.05. A total of 148 µl of culture was dispensed per well in a 96-well plate. To each well, 2 µl of water (control) or ΦCr30  was added. To prevent evaporation, each well was overlaid with 100 µl of mineral oil. OD660 was recorded every 20 min on a BMG Labtech CLARIOstar plate reader with incubation at 30°C with continuous shaking.For polymyxin B sensitivity assays, 660 of 0.2. Cells (1 ml) were aliquoted into glass culture tubes, and 105 PFU of ΦCr30 was added. Cultures were incubated with shaking at 30°C; at various time points, 10 µl of culture was removed and diluted into 1 ml of water-chloroform (9:1 [vol/vol]). Ten microliters of this mixture was used to titer the unbound phage as described above. To measure viral burst size, cells were grown in HIGG–1 µM phosphate and diluted to an OD660 of 0.1. Cells (0.5 ml) were infected with 0.5 × 105 PFU of ΦCr30 and incubated at 30°C for 15 min. The culture was diluted 1,000-fold into HIGG–1 µM phosphate; a 200-µl aliquot was removed every 15 min for titering as described above.Phage adsorption and burst size were measured essentially as previously described . To measE. coli strains were grown overnight in LB medium with 1 mM IPTG to induce protein expression. A total of 500 µl of each strain was collected by centrifugation  and the pellet was resuspended in 100 µl of sample buffer. Protein samples were resolved on a 12% SDS-PAGE gel and stained with Coomassie blue for visualization.C. crescentus cells grown in HIGG–1 µM phosphate (OD660 = 0.5) was collected and washed once in 10 mM HEPES (pH 7.2). Cells were resuspended in 250 µl of TE buffer  and frozen overnight at −20°C. Cells were thawed, treated with 1 µl of DNase (0.5 mg/ml), 20 µl of lysozyme (10 mg/ml), and 3 µl of MgCl2 (1 M), and incubated at room temperature for 15 min. For each sample, 36.25 µl was mixed with 12.5 µl of 4× SDS sample buffer and boiled at 100°C for 10 min. After cooling to room temperature, 1.25 µl of proteinase K (20 mg/ml) was added and samples were incubated at 60°C for 1 h. LPS samples were resolved on a 12% SDS-PAGE gel and stained with a Pro-Q Emerald 300 LPS stain kit according to the manufacturer’s protocol (Thermo Scientific). Images were acquired on a Bio-Rad ChemiDoc MP using UV excitation and a 530-nm emission filter.Lipopolysaccharide (LPS) was purified essentially as previously described . Briefly660 = 0.6) was collected by centrifugation. The cell pellets were washed twice in 5 ml of 10 mM HEPES (pH 7.2), resuspended in 200 µl of 100 mM HEPES (pH 2), and incubated at room temperature for 10 min. Cells were pelleted , and the supernatant containing RsaA was collected and neutralized with 2.8 µl of 10 N NaOH. RsaA samples in 1× sample buffer were resolved on a 7.5% SDS-PAGE gel without heat denaturing and stained with Krypton protein stain (Thermo Scientific). Images were acquired on a Bio-Rad ChemiDoc MP using green light-emitting diode (LED) excitation and a 605-nm emission filter.RsaA was purified essentially as previously described . Briefly10.1128/mBio.00107-19.1TEXT S1Text S1, DOCX file, 0.03 MB.Detailed description of strain construction and supplemental references. Download Copyright © 2019 Stankeviciute et al.2019Stankeviciute et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the