Candida species can vary from mild to severe dependent upon the site of infection, length of exposure, and past medical history. Patients with impaired immune status are at increased risk for chronic fungal infections. Recent epidemiologic studies have revealed the increasing incidence of candidiasis caused by non-albicans species such as Candida parapsilosis. Due to its increasing relevance we chose two distinct C. parapsilosis strains, to describe the cellular innate immune response toward this species. In the first section of our study we compared the interaction of CLIB 214 and GA1 cells with murine and human macrophages. Both strains are commonly used to investigate C. parapsilosis virulence properties. CLIB 214 is a rapidly pseudohyphae-forming strain and GA1 is an isolate that mainly exists in a yeast form. Our results showed, that the phagocyte response was similar in terms of overall uptake, however differences were observed in macrophage migration and engulfment of fungal cells. As C. parapsilosis releases extracellular lipases in order to promote host invasion we further investigated the role of these secreted components during the distinct stages of the phagocytic process. Using a secreted lipase deficient mutant strain and the parental strain GA1 individually and simultaneously, we confirmed that fungal secreted lipases influence the fungi's virulence by detecting altered innate cellular responses. In this study we report that two isolates of a single species can trigger markedly distinct host responses and that lipase secretion plays a role on the cellular level of host–pathogen interactions.Numerous human diseases can be associated with fungal infections either as potential causative agents or as a result of changed immune status due to a primary disease. Fungal infections caused by  Candida species are the most common etiological agents of systemic fungal infections  species  was isolated from the feces of a patient in Puerto Rico, and rapidly forms pseudohyphae producing a rough, concentric colony phenotype on agar  phagocyte aggregation at the site of infection, (2) recognition of pathogen associated molecular patterns (PAMPs) via receptors, (3) internalization of the foreign particles, and (4) digestion of ingested agents through phagosome maturation and activation of hydrolytic enzymes , resulted in significantly decreased virulence as determined in both in vitro and in vivo infection models  formed less complex and thinner biofilms when compared to the C. parapsilosis GA1 parental strain  were maintained on YPD solid medium at 4°C and prepared for experiments as described in our previous work  at 30°C with shaking at 200 rpm. The cells were collected and washed three times with PBS , counted and diluted to the final concentration of 1 × 108/ml. UV-killed fungal strains were prepared using twenty exposures to 20 mJ/cm−2 UV. In order to differentiate between fungal cells after the co-infection, 1 mg/ml FITC  and 50 μg/ml CFW  were used to label yeasts. FITC was dissolved in dimethyl sulfoxide (DMSO) and added to 108/ml cells suspended in 0.05 M carbonate-bicarbonate buffer (pH 9.6). CFW was dissolved in distilled water and added to 108/ml yeasts in PBS. C. parapsilosis cells were stained at room temperature in the dark for 10 min, followed by washing steps three times with PBS and suspended in 1 ml 1X PBS. In dual yeast cell co-infection experiments, mixtures of GA1 and LIP1–LIP2 yeast cells were used in which GA1 was labeled with FITC and the mutant with CFW. Experiments were simultaneously performed using GA1 yeast cells labeled with CFW and mutants with FITC. We found that there was no difference in our experiments whether the GA1 or lipase mutants were labeled with the alternate stain. Although we used both conditions in all experiments, we only reported out the condition where GA1 was labeled with FITC and the mutant with CFW.Wild type 2 prior to and during the experiments. For live video microscopy, similarly to our previous report, 1.2 × 105 macrophages were seeded on eight-well μ-slides  and incubated at 37°C overnight prior to infection with Candida strains. For visualizing phagocytosis Lysotracker Red was used to label the acidic compartments of phagocytes. The original media was replaced with fresh, 300 μl pre-heated supplemented DMEM containing 1 μM Lysotracker Red DND-99  immediately before the experiments.Dulbecco's modified Eagle's medium  supplemented with 2 mM L-glutamine , 10% fetal calf serum , and 200 U/ml penicillin/streptomycin  was used in order to maintain the murine cell line. J774 cells were kept at 37°C, in the presence of 5% CO5 cell/ ml) were then plated on eight-well μ-slides  and incubated at 37°C, 5% CO2 for 6–7 days in serum supplemented DMEM . Shortly before the experiment, similarly to the murine macrophage preparation, the media was replaced with 300 μl fresh, pre-heated supplemented and 1 μM Lysotracker Red DND-99  containing DMEM.For the isolation of human peripheral blood mononuclear cells (PBMCs), a standard protocol was used  4 × 105 /ml J774 murine and 7.5 × 105 /ml human PBMC-derived macrophages on eight-well μ-slides  in 300 μl volumes immediately before the video capture was initiated. During individual infections, the effector/target ratio was 3:1, while in the case of simultaneous infections the ratio of 1.5 GA1: 1.5 lipase mutant: 1 macrophage was used. Images were captured over a 6 h period with a CCD camera attached to an UltraVIEW VoX Spinning disc confocal microscope  using a 40X oil immersion objective. For the comparison of the C. parapsilosis GA1 and CLIB 214 clinical isolates, over a 100 J774 macrophages were monitored individually and evaluated from at least four separate experiments—with 20–40 macrophages analyzed per experiment, dependent on frequency of macrophage–Candida interactions in the respective videos. From PBMC-DM experiments, 60 macrophages were tracked from three separate experiments with 15–20 macrophages analyzed per experiment due to cell size. For the C. parapsilosis GA1 and CpΔΔlip1 − ΔΔlip2 comparison 250 macrophages were selected and monitored from five separate experiments, with 45–55 macrophages followed per experiment, dependent on frequency of macrophage–Candida interactions in the respective videos, in order to make clear assumptions from the co-infection studies.In order to examine the phagocytic processes post infection, a previously described and standardized protocol was used accordingly  was used. Similarly to our previous study, measurements included the macrophage migration toward C. parapsilosis strains. The engulfment time was defined as the time difference between the establishment of the phagocyte-fungal cell contact and the phagocyte membrane enclosing around the fully ingested cell. Full engulfment events were determined when macrophages stretched toward fungal cells, the phagocyte membrane encircled around the yeast cell and the host cell regained its original shape. The prevalence of uptake represents the percentage of uptake events during defined time intervals. Individual events were defined by the starting point of the recognition of fungal cells by macrophages. The percentage of macrophages taking up specific numbers of fungal cell was referred to as distribution of fungal cells per macrophages. Average uptake is defined as the average number of fungal cells taken up by phagocytes that ingested at least one yeast cell over the whole incubation period. The percentage of dead macrophages relative to the visible macrophage population, that ingested at least one C. parapsilosis cell, represents post-ingestion macrophage rupture (PIR) events. Individual PIR events were defined as the disruption of membrane integrity that were visible up until the end of the 6 h of co-culturing period.High throughput analysis with Volocity software 6.3 allowed us to calculate the mean tract velocity of macrophages in the presence of different C. parapsilosis infection. As a postive control, cells were treated with 1% Triton X-100 solution. For LDH detection, the Cytotoxycity Detection kit  was used according to the user's manual. Basal LDH activity of human PBMC-derived macrophages may be due to the presence of undifferentiated monocytic cells or dead cells in the culture before the assay.To determine the concentration of LDH released by macrophages, culture supernatants were collected 6 h after Candida strains.Tracking of individual macrophages and high-throughput migration analysis was achieved by Volocity software 6.3. Extracted data were used to calculate the mean track velocity of phagocytes cultured with t-tests were used and confirmed by One-way ANOVA analysis followed by a Bonferroni's multiple comparison post-hoc tests. GraphPad Prism v 5.0 software was used to determine statistical significance. Significant differences were considered at p-values of ≤ 0.05. Data were pooled for migration analysis, engulfment time and LDH, thus SD-values are shown, percent phagocytosis, average uptake values and data for the fungal cell distribution/macrophages were evaluated as mean of means, thus SEM-values indicate error.For the comparison of the calculated mean values of each of the aspects, student's unpaired, two-tailed C. parapsilosis GA1 or CLIB 214 cells at an effector/target ratio of 1:3 and the phagocytic process was monitored by live cell video microscopy for 6 h  released into supernatant after co-incubation with 4 Figure  or human4 Figure . LDH res4 Figure .C. parapsilosis GA1 or CpΔΔlip1 − ΔΔlip2 individually in a ratio of 3:1. In the case of co-infection, GA1 and CpΔΔlip1 − ΔΔlip2 cells were used in the ratio of 1:1, and the MOI was kept at 3:1 , in comparison with GA1 yeasts (3.09 ± 1.36 min). Mixed cultures of GA1 and lipase mutant yeasts had engagement times that were significantly increased relative to GA1 alone  compared to GA1 (1.79 ± 0.20) or co-infections with GA1 and lipase mutants .We further examined the overall uptake of s Figure . The meaLIP1 and LIP2 genes did not affect host cell damage .As a measure of host cell damage, LDH released by J774 phagocytes was measured from culture supernatant 6 h post-infection. Similar levels of LDH were detected with either individual; or mixed wild type and lipase deletion strains. Thus, the lack of e Figure . SimilarCandida species, C. albicans, C. glabrata, and C. parapsilosis CLIB 214  have been identified in C. parapsilosis  have already been investigated. According to our previous findings, the deletion mutant was killed more efficiently, however, phagocytosed similarly by macrophages when compared to the wild type (Tóth et al., CpΔΔlip1 − ΔΔlip2 lipase mutant and the parental strain GA1. Numerous preceding studies have used co-infection methods to gain insight into virulence attributes (Silva et al., CpΔΔlip1 − ΔΔlip2 yeast cells could be overcome with the secretion of lipases by wild type yeasts during an infection, murine macrophages were challenged with wild type and lipase mutant yeasts individually, and simultaneously. In general, the overall macrophage uptake of mutant cells was more effective, as a higher percentage of phagocytes ingested a greater number of CpΔΔlip1 − ΔΔlip2 cells compared to the wild type yeasts. In correlation with the uptake rates, murine macrophage migration was increased toward mutant cells, indicating enhanced recognition of CpΔΔlip1 − ΔΔlip2 compared to wild type cells. These results suggest that, the lack of secreted lipase results in decreased virulence on the cellular level. Although, our current results are in contrast with a previous study on CpΔΔlip1 − ΔΔlip2 and GA1 phagocytosis comparison (Tóth et al., CpΔΔlip1 − ΔΔlip2 phagocytosis. Nagy et al. have used dendritic cells to study the virulence properties of C. parapsilosis secreted lipases and also found increased phagocytic efficiency (Nagy et al., CpΔΔlip1 − ΔΔlip2, we found no difference in murine macrophage damaging capacity compared to the wild type.Secreted hydrolytic enzymes play a critical role in host invasion by fungi (Ghannoum, CpΔΔlip1 − ΔΔlip2. This result can be explained by the above mentioned hypothesis on an additional deficiency of CpΔΔlip1 − ΔΔlip2 that cannot be rescued with the presence of secreted lipases. Interestingly, co-infection of macrophages with both strains led to significantly increased macrophage migration. As shown above, the presence of the CpΔΔlip1 − ΔΔlip2 cells increased the average track velocity markedly. A possible explanation for this phenomenon is that in case of co-infection, the presence of the mutant cells might have unmasked a potential immune evasion ability in wild type cells. The presence of both strains simultaneously had no further effect on host cell damage compared to the wild type.In conditions when both fungal strains were present, a significant reduction was observed in overall phagocytosis of yeast cells compared to the lipase mutant alone. Thus, lipase deficiency can be partially restored with the presence of the wild type cells that further confirms the role of fungal extracellular lipases during host–pathogen interactions. No difference was detected in yeast cell preference when macrophages were incubated with both types of fungal cells further supporting our hypothesis on secreted component deficiency complementation. The presence of wild type secreted lipases might had a direct influence on fungal cell uptake regardless of the yeast cell types present in the media. However, co-infection with GA1 and lipase mutant yeast cells increased the engulfment time similarly to This study confirmed the role of secreted lipase in virulence, as lack of the secreted component resulted in decreased virulence by altering the dynamics of phagocytosis. Our results further showed that in terms of uptake, lipase deficiency is partially restored when the wild type strain is present, which further exemplifies the role of secreted lipases on phagocytosis.In this report we have shown that two distinct isolates of a single species are able to trigger significantly different host responses and that fungal lipase secretion plays an important role in host–pathogen interactions on the cellular level.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.


New routes are developed to allow introduction of sensitive side-chains and synthesis of challenging proteomimetic sequences. α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein–protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation. These comprise (a) coupling of unfunctionalized monomers followed by subsequent introduction of the amino acid side chain mimicking group  using designed small molecules.e.g.A–D have beeompounds : (i) iteng group  and (b) ng group .30 We recently reported that helix 3 plays the dominant role in mediating potency of recognition but that helix 2, either as a fusion with helix 1 or 3 plays a role in binding /p300 plays a major role in regulating the hypoxic response and as such its inhibition represents an attractive approach to prevent development of new vasculature by hypoxic tumours. The HIF-1α/p300 interaction is substantially mediated by the binding of HIF-1α around the p300 CH1 domain and involves 3 regions which adopt a helical conformation on binding to p300 .29,30 We binding .31 We al mimetic . Motivat800 and Asn804 as shown in 34 Initial attempts to prepare helix 2 mimics -acetamide before final reduction, hydrolysis and deprotection to yield the desired, densely functionalised dimer 5 as shown in 5 an alcohol side chain was used on the N-terminal amino benzoic acid monomer in lieu of a thiol.37 The O- to N-acyl synthetic method was also employed to synthesise the reverse side chain orientation of 5 , we further adapted this approach to permit late stage derivatisation of the trimeric oligobenzamide scaffold .Having demonstrated synthetic feasibility for late stage alkylation of the phenolic position in the course of synthesising dimer scaffold  and rapi10 a series of compounds with cleavable protecting groups was prepared, e.g.6b. Phenolic allyl ethers are readily cleaved via the formation of a π-allyl complex with palladium(0) but are relatively inert to other conditions. This makes then ideal for use in the late stage derivatisation of oligobenzamides. Indeed, the allyl groups in 6a–c were readily cleaved with palladium(0) tetrakis(triphenylphosphine) employing toluene sulfinic acid as a scavenger (6b → 7b)).Using the recently identified proteomimetic inhibitor see  of the H7a–c could then be functionalised by employing Mitsunobu chemistry or alkylation with an appropriate halide yielding functionalized helix mimetics which, upon reduction of the aryl nitro group and ester hydrolysis in one pot, afford final compounds 8a–i (7b → 8e)). This approach was thus employed to prepare a small library of compounds 8a–i with previously unreported side chain combinations. Alcohols and alkyl halides were selected to challenge the synthetic approach (focusing on tertiary amines) rather than a specific intention to develop optimised inhibitors.The phenols nds 8a–i , followi7b with sodium hydride then treating with the iodide 9, the desired functionality could be introduced regioselectively. Final ester hydrolysis was performed in situ by treating the reaction mixture with sodium hydroxide to yield the photo-crosslinking helix mimetic 8j. 8k was synthesized using a similar sequence of steps from compound 6c  were also screened for activity  and is complementary to solution and SPS strategies for helix mimetic assembly useful for library production and initial screening.


The results suggest that CH3NH3PbI3−xClx perovskite is in an antiferroelectric phase at room temperature. The antiferroelectric phase can be switched to ferroelectric phase by the poling treatment and exhibits ferroelectric-like hysteresis P-E loops and dielectric behavior around room temperature; namely, the perovskite can generate a ferroelectric polarization under PSCs operating conditions. Furthermore, we also discuss the implications of ferroelectric polarization on PSCs charge separation.Perovskite solar cells (PSCs) have been attracted scientific interest due to high performance. Some researchers have suggested anomalous behavior of PSCs to the polarizations due to the ion migration or ferroelectric behavior. Experimental results and theoretical calculations have suggested the possibility of ferroelectricity in organic-inorganic perovskite. However, still no studies have been concretely discarded the ferroelectric nature of perovskite absorbers in PSCs. Hysteresis of P-E (polarization-electric field) loops is an important evidence to confirm the ferroelectricity. In this study, P-E loop measurements, in-depth structural study, analyses of dielectric behavior and the phase transitions of CH In the present study, P-E loop measurements and in-depth structural study of CH3NH3PbI3−xClx perovskite were carried out to clarify the doubtful ferroelectric behavior.Experimental results and theoretical calculations have suggested the possibility of ferroelectricity in CH3 and PbO3, depends strongly on their crystal parameters. The crystal structural parameters of the synthesized CH3NH3PbI3−xClx were measured using powder X-ray diffraction (XRD). The all diffraction peaks in the XRD pattern  and c ′ = 6.31899 Å ( = c/2) (c ′/a′ ratio (1.007) of the CH3NH3PbI3−xClx perovskite is greater than 1. And that value of ferroelectric tetragonal BaTiO3 is 1.010c ′/a ′ ratio of the tetragonal CH3NH3PbI3 perovskite towards unity at 327 K, the phase transition temperature from tetragonal to cubic structureIt is pertinent to evaluate lattice parameters to analyze ferroelectric behavior because ferroelectric behavior of the well-known ferroelectric metal oxide perovskites, such as BaTiO pattern  can be itructure  consists( = c/2) . The c ′3NH3PbI3−xClx perovskite can be confirmed from its P-E hysteresis loops  and the coercive field (Ec) values from the P-E loops measured at different applied electric field intensities (Ea) to find a relationship between the applied field and polarization which can correlate to the PSCs I-V measurement conditions. It should be noted that polarization can also arise from conductive effects because our measurements were performed at a high frequency of 2 kHz, the polarization by the movement of ions approaches to zero due to their low mobility (10−9 cm2 V−1 s−1 for I−)2/perovskite and perovskite/HTM2 and HTM compared to the perovskite; namely, low conductivities at TiO2/perovskite and perovskite/HTM interfaces. In the present study, to remove the electron trapping we have used perovskite pellet samples with Au-electrodes on the both sides for the P-E hysteresis measurement. Therefore a large electrons trapping cannot be expected at the Au/Perovskite interface due to the high conductivity of Au. There may be some trapping electrons at the interface of our pellet. However, it could not generate a large polarization like the normal ferroelectric materials in the order of μC/cm2. We think the polarization from the electron trapping must be less than in an order of nC/cm2 in our measurements. Therefore, we believe our results represent mainly ferroelectric polarization. The Pr and Ec gradually increase with increasing the Ea to about 7 kV/cm and then reach saturation (oc) = 0.9 Vc) of CH3NH3PbI3−xClx perovskite, 51 °C (see next section), is close to the room temperature (poling treatment temperature).We estimated the remanent polarization  can be achieved by applying a mechanical pressure (3NH3PbI3−xClx perovskite shows a maximum dielectric constant around Tc (51 °C), similar to the most ferroelectric and antiferroelectric metal oxide perovskites  ion in the PbI6 octahedron along the c′ axis as well as the displacement of CH3NH3+ cation position in the organic-inorganic perovskites similar to the most ferroelectric and antiferroelectric metal oxide perovskites20213NH3PbI3−xClx perovskite is an antiferroelectric material and it can be switched to the ferroelectric phase after poling. The structural studies on the organic-inorganic perovskites have revealed straightening of the Pb–I–Pb bonds with increasing temperature from the tetragonal phase to cubic phase and the distortions of PbI6 octahedra from ideal even for the cubic lattice3NH3PbI3−xClx perovskite, a detail structural analysis is necessary, and identifying the origin of the antiferroelectricity would be a challenging study in the future.The results of neutron diffraction studies have suggested the possibility of formation of either antiferroelectric or ferroelectric domains in CH2 and perovskite/HTM). In the ferroelectric solar cell , the poling treatment is not necessary for ferroelectric semiconductor solar cells, which is different from the conventional ferroelectric solar cells. The ideal photovoltaic material should separate charges as efficiently as possible and transport them independently to the contacts, to minimize recombination between electrons and holes3NH3PbI3−xClx perovskite is an ideal photovoltaic material due to its ferroelectric and semiconducting nature.ar cells . Then thar cells . Therefo3NH3PbI3−xClx perovskite were analyzed. The CH3NH3PbI3−xClx perovskite is an antiferroelectric semiconductor different from the typical semiconducting and ferroelectric materials. The CH3NH3PbI3−xClx perovskite exhibits ferroelectric-like P-E hysteresis loops and dielectric behavior around room temperature. The antiferroelectric CH3NH3PbI3−xClx based PSCs, under working condition, can generate about 1.2 μC/cm2 polarization and it can promote the charge separation. A clear understanding of the perovskite absorber material is imperative for future developments in the ferroelectric semiconductor PSCs and the manipulation of ferroelectrics of the absorber materials is a promising strategy to optimize the cell performances.Ferroelectric hysteresis loops of CHHow to cite this article: Sewvandi, G. A. et al. Antiferroelectric Nature of CH3NH3PbI3−xClx Perovskite and Its Implication for Charge Separation in Perovskite Solar Cells. Sci. Rep.6, 30680; doi: 10.1038/srep30680 (2016).


Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO−3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO−3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 μM NO−3 and 100 μM NO−3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO−3. Activation of denitrification could be achieved by supplementation with as little as 62.5 μM of NO−3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1  further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO−3 followed by a transient increase of NO−2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus.Chronic  Pseudomonas aeruginosa is the most severe complication in cystic fibrosis (CF) patients   and nitric oxide (NO) within sputum samples  in P. aeruginosa isolates  reduction to nitrite (NO−2) contributes directly to energy generation via the proton pumping action of the membrane-bound nitrate reductase (NarGHIJ)  by bacteria  in sputum . The Holloway PAO1 wild type was used as the control for the examinations of the Holloway PAO1 ΔnirS-N and the Holloway PAO1 ΔnosZ mutants. The PAO1 strains grew at similar rates under anoxic conditions.The strains and plasmids used in this study are listed in Table nirS to nirN and nosZ were constructed with the same procedure described previously  and frozen cultures were produced from a single colony.The reporter plasmid insertion was achieved by preparation of electro-competent cells as previously described  were isolated from expectorated sputum of 7 chronically infected CF patients  operon was studied.In this study, we first establish experimental evidence for the ability of physiological levels of NO2 <0.02% routinely monitored with an luminescent dissolved oxygen O2 sensor . Media was sealed with parafilm  and allowed to equilibrate for a minimum of 3 days. Strains were plated from frozen cultures and single colonies were used to initiate normoxic overnight cultures. They were then adjusted to OD600 0.1 and regrown to OD600 0.4 to ensure all cells were in the exponential growth phase. The culture was inoculated by diluting 1:100 in relevant anoxic Luria-Bertani broth (LB) media supplemented with KNO3 (Sigma-Aldrich) to obtain the noted concentrations of NO−3 in 2 ml glass vials , which were sealed inside the anoxic bench before transfer to shaking at 200 rpm. Physiological levels of NO−3 here are considered to be approximately 400 μM with a maximum of 1 mM based on the concentrations of NO−3 detected in CF sputum  by a spectrophotometer  and values obtained were shown to correlate linearly to dilutions of an overnight culture in LB at OD600 values > 0.02. Accordingly, only OD values higher than 0.02 were used in growth rate calculations. Growth rates were calculated as described  with O Widdel, .−3, where our measured growth rate of 0.7 divisions h−1 and final OD600 of 1.0 (data not shown) corresponded well with previously reported growth rates of P. aeruginosa  were studied using qRT-PCR and fluorescent reporter strains, respectively. Reporter strains with the promoter region of the four denitrification reductase operons; 2 for chromophore maturation  was added to the cultures to prevent new protein synthesis  were used for instrument calibration, and flow data were processed and analyzed by Diva (BD Biosciences).Detection of reporter strain fluorescence was performed using a FACSCanto flowcytometer  with a 488 nm argon laser. eGFP was detected in FL-1 with a 502 nm long pass filter and a 530/30 nm band pass emission filter. DsRed-Express fluorescence was collected in FL-2 with a 556 nm long pass filter and 585/42 nm band pass emission filter. To maximize resolution, samples were analyzed at low flow rate corresponding to 10 μl min3. The 3 h time point was chosen as it represented the approximate peak of promoter activity, as seen in the reporter strains. After 3 h, the cultures were mixed with 2 volumes of RNAlater  within the anoxic bench for transcript stabilization and kept at 4°C overnight before freezing at −80°C. The cultures were subsequently thawed on ice, centrifuged to harvest the cells and lysed with 100 μl of 1 mg ml−1 lysozyme  at room temperature for 13 min. Total RNA was extracted with RNeasy Mini Kits  and contaminating chromosomal DNA was removed by treatment with RQ1 RNase-free DNase . RNA quality was detected in a NanoDrop spectrophotometer (Thermo Scientific). cDNA was synthesized from 250 to 500 ng of RNA (depending on the concentration extracted) and High Capacity RNA-to-cDNA Master Mix  with a final reaction volume of 20 μl in a 2720 Thermal Cycler (Applied Biosystems). The conditions consisted of 5 min at 25°C, 30 min at 42°C, 5 min at 85°C and subsequent holding at 4°C. cDNA was frozen at −20°C. Negative controls contained template with the Master Mix replaced with water. cDNA was diluted 1:100 for qPCR.PAO1 was grown in triplicate for 3 h under anoxia with either unsupplemented LB or LB with 400 μM KNO1, nirS, norC, nosRnarK and the reference gene rpoD were designed using Integrated DNA Technologies Primer Quest software (http://www.idtdna.com) using sequences from the PAO1 Genome Sequence, Pseudomonas Genome Database  with initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s.Each PCR mix had a final volume of 20 μl containing 10 μl SYBR Green qPCR Master Mix 2x (Applied Biosystems) 2 μl cDNA, 2 μl of 2 pmol μlrpoD, a housekeeping gene encoding the primary sigma factor for RNA polymerase found to be constitutively expressed in P. aeruginosa  (pore size 0.22 μm) and frozen at −20°C for later measurement. NO−3 and NO−2 were detected in 96-well microtiter plates using a modified Griess nitrite/nitrate colorimetric assay  according to manufacturer's instructions and as previously described . For NO−3 concentration, NO−3 was first converted to NO−2 by incubation with NO−3 reductase and subsequent addition of the Griess reagent to detect total NO−3 plus NO−2. The NO−3 concentration was calculated as the difference between the total NO−2 and NO−3 concentration and the NO−2 concentration. The detection limit of the assay was ~2 μM for NO−2 and ~5 μM for NO−3.For measuring NO−3 and production of NO−2 as well as measurements of N2O gas accumulation in a ΔnosZ mutant strain incapable of the final reduction step in denitrification of N2O to N2. Each small glass vial  containing anoxic planktonic cultures of either PAO1 WT or the ΔnosZ mutant were placed in a 37°C heated rack and kept sealed prior to individual measurements, upon which they were shaken and the lid removed. Immediately after, N2O was detected in the media using an amperometric N2O microsensor . Unless otherwise mentioned, parametric data was analyzed by One-Way ANOVA with Dunnett's multiple comparison test and non-parametric by Friedman's test with Dunn's multiple comparison. P. aeruginosa grown for 20 h in anoxic LB supplemented with physiologically relevant concentrations of NO−3 (0–1 mM of NO−3) were calculated. PAO1  and ≥100 μM (p = 0.0132) NO−3, respectively. Despite the considerable variation between the growth rates of the clinical isolates, there was no significant correlation with the duration of infection as well as no significant difference between the growth rates of the mucoid and non-mucoid clinical isolates.The specific growth rates of 1 Figure  and 12 c1 Figure  displaye−3, where our measured growth rate of 0.7 divisions h−1 and final OD600 of 1.0 (data not shown) corresponded well with previously reported growth rates of P. aeruginosa . One mM NO−3 was required to initiate a significant increase in growth rate from the unsupplemented control in the ΔnirS-N mutant (p = 0.0021).To discern the portion of growth attributable to denitrification, we compared the specific growth rates in anoxic cultures supplemented with NOd Figure . NO−3 su−3 and the accumulation of NO−2 in the sterile filtered growth media of the ΔnirS-N mutant at 20 h confirmed that the ability to reduce NO−3 to NO−2 was present in the ΔnirS-N mutant, but that the mutant failed to further reduce NO−2 as opposed to the total consumption of NO−2 by the WT. Furthemore, we demonstrated very low concentrations of NO−3 and NO−2 pre-inoculation in LB and that the concentration of NO−2 was not affected by NO−3  in comparison to Nor, which showed a stronger peak at 2–3 h (p < 0.0001). Nir and Nos first showed significant upregulated promoter activity after 1 h and continued to be so over 20 h (p < 0.0001 and 0.0273). In all reductases other than the Nos, there were also periods of significant upregulation in the unsupplemented control, but for shorter periods of time and with lower peaks. The transcripts of the denitrification reductase genes in PAO1 grown under anoxia with 400 μM NO−3 hours showed significant upregulation of nirS, norC and nosR after 3 h   Figure .−3. We speculate that this is an explanation for the observed upregulation in our unsupplemented controls. However, the focus of the present study is on the differences due to the additional physiological levels of NO−3 supplementation.We noted that anoxic conditions alone were apparently sufficient to trigger upregulation of some denitrification reductases as previously observed in other planktonic and biofilm studies   Figure . The WT P. aeruginosa is able to thrive under anoxic conditions by performing denitrification. Our initial studies performed with clinical isolates and PAO1 under anoxic conditions showed that physiological concentrations of NO−3 are sufficient to support a significant increase in growth rate, even in cultures supplemented with less NO−3 than the 400 μM applied by Palmer et al.  regulator (Schreiber et al., The Nar product, NO−3 reduction is also able to feed into dissimilatory pathways for ammonia excretion or into assimilatory pathways for incorporation into biomolecules (Zumft, 2O production. The known pathways for N2O production are denitrification and nitrification via oxidation of hydroxylamine to N2O (Khalila et al., P. aeruginosa performs or possesses homologs of the enzymes required for nitrification [PAO1 Genome Sequence, Pseudomonas Genome Database (Winsor et al., 2 (Meyer et al., 2O was produced via the denitrification pathway and therefore acted as a specific marker. In the P. aeruginosa nitrous oxide reductase mutant ΔnosZ, unableto reduce N2O to N2, we found a significant accumulation of N2O in the anoxic growth media with as little NO−3 supplementation as 62.5 μM. This finding further supports that P. aeruginosa employs denitrification to obtain energy for growth at physiological levels of NO−3 in anoxic conditions.As N-oxides are not exclusively utilized in strict denitrification—NOs Zumft, —we also −3. This may relate to adaptations of more flexible nitrate metabolism over time, with denitrification suggested to no longer be essential for growth for some strains at later phases of infection (Oberhardt et al., −3 was unable to stimulate anoxic growth of the late isolate, alternative anoxic growth may occur by arginine fermentation (Vander Wauven et al., Interestingly, one late clinical strain isolated from a patient chronically infected for 35 years, was unable to grow in anoxic conditions with NOP. aeruginosa (Alvarez-Ortega and Harwood, −3 are also able to support growth by denitrification in anoxic conditions, highlighting the flexibility of P. aeruginosa's ability to survive in a range of environments in the CF airways (Worlitzsch et al., −3 may also enhance growth of P. aeruginosa even at micro-oxic conditions since a growth advantage of laboratory strains carrying a lasR mutation, which is associated with severe lung damage in infected CF patients (Hoffman et al., −3 in conditions with limited O2 mass-transfer (Hoffman et al., It has previously been shown that micro-oxic conditions are able to support growth of P. aeruginosa is readily able to adapt to the physiological levels of NO−3 and lack of O2 in the infected CF lungs by employing denitrification. The ability of P. aeruginosa to grow under anoxia has clinical relevance as multiple classes of antibiotics are known to have reduced efficacy in the absence of O2 (Walters et al., P. aeruginosa's observed tolerance to antibiotic treatment in CF patients (Høiby et al., narG and narH resulting in increased susceptibility of P. aeruginosa to fosfomycin and tobramycin combination treatment under anoxic conditions (McCaughey et al., P. aeruginosa as well as the highly pathogenic species Achromobacter xylosoxidans and Burkholderia multivorans in comparison to the less pathogenic Stenotrophomonas maltophilia which is unable to perform denitrification (Kolpen et al., P. aeruginosa's flexible metabolism more broadly, with the denitrification reductases presenting potential targets, and that can be administered to reach the varied lung microenvironments that P. aeruginosa is able to adapt to and persist within.In conclusion, 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.


We present a series of experimental lines of evidence, including dielectric permittivity, heat capacity and thermal conductivity measured down to 66 mK, to reveal the existence of an unusual liquid-like quantum phase in BaFe12O19, characterized by itinerant low-energy excitations with a small gap. The possible quantum liquids of electric dipoles in frustrated dielectrics open up a fresh playground for fundamental physics.Geometric frustration and quantum fluctuations may prohibit the formation of long-range ordering even at the lowest temperature, and therefore liquid-like ground states could be expected. A good example is the quantum spin liquid in frustrated magnets. Geometric frustration and quantum fluctuations can happen beyond magnetic systems. Here we propose that quantum electric-dipole liquids, analogues of quantum spin liquids, could emerge in frustrated dielectrics where antiferroelectrically coupled electric dipoles reside on a triangular lattice. The quantum paraelectric hexaferrite BaFe In magnetic materials, geometry-defined competing interactions between spins combined with quantum fluctuations can present the possibility of quantum liquid states which do not order even as 0K is approached. Here, the authors present an analogue built from electric dipoles on a triangular lattice. Geometric frustration arises on various triangle-based lattices such as one-dimensional 1D) trestle lattice, two-dimensional 2D) triangular and kagome lattices and three-dimensional 3D) B-site spinel and pyrochlore lattices, and are typically investigated in spin systemsD trestleD trianguD B-site 3467810312131415T=0 K, exotic disordered quantum phases, such as a quantum electric-dipole liquid (QEL), could emerge in certain conditions. In fact, some theoretical models proposed for ultracold dipolar particles trapped on 2D frustrated optical lattices have predicted topological quantum phases with fractional excitationsSimilar to the situation of spin lattices in magnets, geometric frustration can occur in lattices made of electric dipoles in dielectrics. In the case of small electric dipoles with significant quantum fluctuations persisting down to 2PO4/NH4H2PO4 (KDP-ADP) family or containing random-site impurities such as KTaO3:Li, which usually result in electric-dipole glasses similar to spin glasses203  and antiferroelectric (AFE) constituents such as the KH203 ref. . It was 12O19 in which small electric dipoles that originated from the off-centre displacement of Fe3+ in the FeO5 bipyramids constitute a 2D triangular lattice. Our experiments including dielectric permittivity, heat capacity and thermal conductivity measured down to 66 mK suggest the existence of a very unusual liquid-like ground state, characterized by itinerant low-energy excitations with a small gap. We consider this nontrivial quantum phase as a possible candidate of the QELs. The quantum liquids of electric dipoles in frustrated dielectrics provide a new playground for fundamental physics and may find applications in quantum information and computation as well.In this work, we demonstrate that both geometric frustration and strong quantum fluctuations can be simultaneously achieved in a unique quantum paraelectric hexaferrite BaFe12O19, belong to a completely new family of quantum paraelectrics5 bipyramids in their crystal structures, such as the W-, Z-, X- and U-type hexaferrites, are also likely candidates of quantum paraelectrics12O19 is one of the most popular magnetic materials with a wide use in magnetic credit cards, bar codes, small motors and low-loss microwave devices12O19 is shown in S block and R block along the c axis. The Fe3+ ions occupy three different kinds of sites: octahedral, tetrahedral and bipyramidal sites. In particular, the FeO5 bipyramids only exist in the middle of the R/R* blocks and form a triangular lattice in the ab plane  electric dipoles on a 2D triangular lattice is practically achieved in BaFe12O19  with long-range entanglement continues to fluctuate  of BaFe12O19 increases steadily with decreasing temperature but remains nearly constant below ∼5.5 K. No dielectric phase transition is observed down to 1.5 K. This dielectric behaviour evidences a quantum paraelectricity, similar to that in SrTiO3. The quantum paraelectric behaviour can be well described by the mean-field Barrett formulaA prerequisite of a QEL is the AFE interaction between neighbouring dipoles. To confirm the AFE coupling in BaFeA is a constant, T0 is proportional to the effective dipole–dipole coupling constant and the positive and negative values correspond to FE and AFE interactions, respectively. T1 represents the tunnelling integral and is a dividing temperature between the low-temperature region where quantum fluctuation is important and the high-temperature region where quantum effect is negligible. M=nμ2/kB, where n is the density of dipoles, μ denotes the local dipolar moment and kB is the Boltzmann constant.where ɛc below 160 K to the Barrett formula, we obtained T0=−22.9(1) K and T1=47.3(1) K. The negative T0 confirms the AFE coupling between electric dipoles. We note that recent first-principle calculations12O19. The relative strength of quantum fluctuations can be estimated by ∼|T1/T0|=2.06, which is likely high enough to favour a liquid ground state rather than an ordered or glass phase. The uniaxial anisotropy is evidenced by comparing the dielectric permittivity along the c axis with that in the ab plane. As seen in the inset of ɛ decreases slowly with decreasing temperature . The absence of a paraelectric behaviour in the ab plane is consistent with the uniaxial electric dipoles along the c axis.After fitting the 12O19 are presented in 3, a moderated electric field is able to drive the quantum paraelectric state into a long-range ordered FE state12O19, an external electric field of 5 KV cm−1 applied along the c axis has no detectable influence on the dielectric permittivity. This inertness to external electric fields may indicate the AFE interaction in BaFe12O19. Moreover, the P–E loop at 2 K , both the electronic and magnon contributions to the thermal dynamics become negligible at very low temperaturesT3 dependence would be expected for both the heat capacity and thermal conductivity. CP) of BaFe12O19 at low temperatures (T<10 K). No sharp anomaly due to a phase transition could be detected down to 0.4 K, in accordance with the quantum paraelectric behaviour. Unfortunately, the heat capacity data become scattered and noisy below ∼1 K, possibly because of the very small values that reach the resolution limit of our equipment. Thus, a quantitative analysis of the heat capacity data is not possible.Since BaFeator see  with lonκ-(BEDT-TTF)2Cu2(CN)3, the thermal conductivity measurements12O19 down to 66 mK.The thermal conductivity provides more reliable and critical information on the low-lying elementary excitations because it is sensitive exclusively to itinerant excitations and is totally insensitive to localized entities that may cause the nuclear Schottky contribution and plague the heat capacity measurements at low temperatures33κ measured in the ab plane as a function of temperature below ∼1 K. κ decreases rapidly with cooling, with a change more than 2 orders from 1 to 100 mK. No anomaly due to a phase transition is observed down to 66 mK. The thermal conductivity divided by temperature as a function of T2 is plotted in κ=βT3, with β=0.098 WK−4 m−1. Nevertheless, there is apparently an extra contribution below ∼650 mK in addition to the normal phonon term, strongly suggesting the existence of abundant itinerant low-energy excitations other than phonons. Moreover, the thermal transport behaviour at the zero temperature limit provides the key information on the nature of these low-lying excitations. As seen in κT−1 in the T→0 K limit tends to vanish rather than having a finite residual value, immediately implying the absence of gapless excitations. Instead, the data at the lowest temperature regime (T<125 mK) can be fitted toκ*=κ−βT3 in the lowest temperature region. The good linearity confirms the validity of Δ=0.16(1) K, which is much smaller than the effective dipole–dipole interaction constant T0 (∼23 K). The exponential behaviour of thermal conductivity at the zero temperature limit is very similar to that observed in the frustrated triangular magnet κ-(BEDT-TTF)2Cu2(CN)3, where a QSL with gapped excitations (Δ=0.46 K) was identifiedThe inset of c axis has no influence on the in-plane thermal conductivity in the lowest temperature range. The inertness of these low-lying excitations to external magnetic fields supports our argument that they stem from electric dipoles rather than spins.To exclude the possibility that these itinerant low-lying excitations may have a magnetic origin, we further studied the influence of magnetic field on the thermal conductivity behaviour. As seen in 12O19 that is a very unique dielectric system because both geometrical frustration and strong quantum fluctuations play an important role, a situation similar to frustrated antiferromagnets where people are looking for QSLs. This unusual ground state is characterized by itinerant low-energy excitations with a tiny gap (Δ=0.16 K) that is 140 times smaller than the effective dipole–dipole interaction constant T0 (∼23 K), a feature against a dipole glass or a classical dipole liquid. Thus, we consider it as a possible candidate of an exotic QEL. As current theoretical models proposed for frustrated spin systems, such as the transverse Ising models on a triangular lattice, are inadequate for the frustrated electric dipoles because the long-range dipole–dipole interactions are quite different from the short-range spin exchange interactions, a quantitative comparison between experiments and theories is not available at this stage. We expect that our experimental findings will stimulate theoretical efforts towards this interesting subject in the future. The present work serves as a start point to draw attention on frustrated quantum electric dipoles in dielectric materials where an abundance of exotic phenomena could be awaiting ahead.We think that the above experimental observations, especially the thermal conductivity at the zero temperature limit, strongly indicate the existence of a nontrivial ground state of electric dipoles in BaFe12O19 were prepared with the flux method and were characterized with X-ray diffraction, as shown in 3, Fe2O3 and fluxing agent Na2CO3 were weighed to a molar ratio of 1:1:1, and then were mixed and well ground. The ground raw powder was put in a Pt crucible and heated to 1,250 °C for 24 h in the air, and then cooled down to 1,100 °C at a rate of 3 °C min−1 and finally quenched to room temperature.The single-crystal samples of BaFef=1 MHz.The dielectric measurements were carried out in a Cryogen-free Superconducting Magnet System  down to 1.5 K. To measure the dielectric permittivity, silver paste was painted on the surfaces of a thin plate of crystal and annealed at 150 °C for ∼30 min to make good electrodes. An Agilent 4980A LCR meter was used to measure the dielectric permittivity with the frequency 3He refrigerator. A thin-plate-shaped sample with mass of 12.7 mg was used for this measurement. The contribution of attendant was measured separately and subtracted from the raw data. Thermal conductivity was measured between 60 mK and 1 K using the conventional steady-state ‘one heater, two thermometers' technique in a 3He–4He dilution refrigerator363 was cut from the as-grown crystals for thermal conductivity measurements. A chip heater and two RuO2 chip sensors are attached to the sample with gold wires. The temperature difference between the two thermometers was controlled to be typically 3% of the sample temperature. To minimize heat leak, superconducting NbTi wires with 15 μm diameter are used as the leads of the chip sensors.Heat capacity measurements were performed down to 0.4 K in a commercial Physical Properties Measurement System (Quantum Design) using a How to cite this article: Shen, S.-P. et al. Quantum electric-dipole liquid on a triangular lattice. Nat. Commun. 7:10569 doi: 10.1038/ncomms10569 (2016).Supplementary Figures 1-3


Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. The conceptualisation of the Singer-Nicholson fluid mosaic model for biological membranes2345677934611121314161214214The picket-fence model for compartmentalisation of phospholipids in the plasma membrane has encountered several obstacles for its full acceptance8192In order to resolve the dilemma regarding actin cytoskeleton-modulated lipid compartmentalised diffusion, we have applied stimulated emission depletion fluorescence correlation spectroscopy (STED-FCS)5222323245523Here, the experimental studies were conducted in two adherent cell types: 1) NRK fibroblasts, and 2) Ink4a/Arf (−/−) mouse embryo fibroblasts (IA32 MEFs)2L, in silico. Molecules were assumed to diffuse freely within compartments with a diffusion coefficient Dfree, while transposing compartment boundaries was possible with a certain “hopping probability”, Phop. The simulated trajectories were transformed into intensity traces, which were auto-correlated to generate in silico FCS curves for different observation spots. These curves were then fitted using standard STED-FCS analysis, which determines the average apparent diffusion coefficient, Dapp, of the molecules as a function of the observation spot diameter, dDapp, on the diameter, d, of the observation spot reveals the spatial constraints of molecular diffusion4To demonstrate the applicability of STED-FCS for detecting compartmentalised diffusion, we first simulated diffusion within a heterogeneous lattice with a characteristic average compartment length, Phop = 1) is characterized by a Dapp that is independent of d, whereas compartmentalised diffusion (Phop < 1) is highlighted by a decrease of Dapp with increasing d. Measurements at small observation spots primarily probe the free diffusion within the compartments (Dapp = Dfree), while measurements at observation spots that are comparable to or larger than the compartments themselves result in a reduced apparent diffusion coefficient, Dapp < Dfree, because the compartment barriers slow down the molecular transits through the observation spot. Specifically for Phop « 1, i.e. strong confinement, compartmentalised diffusion can be clearly distinguished from free diffusion using STED-FCS  at large observation diameters μm2/s, Phop  = 0.1 (±0.01) and L  = 80 (±8)nm, and for IA32 cells, Dfree  = 0.8 (±0.02)μm2/s, Phop  = 0.1 (±0.01) and L  = 150 (±12)nm.Experimental STED-FCS measurements  were coniameters . The expIn order to assess the underlying molecular mechanisms for the observed pattern of compartmentalised diffusion, we systematically performed STED-FCS experiments on NRK and IA32 cells where the 1) actin cytoskeleton was modulated, 2) membrane cholesterol was depleted, or 3) myosin II activity was inhibited .d = 240 nm in both cell types, markedly in NRK cells   (Actin cytoskeleton modulation was achieved by treating cells with either latrunculin B (LatB) or CK-66627RK cells , but wea = 0.06) . The dif = 0.42) .Conversely, blebbistatin inhibition of myosin II had no effect on DPPE diffusion in neither NRK nor IA32 cells . This reTo assess the generality of the observed actin cytoskeleton modulation of lipid diffusion, we also performed STED-FCS measurements closer to the cell body of NRK cells. Unlike IA32 cells, NRK cells feature relatively large cell bodies, where STED-FCS specifically probes the basal plasma membrane . These mL, in unperturbed PtK2 is smaller than the accessible spatial sampling range of STED-FCS, or that the compartment strength, Phop, is weaker than that seen here in the case of NRK cells and IA32 MEFs. Application of our simulation model to the case of PtK2 cells, we now find that a combination of these factors can explain the previous observation of apparent free diffusion of DPPE in PtK2 cells, as our simulation model shows that compartmentalised diffusion with Dfree = 0.7 μm2/s is perceived as free diffusion (for d > 40 nm) with Dapp = 0.4 μm2/s if, for example, L = 25 nm and Phop = 0.25  in these cells is compatible with free diffusion (P = 0.15)  = 0.15) . This reL, i.e. Phop = 0.1. L was found to be about two-fold smaller in NRK cells (L = 80 nm) than in IA32 MEFs (L = 150 nm). The unhindered free diffusion coefficient within the confinement regions was in both cases the same of about D = 0.8 μm2/s. Applying the Saffman-Delbruck modelIn this work, STED-FCS facilitated a minimally invasive observation of fluorescent phospholipid analogue diffusion in the plasma membrane of living cells. Our data showed a clear decrease of the apparent diffusion coefficient with increasing observation spot sizes, suggesting that phospholipids undergo compartmentalised diffusion in the plasma membrane. The confinement strength was observed to be similar in both investigated cell types such that on average one out of ten collisions with the confinement barrier resulted in the escape of the lipid probe from confinement regions of size Using a combination of pharmacological treatments, we showed that this temporal confinement was dependent on the actin cytoskeleton, but not on the plasma membrane cholesterol content. Recent studies have shown that the actin cytoskeleton is not a single structure but is composed of distinctly different sub-structuresUnlike other cytoskeletal structures, the composition of the cell cortex is poorly understood3135262629313638392/s compared to 5.4 μm2/s) and about 2-fold faster long-term diffusion coefficients (0.31 μm2/s compared to 0.17 μm2/s). This indicates that the plasma membrane compartmentalisation in our STED-FCS studies in NRK cells with a smaller and thus potentially less invasive probe (Atto647N label) is much weaker than it was suggested in the previous SPT studies with a much larger and thus potentially more invasive gold probe. Additionally, our results for the long-term diffusion coefficient are in much closer agreement with previous SPT measurements using a comparably minimally invasive dye labelled Cy3-DOPE lipidThe pattern of compartmentalised diffusion observed in our studies is in partial agreement with the original gold probe-based SPT studies by Kusumi and co-workers that first suggested that the diffusion of lipids in the plasma membrane of NRK cells is restricted by the actin cytoskeleton2. Conversely, the very fast short-term diffusion coefficient reported by Kusumi and co-workers is similar to that measured in artificial lipid membranesDfree is fixed to 5.4 μm2/s. STED-FCS measurements at even smaller observation spots (d < 40 nm) would help to define Dfree more precisely .The observed discrepancy between our STED-FCS study and previous SPT studies could be explained by the differences in the lipid probes. For example, gold probes and also related QD probes are linked to lipids via biomolecules that can attach several lipids at the same time. Oligomerised structures are likely to be significantly more sensitive to diffusion barriers, which explains the small long-term diffusion coefficient observed with SPT in combination with gold probes and QDsL = 80 nm) as compared to previous measurements by SPT (L = 230 nm). This difference could be partly explained by the fact that the spatial precision in the SPT measurements is limited by the localisation error such that the size of the compartments will be artificially larger as has been proposed previouslyWe also observed a discrepancy in the mean compartment size in NRK cells whereby our results were 3-fold smaller , we are compelled to infer that plasma membrane compartmentalisation by the actin cytoskeleton is a fundamental cellular process, generic to most cells. The determination of the precise structural and molecular mechanisms by which the cytoskeleton performs lipid compartmentalisation 2] of the diffraction-limited excitation light  and a time-averaged maximum intensity I = 1/2 P/[π(FWHM/2)2 stemming from P = 98 mW for the highest STED power). Calibration of the diameter d(PSTED) of the effective fluorescence observation spots formed by a certain STED power PSTED was performed by STED-FCS measurements of fluorescent lipid analogues in supported lipid bilayers (SLBs), for that such lipid bilayers provide a two-dimensional free diffusing system of molecules, labelled with the same fluorophore used in our cellular experiments. The confocal FWHM being determined by fluorescent beads (d(PSTED = 0)≈240 nm), the other effective diameters d(PSTED≠0) can be calculated using the relation:The STED nanoscope was based on a home-built confocal microscope setup equipped with a 640 nm laser  for excitation with a repetition rate of 45 MHz of the fluorescent label. The STED beam was provided by a Titanium:Sapphire laser system  operating at 780 nm with a repetition rate of 90 MHz. The time interval between the pulses of both lasers was adjusted using a home-built electronic delay unit, where the STED pulses served as the trigger master. The STED laser pulses were stretched from 200 fs to a pulse length of approximately 180 ps using four 30 cm optical SF6 glass rods and a 125 m long polarisation maintaining single-mode fibre (OZ Optics). Fluorescence excitation and collection was realized using an oil immersion objective . The laser beams were spatially overlaid and the fluorescence light filtered by appropriate (dichroic) filters . The doughnut-shaped focal spot of the STED beam featuring a central intensity zero was produced by introducing a phase-modifying plate (RPC Photonics) into the beam path, imprinting on the wave front a helical phase ramp exp(iφ) with 0 ≤ φ ≤ 2π. A λ/4-plate ensured circular polarisation of the STED and excitation beams. The fluorescence was coupled into a multi-mode fibre splitter (Fiber Optic Network Technology) with an aperture size corresponding to 1.4x the magnified excitation spot. The 50:50 split fluorescence signal was then detected by two single-photon counting modules  and the recorded fluorescence counts were further processed by a hardware correlator card . The focal intensity distribution of the excitation and STED light were measured by scanning a scattering gold bead of 80 nm in diameter  using a non-confocal detector . The applied laser powers tD stands for the average transient times through the observation spot as determined by FCS for each given PSTED. The relation above stems from the fact that for absolute two-dimensional free diffusion, the transit time scales proportionally with the size of the observation area.where N ≈ 0.5–2 fluorescent particles in the observation spot for the highest STED power and N ≈ 10–30 fluorescent particles for confocal recording. We fitted all correlation data with a model for two-dimensional diffusion, resulting in different values of the average transit time tD through the observation spot. Each individual measurement resulted in a transit time tD. The correspondent apparent diffusion coefficient Dapp = d2/(8 tD ln(2)) was calculated based on the knowledge of the diameter d of the observation spot, provided by the calibration of the system.The general theoretical background for STED-FCS analysis has been described in detail5Dapp between data representing FWHM = 40 nm and FWHM = 240 nm in the same experiment, as well as for comparison of the mean Dapp between data representing FWHM = 240 nm and control experiment (FWHM = 240 nm data), we used unpaired student’s t-test.For comparison of the mean Supported lipid bilayers (SLBs) were used to calibrate the STED-FCS setup. The SLBs were prepared based on the following procedure: The lipid DOPC  and the fluorescent lipid analogue  were mixed in organic solvents (Chloroform/MeOH 1:1) at a lipid concentration of ~1 mg/ml. The ratio of labelled lipids per non-labelled ones was approximately 1:10,000. 50 μl of such solution were dropped onto a piranha-cleaned  standard microscope cover glass  and spin-coated at 60 Hz for about one minute. The cover glass was then placed in a microscopy chamber and subsequently the dry thin lipid film was rehydrated with 500 μl buffer solution . Such bilayers were stable for several hours.2 in air. Incorporation of the fluorescent lipid analogues (DPPE-Atto647N) into the (presumably outer leaflet of the) plasma membrane of the cells was accomplished via BSA coupling, as in detail outlined before [Refs. IA32, NRK, IA32 2xKD and PtK2 cells were seeded on standard glass coverslips  to a confluence of about 60% and grown at 37 °C in a water-saturated atmosphere of 5% COre Refs. .We assessed the dynamics of DPPE-Atto647N by placing the focused co-centred excitation and STED beams on random positions at the plasma membrane. On the lamellipodium or very close to the edge of the cells, we probed lipid diffusion concurrently in both the apical and basal membranes. That happens because the effective observation volume is decreased only laterally by the STED beam, remaining approximately 700 nm long in the axial direction (for excitation wavelength of 640 nm), thus encompassing both membranes when they are close enough . Especian individual cells (from r > 4 different sample preparations) were then averaged so that the correspondent error bars reflect mostly the variance among cells. We performed experiments in cells with passage number less than 20 and in samples where cells presented normal morphology. Our cells are once per year tested for mycoplasma.Measurements were taken at room temperature and completed before any significant morphological changes in the cell could occur. The duration of all measurements were 10 s; a correlation time longer than two orders of magnitude times the typical transient time of the labelled lipids through the confocal observation area. For each FWHM (or STED power) the average and standard deviation of the apparent diffusion coefficient was calculated in the following way. First, an average value was determined from 5-8 measurement on different positions of a single cell. The averaged values obtained from As detailed previously5The cells were treated with 1 U/ml Streptomyces spec. COase (Sigma-Aldrich) in HEPES buffered DMEM (HDMEM) (and washed afterwards in HDMEM) for 30 min under culture conditions. Treatment was performed before the insertion of the fluorescent lipid analogues into the plasma membrane.Treatment with 15 μM Blebbistatin (EMD Millipore) in HDMEM (and washed afterwards in HDMEM) for 2 h under culture conditions.Treatment with 1 μM (100 nM) Latrunculin B (Sigma-Aldrich) in HDMEM (and washed afterwards in HDMEM) for 15 min under culture conditions was performed for IA32 cells (NRK cells). NRK cells were found to be more sensitive to this drug treatment than IA32 cells, in a way that for these cells reduction of Latrunculin B concentration to 100 nM produced similar results to 1 μM. Treatment was performed before the insertion of the fluorescent lipid analogues into the plasma membrane.Treatment with 100 μM CK-666 (EMD Millipore) in HDMEM (and washed afterwards in HDMEM) for 4 h under culture conditions. Treatment was performed before the insertion of the fluorescent lipid analogues into the plasma membrane. In addition, insertion of fluorescent lipid analogues and STED-FCS measurements were carried out in 100 μM CK-666 in HDMEM.Phop. This was implemented in the following way: If free diffusion with diffusion constant Dfree would have led the lipid to cross the compartment boundary, the effective transposition would have taken place in only a fraction of such cases (being defined by Phop) while in all other cases the molecule would remain diffusing in the original compartment. The simulation area was a circle of 3 μm diameter and the compartmentalisation of this area was implemented as a Voronoi mesh on a uniform random distribution of seed points. We defined the square root of the average compartment area as the average compartment size or length L. The average compartment size, the hopping probability and the free diffusion coefficient completely described our simulation model. In most cases a simulation placed 100 independent molecules in the simulation area and took a time span of 200 s with 20 μs time steps.Monte Carlo simulations were performed using custom written routines in Matlab. In these simulations, we generated fluorescence time traces of 2-dimensional diffusion of fluorescent molecules through an observation spot with a Gaussian-shaped fluorescence detection profile. The area explored by diffusion comprised randomly sized compartments. Within a compartment the molecules were assumed to diffuse freely while crossing from one compartment to another was only possible with a given “hopping probability” tD was converted to an apparent diffusion coefficient using Dapp = d2/(8 tD ln(2)) with diameter d of the observation spot given as the FWHM.The simulated fluorescence time traces were auto-correlated and the correlation curves were fitted to the aforementioned two-dimensional diffusion model. The transient time D(d) dependence was achieved by performing repeated simulations on iteratively finer spaced parameter grids based on the minimisation of the squared distance of the mean apparent diffusion coefficients to the measured values. After reaching an approximate accuracy of 1 × 10−10 cm2/s in Dfree, 0.01 in Phop and 5 nm in L, the program stopped running the iterations and displayed the most pertinent set of fitted parameters achieved.Fitting of the measured hopP and L was observed with the axis of largest error being the ratio of both.Confidence intervals on the best model parameters were calculated by applying the Bootstrap methodHow to cite this article: Andrade, D. M. et al. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS. Sci. Rep.5, 11454; doi: 10.1038/srep11454 (2015).


This work presents new approach to tailor degradation behavior of iron by incorporating biodegradable polymers into the metal. Porous pure iron (PPI) was vacuum infiltrated by poly(lactic-co-glycolic acid) (PLGA) to form fully dense PLGA-infiltrated porous iron (PIPI) and dip coated into the PLGA to form partially dense PLGA-coated porous iron (PCPI). Results showed that compressive strength and toughness of the PIPI and PCPI were higher compared to PPI. A strong interfacial interaction was developed between the PLGA layer and the iron surface. Degradation rate of PIPI and PCPI was higher than that of PPI due to the effect of PLGA hydrolysis. The fast degradation of PIPI did not affect the viability of human fibroblast cells. Finally, this work discusses a degradation mechanism for PIPI and the effect of PLGA incorporation in accelerating the degradation of iron.Iron and its alloy have been proposed as biodegradable metals for temporary medical implants. However, the formation of iron oxide and iron phosphate on their surface slows down their degradation kinetics in both  Biodegradable metals have been studied as new potential materials for load-bearing temporary medical implants where they support the healing process of diseased tissue and progressively degrade thereafter22 evolution and slower decrement of compressive yield strength in immersion tests using simulated body fluid (SBF)161819In vitro cytotoxicity assessment on porous Fe-Mg, pure iron and Fe-CNTs manufactured by replication method showed a small proliferation effect towards osteoblast21622222426Recently, porous biodegradable metals have also been viewed as potential materials for hard tissue scaffolds578101112132O3 created more phase/grain boundaries which acted as active sites for accelerating degradationHowever, degradation rate of porous iron is still considered too slow and unmatched with the tissue healing period232823273032co-glycolic acid) on bulk magnesium and its alloys, in an attempt to slow down their degradation, has generally turn to the opposite results38394041co-glycolic acid) or PLGA degrades quite rapidly. It takes 1-2 months for a complete degradation depending on the ratio of lactic to glycolic acidsin vitro and in vivo444546Above all, the reason for slow degradation kinetics of iron is the formation of passive iron oxide and phosphate layers. Acidic condition has been known to greatly escalate the dissolution and solubility of iron degradation products36−1 of PLGA spectrum assigned to carbonyl (C=O) stretching, one of the characteristic peaks for PLGA  compared to those of PCPI (0.1–0.15 strain) and PIPI (0.08–0.14 strain). The plateau region refers to the possible events of buckling, yield and fractures of cell walls56The PCPI possesses slightly higher strength compared to PIPI and PPI . Meanwhi102+ concentration and degradation rate (6.42 mm/year) followed by PCPI (0.76 mm/year) and PPI (0.33 mm/year). Higher weight loss in PIPI can be attributed to the rapid degradation (hydrolysis) of PLGA. In association with immersion test, pH measurement shows a decreasing value from 7.5 to 5.82 and to 5.18 for pure PLGA samples with equivalent weight to that in PCPI and in PIPI samples (− ions samples . The TGA samples  shows thcorr) of the samples increases in order of PPI < PCPI < PIPI. The PIPI possess the highest corrosion rate (0.72 mm/year) compared to that of PCPI (0.42 mm/year) and PPI (0.11 mm/year), a similar trend of degradation rate as that for immersion test. The corrosion potential of PIPI is the lowest among others indicating higher corrosion susceptibility for the PIPI. The shifting of corrosion potentials to more negative values indicates that the polarization are mostly occurred at the cathode and hence the corrosion rates of iron in PCPI and PIPI are cathodically controlled61+ as cathodic reaction, together with common oxygen reduction. The exchange of icorr in H+ reduction is much higher than that in oxygen reduction, and results in higher icorr and consequently the corrosion rate2 from 2.33 μA/cm2 at day 1 postulating that the passive oxide layer resided on the iron surface and hence slow down the degradation rate of iron. PCPI and PIPI had the constant current densities of 11.77 and 17.79 μA/cm2 respectively. These values are still much higher than those at day 1, which are 5.18 and 9.84 μA/cm2 for PCPI and PIPI respectively. This indicates that the degradation rate of iron in PCPI and PIPI have been accelerated in the presence of PLGA.As shown by the potentiodynamic polarization curves in ct) or so-called polarization resistance (Rp) and by the double layer capacitance (Cdl) whose values depend on the increase of wetted metal area. PPI sample showed a larger Rp , and only two peaks on PCPI and PIPI (3436 cm−1 and 1630 cm−1). A broad peak at 3422 cm−1 on PPI indicates the existence of b-FeOOH or akaganéite in the degradation products68−1 connotes an O-H stretching due to adsorbed water in the degraded pure iron7071−1 implies the existence of γ-FeOOH or lepidocrocite. Two medium intensity peaks at 900 cm−1 and 744 cm−1 ascertain the existence of α-FeOOH (goethite) and γ-FeOOH (lepidocrocite), respectively7374−1 reveals Fe-O stretching vibration in magnetite, Fe3O4−1 indicates the O-H stretching vibration of hydroxyl end groups in the degradation products−1 is assigned to O-H stretching due to the presence of water molecules in the degradation products (hydration). The band at 3436 cm−1 signifies the presence of the hydroxyl group (OH) proving the hydrolysis of PLGA in the PCPI and PIPI. It is known that the ester group in the PLGA hydrolyzed into OH and carboxyl (RCOOH) end group through the hydrolysis which leads to an overall increase of OH groups in the degrading PLGA77−1 peaks at week 4 is relatively higher for both of PCPI and PIPI, compared to those at week 2. This obviously indicates that more hydrolysis had taken place in samples at week 4 as OH groups increased with the progression of immersion time. In addition, it can be postulated here that the degradations of iron in the PCPI and PIPI are greatly proportional to the rate of the PLGA hydrolysis.The IR spectra  indicate3O4, Fe2O3.H2O, Ca3(PO4)2, Ca2P2O7, α-FeOOH and (MgFe)3(PO4)2.8H2O. The low solubility Fe3O4, which was formed at the early corrosion process due to insufficient oxygen supply to the deposited Fe(OH)2, resides at the innermost degradation layer in contact with the iron substrate and impedes further degradation of iron3(PO4)2, Ca2P2O7 and (MgFe)3(PO4)2.8H2O were formed from the precipitation of PBS component and reside on the PLGA’s outermost surface, as postulated by previous study3 which is resistant to mass transferThe XRD patterns on the surface of PPI and PIPI samples after 4 weeks of degradation  identifyIn a neutral aqueous environment, generally iron (PPI) degrades via the following reactions:2 could be further air-oxidized to form Fe3O42 triggers the formation of Fe3O4 on the PPI surface. Hence, we can assume that some parts of the PPI's struts have been covered by tightly-adhered Fe(OH)2 which suppresses oxygen diffusion onto the iron surface. Further corrosion of iron is therefore inhibited as reported elsewhere2330792 layer as hydroxide layers is more soluble in acidic environment3637+ through a heterolytic cleavage of OH bond. The H+ ions react with the electrons at the iron surface to form hydrogen gas (hydrogen evolution) allowing a continuous passage of an equivalent quantity of Fe2+ into the solution. The hydrogen evolution disrupt the formation of inhibition layer facilitating oxygen diffusion onto the iron surface and enhance iron degradation+ reduction is relatively one-order higher than that of the oxygen reduction  for iron electrode+ is reduced on the iron surface and more electrons are required to complete the reaction. Higher rate of electron supply requires higher rate of iron dissolution (Fe2+). The tendency of H+ to be reduced at the iron surface is more prominent as H+ ions are much easier to diffuse from the electrolyte onto the iron surface as the diffusion coefficient of hydrogen is bigger than oxygen2) leading to hydrogen blistering and adding an extra aid to the overall iron degradation. Due to the concentration gradient (more hydrogen on the surface), the hydrogen atoms were adsorbed and absorbed and diffuses into the bulk of the metal84848682The Fe(OH)+ and thus increases the hydrogen adsorption on the iron surface that consequently promotes its absorption into the bulk. As also proved by PDP result in + ion . Hence, the more H+ attracted onto iron surfaces, the more tendency iron will degrade further. Strong interfacial interaction between PLGA and iron surface could be a significant catalyst for continuous H+ supply on iron surface through the hydrolysis of the PLGA.8489905 cells) compared to PPI and control after 24 h incubation  structure in the forms of PLGA-infiltrated porous iron (PIPI) and PLGA-coated porous iron (PCPI). The compressive strength of PCPI is higher than PIPI and PPI with the values fall within the range of that of cancellous bone. A stronger interfacial interaction between PLGA and iron surface resulted into a faster degradation of both PCPI and PIPI. Degradation rate of PIPI is faster than PCPI and PPI as measured by weight loss and Feco-glycolic acid) (PLGA) granules  were used as the raw materials. The PLGA granules were dissolved in chloroform to produce PLGA solution (5% w/v). The PLGA solution was infiltrated into the PPI at vacuum pressure of −0.07 MPa for 20 min to produce samples of fully dense PLGA-infiltrated porous iron (PIPI). Samples of partially dense PLGA-coated porous iron (PCPI) were produced by dipping the PPI into the PLGA solution using a dip coating machine  at down and withdraw speeds of 200 mm/min for 1 cycle at dwelling time of 8 s. The residual solvent on the samples were let to evaporate at room temperature for 48 h.Two sets of porous pure iron (PPI) sheets  and poly and the decomposition were done by the Shirley-type background removal.Microstructural analysis and elemental mapping were done on the polished and gold coated PIPI and PCPI samples using a scanning electron microscope  coupled with an energy-dispersive X-ray spectroscopy . Prior to the EDS mapping, the PIPI were cross-sectioned and mechanically ground using silicon carbide SiC papers up to 1200 grit. The interfacial interaction between the iron surface and the PLGA was investigated using an attenuate transform infrared-Fourier transform infrared spectroscopy  and further confirmed by a thermogravimetric analyzer and differential thermal analyzer  and an X-ray photoelectron spectroscopy . The TGA-DTA analysis were run from room temperature to 700 °C at a heating rate of 10 °C/min in nitrogen environment with a purging rate of 10 ml/min. The XPS analysis was conducted using an incident X-ray radiation Mg KαCompressive tests were done on five samples of 450 μm and 800 μm PPI, PIPI and PCPI (specimen dimension = 15 × 12.5 × 1.6 mm) at 0.001 mm/s cross-head speed with a 1 kN load cell using a universal testing machine . The test and the determination of compressive strength, yield strength and modulus of elasticity were done by following the ASTM D1621 and ISO 844 standards. The compressive strengths were computed as the first maximum on the stress-strain curve while the yield strengths were determined at 0.2% offset strain. The values of modulus of elasticity were measured as the slopes of stress-strain curves in the elastic deformation region, while toughness was determined by calculating the area under the compression curves.Degradation tests were carried out by using five different approaches: weight loss complimented with ion release measurement as a direct indicator of degradation rate, then potentiodynamic polarization (PDP), potentiostatic polarization (PSP) and electrochemical impedance spectroscopy (EIS) to measure corrosion current and the effect of PLGA on the charge transfer. Static immersion test was performed on PPI, PCPI and PIPI samples where each 5 specimens (dimension = 6 × 6 × 1.6 mm) were immersed in 30 ml of phosphate buffered solution  in a 50 ml glass beaker. The temperature and pH were monitored and maintained at 37 ± 0.5 °C and 7.4 by placing the beakers in an isothermal water bath and by refreshing the solution every 24 h. Weight loss was measured at week 1, 2 and 4 on at least 5 specimens from each group. The specimens were rinsed in deionized water and ethanol then brushed gently as specified by the ASTM G1 standard, followed by air and vacuum drying for 48 h to completely remove all degradation products before weighing. Degradation rate was calculated by using the following formula:2), t = time of exposure (h) and ρ = density (g/cm3). The density was determined via Archimedes principle method where the values of 1.89 g/cm3, 1.12 g/cm3 and 1.26 g/cm3 for PPI, PCPI and PIPI respectively, were used.where CR = corrosion rate (mm/year), W = weight loss (g), A = area  was obtained. Corrosion current was determined using Tafel extrapolation and corrosion rate were measured using the following formula:icorr = corrosion current density (μA/cm2), EW = equivalent weight (28 g/eq) and ρ = density.The PDP tests were carried out using a standard three-electrode system in 250 mL of PBS with graphite rod as the auxiliary and Ag/AgCl (KCl 3.5 M) as the reference electrodes connected to a potentiostat . The working electrode  has 0.502 cmThe PSP tests were conducted using similar instrument set-up and set of specimens as for the PDP tests but by using a constant stepping potential of −0.5 V for 18 hours after 7 days immersion in 250 mL PBS solution at 37 ± 0.5 °C and the trends in the current densities were monitored. Meanwhile, the EIS experiments were performed on similar set of specimensas for the PDP tests up to 2 hours in 250 mL PBS at room temperature. VersaStudio software  was used for data acquisition at open circuit potential over a frequency range of 100 kHz down to 10 mHz using a peak-to-peak 10 mV sinusoidal perturbation. Additional electrochemical tests were conducted to evaluate the susceptibility of hydrogen atom adsorption onto iron surface by using open circuit potential (OCP) method. Similar instrument set-up and set of specimens as for the PDP tests was used and the tests were conducted at 37 ± 0.5 °C for 30 minutes in 250 mL PBS after day 1, day 3 and day 7 of immersion.2+) concentration. Surface morphology of the degraded samples was analyzed using the SEM/EDS after week 1 and 4 of immersion. Elemental composition profile across the iron’s substrate and degradation layer was mapped using EDS profiling while an X-ray diffractometer  was used to determine the phases residing in the 4 week degraded samples. The XRD data were analyzed using X’Pert HighScore software . Fourier transform infrared spectroscopy  was used to characterize the changes in chemical structure of degraded samples after week 2 and 4 of immersion. The FTIR absorbance spectra were obtained with 32 scans per sample over the range of 4000 to 400 cm−1 and then subtracted from the background ratio and plotted using OriginPro . The FTIR was also used to ascertain the formation of common degradation products of iron and the changes in O-H and carboxylic end group of the degraded PCPI and PIPI samples.Samples of 6 ml of the test solution from each group after immersion tests were analyzed using a graphite furnace atomic absorption spectrophotometer  to determine ferrous ion  and placed in a 6-well plate. Possible effect of chlorinated solvent residue, which may inhibit cell proliferation as found in our preliminary studies, was diminished by continuous stirring of PLGA solution during PIPI preparation to evaporate all the remaining solvent. The control consists of seeded cells and medium only without specimens. Six replicates were used for each group. Two mL of MEM enriched with 10% fetal bovine serum was added to each well. Cultures were maintained in the incubator up to 72 h for cell attachment analysis while cell adhesion was investigated after 24 h. Subsequently, cell proliferation analysis after 24 and 72 h were also performed using MTT . Five mg/ml MTT -2,5-diphenyl tetrazolium) solution was prepared by dissolving MTT in PBS. 100 μl MTT solution was added to each well to form formazan by mitochondrial dehydrogenase. After 4 h of incubation, 1 ml of DMSO was added and shaken gently for 10 min to dissolve the formazan crystals. 200 μl of supernatant was transferred to 96-well plates and three data points were obtained from each sample. The optical density was determined with a microplate reader  at a wavelength 570 nm. All results were expressed as mean ± standard deviation (SD) of 6 replicates. The SigmaPlotTM software  and student’s t-test was used for statistical analysis and multiple comparison and significance level was determined at p < 0.05.Human skin fibroblast cells (HSF 1184) were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% (v/v) COHow to cite this article: Yusop, A. H. M. et al. Controlling the degradation kinetics of porous iron by poly(lactic-co-glycolic acid) infiltration for use as temporary medical implants. Sci. Rep. 5, 11194; doi: 10.1038/srep11194 (2015).