Porous silicon (pSi) continues to receive considerable interest for use in applications ranging from sensors, biological scaffolds, therapeutic delivery systems to theranostics. Critical to all of these applications is pSi degradation and stabilization in biological media. Here we report on progress towards the development of a mechanistic understanding for the dissolution behavior of native (unoxidized) and thermally oxidized (200–600 °C) pSi microparticles. Fourier transform infrared (FTIR) spectroscopy was used to characterize the pSi surface chemistry after thermal oxidation. PSi dissolution was assessed using a USP method II apparatus by monitoring the production of orthosilicic acid, and the influence of gastro-intestinal (GI) fluids were examined. Fitting pSi dissolution kinetics with a sum of the exponential model demonstrated that the dissolution process strongly correlates with the three surface hydride species and their relative reactivity, and was supported by the observed FTIR spectral changes of pSi during dissolution. Finally, the presence of GI proteins was shown to hamper pSi dissolution by adsorption to the pSi surface acting as a barrier preventing water attack. These findings are significant in the optimal design of pSi particles for oral delivery and other controlled drug delivery applications.  More than 20 years after the initial discovery of porous silicon (pSi) photoluminescence by Professor Canham, and his subsequent work on the fabrication of bioactive pSi , there rMany biomedical applications of pSi are influenced and dependent on the balance between dissolution/erosion versus oxidation of the silicon matrix that occurs in aqueous bio-relevant environments. The Si–Si and Si–H bonds are intrinsically weaker than their oxidized analogues, Si–O–Si and Si–OH . When naPrevious pSi dissolution studies have typically involved direct visualization/monitoring of a planar pSi wafer surface using techniques such as atomic force microscope (AFM), scanning electron microscope (SEM) ,31,32, pWe have previously reported on the use of thermally oxidized pSi microparticles for oral delivery of poorly water-soluble non-steroidal anti-inflammatory drugs ,40. In t2/g, and an average particle size of 49.4 µm  as determined by a laser diffraction . PSi thermal oxidation was performed in a clean oven by heating in air at 40 K min−1 to a specific oxidation temperature in the range 473–873 K for 1 h, before convective cooling to ambient temperature.PSi powder and wafer samples (p-type silicon) used in this investigation were supplied by pSiMedica Ltd.  and showed a porosity of ~70%, a specific surface area (SSA) of 310.2 m® buffer solution pH 7.2 aminomethane HCl), pancreatin from porcine pancreas  and pepsin , were purchased from Sigma-Aldrich Australia Pty Ltd, Sydney, Australia. All the other chemicals were analytical grade used as received. High purity water  was used throughout the experiments.Tween 80 (Polysorbate 80) and sodium dodecyl sulfate (SDS) were purchased from Chem-Supply Pty Ltd., Adelaide, Australia. Trizma® syringe filter . The aliquots were analyzed to determine the concentration of silicic acid using a validated molybdenum blue spectroscopic assay . For dissolution experiments performed in phosphate buffer solution (pH 6.8), the samples were analyzed using ICP-AES , since phosphate interferes with the molybdenum blue assay. All the experiments were conducted in duplicate unless otherwise indicated.pSi dissolution was conducted with a VanKel™ (VK 6010) dissolution test station attached with apparatus USP II  at 37.0 °C (± 0.5). PSi particles  were pre-wetted with 200 µL of ethanol and then transferred into 500 mL of 0.05 M Tris buffer solution at pH 7.2 . At predetermined time points, aliquots of media (5 mL) were collected and filtered with a Millex−1 resolution. Additionally, solid residues from the dissolution study were extracted, thoroughly rinsed with Milli-Q water and then dried at 105 °C for 2 h in an oven prior to FTIR analysis.Infrared transmission spectra of pSi particle samples were obtained with a Nicolet Magna-IR 750™ FTIR spectrometer equipped with a liquid nitrogen-cooled mercury-cadmium-telluride (MCT) detector . The particles were pressed into KBr discs and 256 scan spectra were recorded with a 4 cmWater contact angle measurements were performed on pSi wafers using a static sessile drop method . The wafers were either unoxidized or thermally oxidized using equivalent conditions to those used for the pSi microparticles.Thermal oxidation of pSi particles was carried out at 200, 300, 400, 500, and 600 °C for 1 h in a clean furnace, open to the atmosphere. The surface area and porous nature of the thermally oxidized pSi samples are presented in −1 was observed. These absorption bands were assigned to Si-Hx stretching modes  [−1 indicated that dihydride (SiH2) was the more predominant surface species, in agreement with previous reports [Upon thermal oxidation of the pSi particles, there is a reduction in the surface area and mesopore volume; this is due to the increased molecular volume of silicon oxidation products, causing constriction of the mesoporous pathways within the pSi. The changing Si speciation upon thermal oxidation was monitored using FTIR spectroscopy, with the IR spectra for both the native and thermally oxidized pSi shown in  to low) ,42, the  reports ,44. Upon reports ,46.−1), a sharp absorption band at 615 cm−1 with a shoulder at 660 cm−1 was observed for native and 200 °C oxidized pSi samples, attributed to Si–Si crystal mode and Si–H2 wagging modes, respectively [−1 was observed with increasing oxidation temperature of the pSi, which was assigned as an oxidised O–SiHx deformation mode [−1 was attributed to a Si–H2 scissoring mode, while the broad band at 1030 cm-1 was attributed to Si–O–Si asymmetric bending. The increased intensity of the 1030 cm−1 band and the decreased intensity of absorption (Si–H2 scissoring) implies that mild oxidation had occurred. This feature became more pronounced with increasing oxidation temperature (>300 °C). The presence of this band (1030 cm−1) in samples not exposed to high temperatures indicates that oxidation of the pSi sample was slowly occurring under ambient conditions. Finally, a band was observed at 2250 cm−1, associated with the backbond oxidized Si–Hx, i.e., (Si–O–)Si–Hx [In the finger print region Si–Hx .−1 disappeared; these bands were associated with Si–H2 wagging, scissoring, and Si–Hx stretching modes. The band at 2250 and 2205 cm−1 due to the backbond oxidized Si–Hx stretching became more profound. These absorptions could be precisely assigned to OxSiHx groups with oxygen-saturated state, i.e., O3SiH (2250 cm−1), O2SiH2 (2200 cm−1), OSiH3 (2160 cm−1) according to the literature [−1 became more intense with a growing protruded shoulder compared to that at 200 °C, in agreement with previous observation [−1 associated with bulk Si–Si decreased, indicating oxidation had started altering the Si–Si “skeleton” at 300 °C, something that has been referred to previously as a threshold temperature for silicon thermal oxidation [−1 had increased in intensity. The spectra of the thermally oxidized pSi particles at 300 °C and above displayed more extensive evidence of oxidation characteristics. The bands at 660, 905, and 2075–2140 cmterature . A broadervation . The banxidation ,47. The −1 due to the asymmetric and symmetric mode of Si–O, which is often observed with fully oxidized silicon; its intensity further increased with oxidation at 600 and 800 °C (data not shown). The single band at 2250 cm−1 instead of the duplet and triplet peaks from pSi sample oxidized at 300–400 °C indicated that there was only SiH with fully oxidized Si–Si backbonds  on pSi surfaces. The phenomenon of Si–Si backbond oxidation can be explained by bond energy calculations, i.e., the bond strength of surface hydride (Si–H) increases with the increase of oxidation degree of its Si–Si backbond. Thus, the survival of the surface Si–H bond at 500 °C would be the one that has three oxidized backbonds, i.e., H–Si(O)3 [The pSi samples treated at 500–600 °C showed more significant oxidation features in their infrared spectra. For oxidation at >500 °C, a new absorption band appeared at 800–790 cmH–Si(O)3 . EventuaFurthermore, there is a strong correlation between pSi surface chemistry and the water contact angle, as shown in In summary, the thermal oxidation of pSi particles in this study follows the backbond oxidation phenomenon that has previously been observed with the oxidation of hydride passive silicon wafers ,47,51,52w/w dissolved before the first sampling time point at 5 min. In comparison, OXpSi-200 °C dissolved 37% of its initial mass in the first 10 min. The dissolution was accompanied by bubbling hydrogen gas due to the hydrolysis of hydrogen passivated surfaces [Dissolution profiles in Tris buffer solution at pH 7.2 for pSi particles of different levels of oxidation are presented in surfaces ,34. The Dissolution profiles for pSi particles oxidized at temperatures >300 °C are shown in -1) and Si–H2 wagging modes for pSi samples thermally oxidized at increased temperatures correlates well with the extent (%) of pSi dissolution observed after 8 h. This highlights the critical dependence of pSi dissolution on the availability of unoxidized surface hydride species. Similarly, in R2 = 0.99) relationship was observed between the integrated absorbance due to Si–O stretching and dissolution time. This indicates that oxidation of the pSi particles in the dissolution media follows zero order kinetics. Given that ~80% pSi dissolution was observed in the first 30 min, i.e., when the extent of pSi oxidation remains relatively low, the pSi dissolution was not significantly affected by the concurrent oxidation occurring.In order to further elucidate the influence of pSi oxidation on the dissolution rate, a parallel experiment was set up, where, at specified time points the dissolution was terminated, and the residual pSi particles were collected and examined with FTIR. The infrared spectra (not shown), indicated that once in contact with the aqueous medium, the pSi simultaneously undergoes both dissolution and oxidation. The proposed mechanism for pSi oxidation in water is oxygen attack of the Si–Si backbond, analogous to pSi oxidation in air , where tφ ≈ 10 nm) in addition to adsorbing at the surface. It has previously been reported that proteins such as albumin, papain, and insulin are able to interact with pSi, as well as being able to penetrate into the porous network [Dissolution kinetics of unoxidized pSi microparticles in simulated gastric fluid (SGF) with/without pepsin are given in  network ,57,58.2C(CH2OH)3) from Tris buffer might act as a catalyst to hydrolyze surface hydrides of pSi, such catalytic effects of organic amines on pSi hydrolysis were observed previously [Dissolution kinetics of unoxidized pSi microparticles in simulated intestinal fluid (SIF) with/without pancreatin are given in eviously .In the presence of pancreatin, fast dissolution was observed in the first 2 h, with ~50% of the initial mass dissolved; this was lower than that without pancreatin. Pancreatin is a mixture of the digestive enzymes including trypsin , amylase  and lipase  . At neutpSi dissolution (as described in Equations (1) and (2)) occurs via a nucleophilic substitution mechanism. Given that the dissolution media volume was far in excess of the sample mass, it can be assumed that pSi dissolution follows pseudo-first order kinetics. However, this was shown not to be the case since a plot of semi-log pSi % remaining versus time data was not linear (data not shown), and more complex kinetics are apparent. The pSi surface is comprised of monohydrides, dihydrides, and trihydrides, each with highly varied activities in aqueous solution. As reported in the literature, the reaction of a vertical dihydride site is five times faster than a terrace monohydride, but two times slower than a kink site (a distorted monohydride) . In addik is the rate constant tC, ∞A and ∞B are concentration of silicic acid at time, t and ∞.P indicates the relative fraction of fast (fP) and slow (sP) dissolving species, and fP + sP =1.The presence of various reaction sites and the parallel oxidation processes result in more complex dissolution kinetics. The total silicic acid is better described as a sum of silicic acid from each surface species reacted with water. Thus, for the reaction of a surface species, with respect to silicic acid,fP + mP + sP = 1. The left term in Equation (7) is equivalent to the ratio of the remaining pSi on the assumptions that (a) pSi will be completely converted to silicic acid at infinity time, and (b) there is no side reaction nor reverse reaction, and silicic acid is the only product. This is rational according to the reaction mechanism described earlier since all surface hydrides react with water to form silicic acid, a new layer of hydride would be reconstructed and ready to next cycle of reactions.This multi-exponential  approach was used by Truesdale et al. to describe biosilica dissolution data . The modAs shown in The third fraction from OXpSi-200 accounts for 44% of the sample mass, which dissolved with a rate constant similar to those of pSi oxidized at 300 and 400 °C. The difference between dissolution observed from OXpSi-200 and pSi clearly indicated changes in their physicochemical properties. First, oxidation has eliminated the highly reactive dangling bonds and trihydrides (only one Si–Si backbond attached), which contributes to the initial fast dissolution phase. This is in agreement with the observation from the study of heat annealing of a silicon wafer, from which the number of dangling bonds decreased significantly at 200 °C , while tkm = 0.4 h−1, which is double the original value. Similarly, better fitting for the OXpSi-200 data (dash line), was achieved with km = 0.92 h−1 and the fractions of medium and slow reactions, Pm = 0.28 and Ps = 0.38. For pSi dissolution, including the 24 h point was the major attribute to the underestimated rate constant . After 8 h, ~90% of pSi dissolved, generating a silicic acid concentration of ~3.2 mM, i.e., just above its saturated concentration (2 mM at 40 °C ). The consequent polymerization of silicic acid could decrease the rate of pSi dissolution. For OXpSi-200, the fraction of slow reaction included ~30% of the total sample mass of undissolved pSi, which might cause overestimated dissolution.The fits from the simulated data calculated from Equation (7) using the variables given in Dissolution kinetics of the pSi samples oxidized at ≥300 °C are presented in - ions [A positive correlation between the dissolution rate constant and surface properties (IR absorption) of pSi was demonstrated in - ions ,65.Our dissolution data showed that the fast dissolution fraction of pSi oxidized at 200 °C, which predominantly is due to surface hydrides (Si–SiHx) was ~1000 times faster than the dissolution of pSi oxidized at 300 °C, which consists of oxidized hydrides (Si–O–SiHx). Dissolution of pSi samples oxidised at ≥300 °C were similar, because all the surface hydrides were in various intermediate oxidation states, to release a Si atom (in silicic acid form), i.e., breaking not only Si–Si bonds but also breaking the newly forming Si–O–Si backbonds which becomes a rate-limiting step for the pSi dissolution. Also, it is worthy of note that the formation of Si–O–Si bonds due to thermal oxidation can cause geometric changes of the pSi lattice resulting in both negative and positive impacts to dissolution. In the former case, expanded crystalline lattice results in reduced surface area and pore volume ; while iThe dissolution kinetics of pSi microparticles can be extensively controlled by thermal oxidation (200–600 °C). Unoxidized pSi and OXpSi-200 dissolved fastest at pH 7.2, which was attributed to the presence of the different surface hydride groups which exhibit different reaction rates. Thermal oxidation has a significant impact on the pSi dissolution mechanism, and pSi oxidized at above 300 °C following pseudo-first order dissolved kinetics, this is due to the rate-limiting step of breaking oxidized backbonds (Si–O–Si). We have successfully developed an exponential kinetic model to describe pSi dissolution, which accounts for the influence of Si speciation. Finally, we investigated the influence of simulated gastric and intestinal fluids on unoxidized pSi dissolution, and showed the presence of GI proteins reduces pSi dissolution; this is presumed do to surface adsorption to pSi and protection against water attack of the Si surface species. These findings provide insight into key considerations for future formulation optimization of specific pSi based biomaterials, particularly for their use in oral drug delivery.


TMSB4X (gene encoding Tβ4) expression in a cell line with high TMSB4X expression. We found out that Tβ4 is not only a component of focal adhesions (FAs) and interacts with several FAs components but also regulates FAs formation. We demonstrate that Tβ4 level has an impact on FAs’ number and morphology. Moreover, manipulation with TMSB4X expression resulted in changes in cells’ motility on non-coated and MatrigelTM (resembling basement membrane composition)-coated surfaces and drastically decreased invasion abilities of the cells. Additionally, a correlation between Tβ4 expression level and exhibition of mesenchymal-like [epithelial-mesenchymal transition (EMT)] features was discovered. Cells with lowered TMSB4X expression were less EMT-progressed than control cells. Summarizing, obtained results show that Tβ4 by regulating melanoma cells’ adhesion has an impact on motility features and EMT. Our study not only contributes to a better understanding of the processes underlying melanoma cells’ capacity to create metastases but also highlights Tβ4 as a potential target for melanoma management therapy.Thymosin β4 (Tβ4), a multifunctional 44-amino acid polypeptide and a member of actin-binding proteins (ABPs), plays an important role in developmental processes and wound healing. In recent years an increasing number of data has been published suggesting Tβ4’s involvement in tumorigenesis. However, Tβ4’s role in melanoma tumor development still remains to be elucidated. In our study we demonstrate that Tβ4 is crucial for melanoma adhesion and invasion. For the purpose of our research we tested melanoma cell lines differing in invasive potential. Moreover, we applied shRNAs to silence  The crucial event during melanoma progression is a transition from the superficial spreading stage to invasive propagation to the dermis . In ordeTMSB4X (gene encoding Tβ4) is correlated with patients’ poor prognosis in some types of tumors  showed that among three Tβs present in human in WM1341D cells Tβ4 is a dominant version of Tβs, although Tβ15 is expressed at a relatively high level too (TMSB10 was expressed at a very low level.According to Oncomine database1  the TMSBand Tβ15  were recand Tβ15 . We clonevel too . In the TMSB4X at higher level when compared to cell lines isolated from primary tumor sites (TMSB4X expression. These observations correlated with invasion capacities of tested cells. The invasion assay was performed using TranswellTM filters coated with MatrigelTM (a mixture of ECM proteins which mimic the basement membrane composition). This assay imitates penetration of the basement membrane by cells during melanoma progression from RPG to VPG. The cells with invasion potential are capable to protrude through MatrigelTM and overcome the pores in Transwell filter’s membrane. The percent of invading A375 cells was three times higher comparing to WM1341D cells, whereas among Hs294T cells there were seven times more invading cells than in the case of WM9 cells  at the tips of stress fibers . Next weor sites . InteresM9 cells . For furDue to the lack of specific Tβ4-recognizing antibodies we obtained DNA constructs , which wTM-coated surface in comparison to A375 cells  revealed that the layer of MatrigelTM stays after scratching  and VIM (gene encoding vimentin) expression levels exhibited positive correlation with TMSB4X expression in human melanoma patients  is a key factor in motility assays and finally Tβ4 influences cells protrusion on resembling basement membrane composition. That is why it is not surprising that invasion of cells with lowered Tβ4 level was impaired. Diminished 3D migration is hence plausible, since it was previously shown that increased Tβ4 level in endothelial cells induced expression of matrix metalloproteinases  expression. At the same time we observed a higher level of ZO-1 in A375 cells than in WM1341D cells. Recent studies suggest that EMT in cancer cells is a dynamic and reversible process manifested by the spectrum of intermediate states between epithelial and mesenchymal phenotypes. It was shown that tumor cells with “hybrid” epithelial/mesenchymal phenotype are more invasive than fully mesenchymal . FurtherTMSB4X expression and EMT progression. In colorectal carcinoma it was demonstrated that Tβ4 triggers EMT transition by ILK upregulation  and Hs294T (HTB-140TM) cell lines were obtained from the ATCC®. WM1341D and WM9 cell lines were bought from Rockland Immunochemicals. The cell culture conditions are described elsewhere (All tested cell lines were of human origin and were authenticated by ATCClsewhere .The assay is described elsewhere . In some® ZOOM System (Essen BioScience). 1000 cells per well were seeded into 96-well IncuCyte® ImageLock plates (Essen BioScience) and image sets have been collected every 2 h for 72 h. The single cell distance and trajectory have been analyzed using Manual Tracking plug-in for ImageJ  for 10 cells from three separate experiments. Directionality was calculated as it is described elsewhere (2D spontaneous migration was monitored using IncuCytelsewhere .® WoundMaker (96-pin woundmaking tool) unified scratches were made. Collective cell migration was performed with the use of IncuCyte® Live Cell Analysis Imaging System (with data collection every 2 h over 48 h) and analyzed with IncuCyte® software. Results were presented as a percent of scratch overgrown area in time. To establish if Wound Maker is able to remove MatrigelTM from coated surface, a confluent monolayer of cells growing on MatrigelTM coated wells was scratched. Cells and surfaces of wells were immunostained using anti-pan-laminin antibodies and fluorescently labeled phalloidin in order to visualize laminin and F-actin.Wound healing assay was performed as described. Cells were seeded onto Essen Bioscience 96-well ImageLock plates and cultured until confluence was reached. With the use of IncuCyte® 568-labeled phalloidin and Hoechst 33342 were purchased from Invitrogen. Photos and Z-stacks were taken using the Olympus FluoView 500 and Zeiss LSM510 confocal laser scanning microscopes. The number of FAs was semi-automatically calculated using ImageJ software, according to a procedure described elsewhere  and the DNA was digested with DNase I (Sigma-Aldrich) according to manufacturer’s protocol. cDNA was obtained by reversed transcription of 0.5 μg total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For qPCR reaction PowerUpTM SYBRTM Green Master Mix (Thermo Fisher Scientific) was used according to manufacturer’s protocol. Applied Biosystems StepOneTM was used for qPCR performance. For quantification, the samples were normalized against the expression of a housekeeping gene – HPRT1 (hypoxanthine phosphoribosyltransferase 1). Primers are listed in RNA from cells was isolated using GenEluteTMSB4X gene silencing were obtained from Sigma-Aldrich – five MISSION pLKO.1-puro TMSB4X shRNA vectors and a control MISSION pLKO.1 puro Non-Target shRNA. Sequences are listed in PCR products were cloned into a vector using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Primers and vectors are listed in The cells were transfected with expression vectors using 2 mg/ml polyethyleneimine (PEI) solution (Sigma-Aldrich). In order to introduce shRNA vectors into cells Neon Transfection System (Thermo Fisher Scientific) was used. Applied pulse parameters were: 1100 V, 40 ms. Stable cell lines were obtained by 1 μg/ml puromycin selection.X = 3 and sigma Y = 3 were used and for ANALYZE PARTICLES command following parameters were used: size = 0–infinity and circularity = 0–1.0.The assay was performed according to manufacturer’s protocol (Sigma-Aldrich). For details see 2 MatrigelTM or without coating surface in serum-free medium containing 0.5% BSA, 2 mM MgCl2 and 2 mM CaCl2. After incubation (1 h at 37°C) unbound cells were removed by five D-PBS washes and the adhered cells were analyzed with a MTT assay (Sigma-Aldrich) according to manufacturer’s protocol.The assay was performed as described elsewhere . 35000 cn = 70), GSE4587 (n = 15), and GSE8401 (n = 83). Each data set was background corrected and normalized using Robust Multichip Average algorithm (RMA) and subsequently compiled and adjusted for batch effect using ComBat  were obtained from Gene Omnibus (GEO). The datasets included: GSE3189 (g ComBat . Probes lgorithm . Preproct-test or ANOVA (one-way or two-way) with post hoc Tukey HSD were applicable, which were performed in GraphPad Prism 7. The significance level was set at ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, or ∗∗∗∗P < 0.0001. Graphs were plotted in GraphPad Prism 7.All data are given as means ± standard deviations (SD) are representative of at least three independent experiments. Their significance was determined using either the two-tailed, unpaired Student’s Thymosin β4 is a predominantly expressed thymosin β in human melanoma cells and is a constituent of FA. This polypeptide regulates formation of FAs in melanoma cells by influencing their morphology and number. Moreover, it has significant impact on adhesion and protrusive abilities under 2D and 3D conditions of melanoma cells. Tβ4 level decides about progression of EMT of melanoma cells.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.AM partially designed the study, performed the experiments, contributed to the preparation of the figures, and took part in writing the manuscript. NM, EMa, and EMr performed some experiments and contributed to the preparation of the figures. AJM designed the study, gained funding for the study, performed the experiments, contributed to the preparation of the figures, and took part in writing the manuscript. DN discussed the manuscript. All authors reviewed the results and approved the final version of 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.


Drosophila melanogaster GSTE1 and GSTE7 in chemosensory male tissues and the expression level of the mRNAs coding for the same enzymes after a PEITC exposure in food.The data presented in this article are related to the research article entitled “Characterization of a Drosophila glutathione transferase involved in isothiocyanate detoxification.”   Specifications TableValue of the data•GSTE1 and GSTE7 mRNA are expressed in male chemosensory tissues.The data presented in this article show that •GSTE1 and GSTE7 mRNA expression is significantly higher in antennae and palps compared to heads. GSTE1 mRNA expression is higher in labellum and forelegs compared to Drosophila heads.•GSTE7 mRNA expression in taste organs but did not significantly change GSTE1 mRNA expression in chemosensory tissues.A three day-long exposure to food containing PEITC led to a significant increase of 1GSTE1 and GSTE7 mRNA expression in Drosophila male chemosensory organs and are related to the research article entitled “Characterization of a Drosophila glutathione transferase involved in isothiocyanate detoxification.”  GSTE1 and GSTE7 showed a higher expression level in olfactory organs  compared to fly heads  wild-type male flies. Flies were reared on standard yeast/cornmeal/agar medium in a humidified, temperature-controlled incubator at 25 °C under a 12 h light: 12 h dark cycle.PEITC (CAS no. 2257-09-2) was dissolved in ethanol  and added to the media at 50 °C . A similar volume of ethanol was added for both the experimental and drug-free control tests. Flies were transferred to experimental treatments at a density of 10 per vial. 30 flies were used in each treatment and they were exposed to experimental treatments during 3 successive days.2.2Total RNA was extracted using Isol RNA Lysis reagent (5Prime) and was treated with RNAse-free DNAse  to avoid genomic DNA contamination. Total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit . The qPCR reactions were carried out on a MyIQ  using the IQ SYBR Green SuperMix . Each reaction was performed in triplicate. All results were normalized relatively to the tubulin and rp-49 mRNA levels and the relative amount of mRNAs were calculated using the ∆∆Ct method.p <0.05, ** p <0.01, *** p <0.001) All error bars represent SEMs. REST Software was used to compare qPCR sets of data. Asterisks indicate the level of statistical significance (*


Scientific Reports 10.1038/s41598-019-46668-w, published online 15 July 2019Correction to: The original version of this article contained a typographical error in the abstract.“Principal component analysis revealed that the Venus flytrap domain (VFD) of T1R2 monomer was adapted by the induced-fit mechanism to accommodate the focused polyols, in which α-helical residues 233–268 moved significantly closer to stabilize ligands.”now reads:“Principal component analysis revealed that the Venus flytrap domain (VFD) of T1R2 monomer was adapted by the induced-fit mechanism to accommodate the focused polyols, in which residues 233–268 moved significantly closer to stabilize ligands.”Additionally, the original version of this article contained a typographical error in the Results section under the subheading ‘Essential dynamics of the T1R2-T1R3 STR upon polyols complexation’.“Moreover, binding of these polyols stimulated the direction of motion of α-helical residues 233–268 (purple dashed circle), located near to the binding pocket, to become significantly closer to xylitol and sorbitol, resulting in a compact molecular shape.”now reads:“Moreover, binding of these polyols stimulated the direction of motion of residues 233–268 (purple dashed circle), located near to the binding pocket, to become significantly closer to xylitol and sorbitol, resulting in a compact molecular shape.”The original version of this article also contained a typographical error in the second paragraph of the discussion section.“Furthermore, binding of the two polyols converted the direction of motion of not only amino acids within a spherical radius of 5 Å but also residues 233–268, which is an α-helix element located near to the binding pocket, to become significantly closer to the ligands.”now reads:“Furthermore, binding of the two polyols converted the direction of motion of not only amino acids within a spherical radius of 5 Å but also residues 233–268, which are near to the binding pocket, to become significantly closer to the ligands.”Furthermore, in Figure 5B the key was incorrect.“Model 2_MM/PBSA”now reads“Model 2_MM/GBSA”.These errors have now been corrected in the HTML and PDF versions of the article.Lastly, in Figure 6A, the labelling for the residue N143 was missing. This has now been corrected in the PDF of the article. The HTML was correct at the time of publication.


By harnessing the dramatic optical contrast of GST, we realize broadband (Δλ/λ ~ 15%) mode shifting between an electric dipole resonance and an anapole state. Active control of higher-order anapoles and multimodal tuning are also investigated, which make the structured GST serve as a multispectral optical switch with high extinction contrasts (>6 dB). With all these findings, our study provides a new direction for realizing active nanophotonic devices.High-index dielectric nanoparticles supporting a distinct series of Mie resonances have enabled a new class of optical antennas with unprecedented functionalities. The great wealth of multipolar responses has not only brought in new physical insight but also spurred practical applications. However, how to make such a colorful resonance palette actively tunable is still elusive. Here, we demonstrate that the structured phase-change alloy Ge 2Sb2Te5 can support multipolar Mie resonances and demonstrate the spectral shifting between electric dipole and anapole modes.High-index dielectric nanoparticles support a great wealth of multipolar responses but active tunability is still elusive. Here, the authors report how structured Ge The intention to control and manipulate light by fully exploiting the advantages from scattering resonances, particularly at the nanometer scale, has stimulated the emergence of modern nanophotonics3 and spawned a myriad of applications ranging from biochemistry to information technology4. In this context, low-loss, high-index dielectric or semiconductor nanostructures featuring a diverse set of optical resonances are currently in the spotlight of research as they can serve as versatile and CMOS-compatible building blocks for various photonic devices9. Besides the practical advances, studies on dielectric nanoresonators have also brought new insight into fundamental physics. Recent experimental investigations on the scattering response of Si nanoparticles have not only shown conventional radiant modes such as magnetic dipole (MD) or electric dipole (ED) resonances12, but also revealed the underlying physics of an intriguing scattering “dark state”, i.e., anapole state, characterized by a pronounced minimum in the scattering spectra and an associated maximum in the near-field energy15. Such a suppressed scattering state stems from two antiphased electric and toroidal dipole moments, whose radiation patterns are identical to each other and thereby interfere destructively in the far field. This unique behavior of anapole states shortly unveil its tantalizing potential in many scenarios such as cloaking17, nanoscale lasers18, field enhancements20, energy guiding21, harmonic generations24 and metamaterials27.Ever since the seminal work of Mie35. For instance, a pioneering work using liquid crystals as embedding media29 generates a maximum spectral shift Δλ ≈ 40 nm at resonance wavelength λ ≈ 1550 nm, corresponding to a relative resonance tuning Δλ/λ merely 2.6%. A very recent study utilizing the thermo-optic effect of Si achieves a resonance shift Δλ ≈ 30 nm at wavelength λ ≈ 1500 nm under an external temperature around 300 °C33. Indeed, given the multitude of resonances and follow-up functionalities offered by high-index dielectrics, tuning one or a few spectral peaks with limited ranges does not sufficiently employ all the benefits from such a fruitful playground. While attempts are also being made to obtain wideband tunability spanning over one linewidth38, active mode switching between or among different dielectric resonances is still an unexplored conundrum.However, despite the great wealth of optical resonances and rendered interesting phenomena, how to actively tune these responses and further switch among them remains a daunting challenge. This is because the induced near fields of dielectric nanoparticles, unlike their plasmonic counterparts, are mainly inside the structures and thereby only mildly sensitive to the change of external environments. For the same reason, the majority of research to date still focuses on passive structures, whose functionalities are set in during fabrication and cannot be altered afterward. Whereas there is a growing recognition of the need to realize active dielectric components, most of the published reports so far only display modest resonance shifts2Sb2Te5 . Owing to its striking electrical and optical contrasts between amorphous and crystalline phases, GST has been widely used in commercial memory applications and was recently introduced into the nanophotonics community39. In contrast to the aforementioned tuning mechanisms such as using liquid crystals or temperature tuning, GST affords a different, non-volatile approach where the induced optical change remains stable even after the removal of external stimuli. So far, most of the research employs GST in the form of thin films functioning as surrounding media for metallic structures49. For instance, Chen et al. realized stepwise tuning of the lattice resonance in a hybrid plasmonic system consisting of a gold disk array and an underneath GST thin film40. The characteristics of such configuration thus are still dominated by lossy plasmonic resonances. Although there are also exciting developments using GST itself as integrated optical constituents55, detailed investigations on the fundamental optical properties of GST nanostructures are surprisingly lacking56. In fact, among all the phase-change chalcogenides, GST features one of the highest refractive indices in its amorphous states57, which satisfies the essential prerequisite for constructing dielectric nanoantennas with strong Mie resonances. Here, we perform a thorough multipole analysis to examine the optical response of standalone GST nanostructures both theoretically and experimentally. For the first time, we demonstrate that the high refractive index and the low loss of GST empower its nanostructures to support MD, ED and anapole states, in a similar manner as other enticing dielectrics such as Si and Ge. Meanwhile, the distinctive tunability of GST makes all these resonances actively controllable. By exploiting the intermediate phases of GST, we show progressive mode shifting between scattering bright and dark states over an extremely broadband region (Δλ/λ ~ 15%). Multimodal shifting among higher-order ED and anapole states is also manifested, naturally making the investigated GST structures function as a multispectral optical switch with high extinction contrasts (>6 dB) as well as multi-level control abilities. Hence, by discovering the concealed portfolio of actively controllable resonances in GST nanostructures, our findings establish a new basis for designing active optical components and pave the way towards metadevices with tunability on demand58.In this work, we realize broadband and controllable mode shifting between distinct scattering states by structuring the phase change alloy Ge57. Fig. p generates a considerable dipolar radiation, giving rise to a scattering bright state. By contrast, the anapole mode features an induced poloidal current of electric fields inside the particle, associated with a torus of circulating magnetic fields17. Such an intricate field distribution is characterized by a significant suppression of far-field scattering. Therefore, this phenomenon is also commonly referred to as a scattering dark state. Based on the Mie theory, the spectral positions of these two modes are heavily dependent on the refractive index of the particle. Therefore, shifting between the modes could be realized by introducing a delicate amount of index change to the GST sphere. To this end, thermal, electrical, and optical stimuli could be utilized to induce controllable phase transformations in the GST material, making it possible to achieve not only the transition between amorphous and crystalline states but also among intermediate  levels39. The scattering response of a GST sphere with a fixed radius R = 450 nm and varying crystallinities C is depicted in Fig. C = 25%, a progressive shifting between scattering maxima and minima can be readily observed. A detailed multipole analysis further shows that the scattering maxima and minima are ambiguously attributed to the ED and anapole states, respectively  are also distinctly manifested. The prolific multipolar effects of the GST sphere come from its notably high index  shown in Fig. To analyze the electromagnetic response of GST nanostructures, we start our investigation by considering the most general case for analytical treatments: a GST sphere situated in the vacuum (see Methods). Here we focus on the mid-infrared range given the high refractive index, substantial optical contrast, and relatively low loss of the GST materialely Fig. . In partx-axis with the electric fields polarized along the z–direction. At MD resonance . An appreciable scattering process also occurs, as shown in Fig. Ez . Therefore, the spectral positions of the two modes are determined by both the crystallinity C and the geometric size R. In this regard, we first consider a GST sphere with an invariant radius (R = 450 nm) and continuously change its crystallinity C. The two-dimensional map of its scattering efficiency Qscat is plotted in Fig. C. To precisely identify the position of ED and anapole states, we further analytically obtain the conditions for the two modes by solving the following sets of equations and inequalities: (1) |a1|′ = 0, |a1|′′ < 0 for the ED resonance, and (2) |a1|′ = 0, |a1|′′ > 0 for the anapole state. With a fixed R = 450 nm, the solution set  is shown in Fig. λ > 700 nm along the x-axis, corresponding to a fractional bandwidth Δλ/λ > 20%. Similarly, the height of the area along the y-axis indicates the amount of a phase change needed to implement the mode shifting. Interestingly, such an amount is nearly constant over the whole spectral range, meaning that the presented mode shifting functionality (from the ED to the anapole state) can be attained by simply introducing a fixed phase change (ΔC ≈ 25%) to the GST nanosphere with an arbitrary crystallinity C below 75%.Next, we examine the spectral response and the tuning range of the mode shifting effect. Since the ED resonance and the anapole state are related to the maximum and the minimum of the ED contribution, they are only related to the multipole coefficient R and investigate the scattering contrast of these spheres at two distinct crystalline phases: amorphous (C = 0%) and an intermediate phase (C = 25%). The scattering contrast Qconstrast is defined as the ratio between the scattering efficiencies of the spheres at the two phases. The 2D scattering maps of each phase are provided in Supplementary Note Qconstrast . Therefore, given a cluster of GST spheres with various radii, by introducing a fixed amount of phase change (here ΔC = 25%), all the structures possessing different ED resonance wavelengths would exhibit the same functionality shifting from the ED to the corresponding anapole state. Thus, the mode shifting effects in GST nanostructures can be maintained in a broadband range regardless of the original resonance wavelengths. Such a nearly “non-dispersive” behavior may find its applications in many interesting aspects. For instance, a major challenge nowadays to realizing actively tunable metasurfaces lies in the fact that metasurfaces are usually composed of plasmonic or dispersive meta-atoms with different sizes and different resonant wavelengths. Therefore, a uniform optical change across the interface does not guarantee that the metasurface can sustain its important functions  after active tuning. By contrast, GST resonators with nearly non-dispersive behaviors may provide a promising solution to overcome this issue.Then we study the impacts of the geometric size on the mode shifting effect. To this end, we consider the spheres with different radii ase C = 2%. The scR to describe the geometric feature of the disks. An SEM image of fabricated GST nanodisk array with R = 1 μm is shown in Fig. g of 3 μm was chosen to avoid coupling between adjacent nanostructures. The influences of the pitch size, the absorption loss of the GST material, and the substrate CaF2 are thoroughly examined and can be referred to Supplementary Note To verify the proposed concepts, we then perform experiments with GST nanostructures. Given the ease of fabrication and convenience for observing anapole states, truncated GST disks were fabricated by using E-beam lithography, magnet sputtering deposition, and standard lift-off process (see Methods). The geometric profile of the fabricated sample was measured by atomic force microscopy  oblique incident angle provided by the applied objective, lattice effect, or a slight overestimation of the material loss used in the simulation. Multipole decomposition of the spectral response (see Methods) further clearly points out that the extinction maxima and minima are exactly correlated with the ED and anapole states, respectively. In particular, we find that, once a phase change of ΔC = 50% is introduced , the GST disks would always undergo a shift between the ED and the first-order anapole state (A1). Such a mode shifting effect thus can be achieved in a broadband region over 600 nm , which is remarkably consistent with our previous theoretical investigations on the GST spheres.To introduce different amounts of phase change and realize intermediate phases of GST, here we applied thermal stimuli by heating the sample on a hotplate at a fixed temperature 145 °C but with different amounts of time. The extinction spectra of the GST disks after different annealing time are presented in Fig. x-axis, indicating a clear combination of the A1 state and an accompanied standing wave character. This phenomenon can be explained by the generation of hybrid Mie–Fabry–Perot modes26 or the superposition of several internal modes59. Hence, the A2 state possesses a stronger field confinement within the disk volume compared to the A1 state, which leads to a higher concentration of internal energy19. Therefore, such higher-order anapoles could exhibit their unique advantages over their fundamental counterparts, particularly in scenarios such as harmonic generation23 and field enhancement20. Interestingly, we find that the shifting between the A1 and the A2 states can be also realized by changing a GST disk from its amorphous phase to its crystalline phase  supported by the 100%–cGST disk. The near-field distributions of the ED, A1, and A2 states are depicted in Fig. ase Fig. .C varying from 0% to 100%. Scattering bright and dark states appear alternately across the spectra, indicating the existence of higher-order ED  and anapole states  are plotted in Fig. C = 50% is introduced, all the disks exhibit the same mode shifting response from the ED1 to the A1 states, despite their different ED1 resonance wavelengths λED1 ranging from 3.2 μm to 4.6 μm. Similarly, when a phase change ΔC = 100% is introduced , all the disks with different radii shift their A1 states to corresponding A2 states at different wavelengths λA1 spanning from 2.7 μm to 3.7 μm. Hence, for both aforementioned mode shifting effects (ED1 to A1 and A1 to A2), nearly non-dispersive responses over 1 μm are demonstrated. Once again, these results substantiate our previous theoretical investigations on GST spheres. It is also worth noting that other multimodal responses such as the shifting from A1 to ED2 and from ED2 to A2 are also sustained for all the disk sizes in a broadband region.Next, we investigate GST disks with different radii 41, here the multimodal tuning and shifting behavior of the GST disks endow themselves with a multispectral capability. As we can see from Fig. C = 0% to C = 50%, the blue curve), the GST disks will undergo three disparate switching at three different wavelengths simultaneously. Both “on” and “off” states of the optical switch thus could be achieved at the same time in different spectral regions. A high contrast ratio of 6 dB is obtained without any further geometry optimization. Moreover, due to the broadband response of the switching functionalities, such a multispectral characteristic is sustained for different crystalline phases of the GST material , the dielectric constant εGST of GST can be estimated by using the effective medium theories (EMTs). Out of various EMTs, here we choose to use the Lorentz-Lorenz relation as it is so far one of the most widely used approaches in the simulation of hybrid and isolated GST nanostructures56. Comparing to other EMTs such as the Maxwell-Garnett approximation60, the maximum difference in the real part of the refractive index Δn0 between different EMTs was found to be smaller than 5%. Therefore, our estimation is well within the acceptance of EMT approximation. The Lorentz-Lorenz relation can be expressed as followsεaGST and εcGST are the permittivities of amorphous and crystalline GST, respectively. Therefore, by applying Eq.  at every discretized points r (coordinate respective to the disk’s center) inside the disks. By defining the polarization current J(r) = −iωε0[εr(r) − 1]E(r), the electric 63:E0 is the electric field amplitude of the incident plane wave; η is the impedance of free space; jl(kr) is the spherical Bessel function of the first kind and Cscat of the GST disks can be written as the sum of partial contributions from these derived multipoles:We performed three-dimensional FDTD simulations with a commercial software package . The optical constants of the GST disks with different crystallinities was determined by the experimental ellipsometric data  was spun onto the PMMA film and baked for 2 min at 90 °C. This coating is used for the dissipation of e-beam charges on insulating substrates. The PMMA was exposed to define a nanohole array by E-beam lithography. All e-beam patterning was performed by SEM, which is equipped with a Raith Elphy Quantum lithography system. The conductive layer was dissolved in DI water for 1 min and then the PMMA was developed in the developer (AR 600-56) for 3 min followed by rinsing in IPA. After the development, a 220-nm-thick GST film was then deposited onto the sample by magnetic sputtering with 50 W DC sputtering power while the substrate temperature was kept at room temperature. The deposited GST thin film was in its amorphous phase. The GST nanodisk array was realized after lift-off by ultra-sonic processing in acetone for 1 min.A 280-nm-thick PMMA (950K AR-P 672.11) was spun onto CaF2, corresponding to ~64 disks in the array with a 3 μm gap distance. All the measurements represent an average of 16 scans taken at a resolution of 4 cm−1. To determine the transmittance (T) of the GST arrays, air was used as the reference. Experimental extinction spectra were then derived as 1 − T. A 15× Schwarzschild objective is applied, which operates at ~16.7° off-normal to the surface of the sample and has a collection cone apex angle of ±7°. Given the small incident angle of the objective and the large diameter-to-thickness ratio of the GST disks, the fabricated structures in our study do not exhibit strong angular dispersion. Phase transformation of the GST material was induced by baking the samples on a hot plate which maintained a temperature of 145 °C. To ensure a systematic optical characterization of each sample, the phase-changing process and transmission measurement were implemented progressively. After being heated for 1 min, the sample was cooled down naturally and then transmission spectra were measured. Another cycle of the annealing process and the extinction measurement would be carried out thereafter until the GST disks were fully crystallized. The scanning electron microscope images were taken by Zeiss Ultra55 and the atomic force microscope (AFM) images were taken by VEECO Multimode.Transmission spectra were measured by using an infrared microscope (Hyperion1000) coupled to a Fourier transform infrared spectrometer . The detector used in the measurement is an MCT detector integrated into the microscope. The size of the probed area is 40 × 40 μmSupplementary InformationPeer Review File


Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10–100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F1F0 ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as  During exponential growth, persister cells (designated type II) arise at very low frequencies whereas upon entry into stationary growth phase the fraction of persister cells (type I) increases substantially4. Commonly, persister cells are considered to be in a state of dormancy and several mechanisms have been proposed to explain the phenomenon. In Escherichia coli and Salmonella, dormancy was previously proposed to be stochastically induced by the action of the toxin-antitoxin (TA) systems that compromise key cellular functions and are activated by ppGpp or by the SOS response10. However, this model was recently disproven12. In fact, conditions that halt transcription, translation or ATP synthesis dramatically increase persister frequency from 0.01% to up to 10–100%13. Particularly, the cellular ATP level is important as depletion of ATP with arsenate increases the number of persister cells both in E. coli and in the human pathogen, Staphylococcus aureus14. In the latter organism, neither the stringent response nor TA loci contribute to persistence but rather persister cells express stationary phase marker genes, such as the capsular polysaccharide operon, suggesting that they are cells that during exponential growth have prematurely entered stationary phase with low ATP levels4.Persister cells are phenotypic variants present in the bacterial populations that in the absence of heritable mutations or antibiotic resistance genes display remarkable antibiotic tolerance. They are detected as survivors when exposed to very high antibiotic concentrations often reaching 100 times the minimal inhibitory concentration (MIC) and they are observed in both exponential and stationary growth phases for multiple bacterial species and with several antibioticsE. coli, fumarate accumulation and perturbations of the TCA cycle as well as the electron transport chain has been linked with increased persistence while others have found that inactivation of sucB, encoding α-ketoglutarate dehydrogenase and other genes of the TCA cycle reduced persister formation17. Tolerance to aminoglycosides is provided by low proton motive force (PMF) as persister cells are eradicated by conditions that enhance PMF and facilitates aminoglycoside uptake19. The varying contributions of metabolism to persister formation may in part be explained by different experimental setups but also by the significant differences in metabolism between organisms and growth conditions13. For example, in Gram-negative bacteria which are growing under laboratory conditions, such as E. coli, ATP is produced via oxidative phosphorylation driven by products generated in the TCA cycle while the low-passage counterpart produces ATP by substrate level phosphorylation; in the Gram-positive bacteria, S. aureus, the TCA cycle is repressed during exponential growth where glucose is metabolized to acetate with ATP generated by substrate level phosphorylation20.Although the central role of bacterial metabolism in bacterial persistence has been documented by several reports, a uniform picture of the contribution has not emerged. In S. aureus is known to give rise to a diverse range of serious diseases such as endocarditis, osteomyelitis and chronic and re-occurring infections that are difficult to treat with antibiotics. The clinical relevance of persister cells has been demonstrated by infection of a chronic wound model with S. aureus that could be eradicated with an acyldepsipeptide antibiotic, ADEP4 that killed persister cells22. With most studies of persisters having focused on the exponential growth phase we set out to determine if the already high level of persisters observed for S. aureus in stationary growth phase may be increased even further and if so which genes are contributing. For this purpose, we have taken a transposon library approach to identify genes that either by inactivation or overproduction increase persister cell frequency in stationary phase23. During enrichment, we found that inactivation of either sucA or sucB encoding subunits of α-ketoglutarate dehydrogenase dramatically increased persister cell formation as did inactivation of other enzymes of the TCA cycle or atpA gene. Commonly, all the mutants with enhanced persister levels displayed reduced membrane potential that contributes to PMF indicating that in stationary phase membrane potential becomes critical for persister cell formation or survival.S. aureus, we constructed a high-density transposon (Tnp) insertion library in strain Newman employing the bacteriophage-based mariner transposon delivery system that allowed both gene inactivation and gene overexpression from an outward-facing promoter in the transposon element24. The coverage of Tnp insertions were assessed in 10 random library isolates and the results revealed that the 10 isolates carrying insertions at 10 independent sites were evenly distributed throughout the genome indicating random Tnp insertion  it was inserted in sucB. sucA and sucB encode subunits of α-ketoglutarate dehydrogenase, which is a key enzyme of the TCA cycle that catalyses the conversion of α-ketoglutarate to succinyl-CoA with the concomitant production of NADH and CO2 in an irreversible reaction26.To determine if individual clones were enriched by repeated exposure of the Tnp library to high antibiotic concentrations, six colonies (named as L1-L6) were randomly picked by the end of the fourth round of enrichment to examine persister formation in individual clones. When treated with ciprofloxacin, all the six isolates exhibited persister frequencies between 20–60% compared to approximately 1% for the wild type Fig. . The trasucA deletion mutant (ΔsucA) was constructed. The ΔsucA mutant yielded more than 30% persisters when exposed to 100 times MIC of ciprofloxacin  agreeing with the notion that in S. aureus, TCA cycle activity is low during exponential growth but de-repressed upon entry into stationary phase20.To rule out the possibility that secondary mutations within the library isolates contribute to the persister phenotype, the Tnp insertion mutations were transduced for all six clones to naïve wild type Newman cells to generate transductants, L11, L21-L61, carrying the individual mutations of the original library clones. All transductants yielded more than 30% persisters in comparison to 1% for the wild type when exposed to lethal concentrations of ciprofloxacin Fig. . To corr9 to 104 CFU/ml after the 7-day treatment with 100 × MIC of oxacillin. Furthermore, the MICs of all library transductants towards ciprofloxacin and oxacillin were identical to that of wild type cells  paralleling that observed for the sucA and sucB mutants, when compared with wild type cells (<1%) in stationary growth phase  of sucA mutant cells was higher than observed for the other TCA cycle mutants  which have mutations of either sucA or sucB within TCA cycle  or isocitrate dehydrogenase (citC), may not affect the downstream metabolic activity of TCA cycle including ATP production26.Recently, it was demonstrated that lls Fig.  even tholls Fig. . This phS. aureus, but in stationary phase oxidative phosphorylation and the ATP synthase could contribute20. Therefore, we examined a mutant lacking atpA that encodes the α-subunit of F1F0 ATP synthase. As has been observed previously for other organisms we found that there was no significant reduction in ATP level of the atpA mutant when cultivated in rich medium  and flow cytometry we consistently found that all TCA cycle mutants which exhibited increased persister cell frequencies displayed reduced membrane potential  as expected it enhanced persister cell formation  characterizes S. aureus persister cells.Inactivation of the TCA cycle is likely to reduce the production of reducing equivalents that enters the electron transport chain and contributes to the PMFial Fig. . Importalls Fig. . This reion Fig. 31–33. CoS. aureus TCA cycle activity is de-repressed in stationary phase20. To examine if this de-repression correlates with expression of TCA cycle genes, we fused the sucA promoter to the cfp reporter gene and monitored expression in exponential and stationary phase single cells  mutant survived levofloxacin equally well as the wild type cells within a 5 days period. In contrast, when the persister assay was repeated with our standard protocol and using levofloxacin, both mutants (sucA and sdhA) exhibited higher persister frequencies compared to wild type cells (data not shown). Thus, we conclude that while the difference is not due to the antibiotics (ciprofloxacin vs. levofloxacin); there is a difference between the outcomes from the two protocols. We do, however, not see the same effect from an sdhA mutant in our strain background when using Wang’s persister assay protocol36, suggesting that strain choice is also important. Moreover, we have replicated our findings in different strain backgrounds  at 37 °C and 200 rpm of shaking in 12-ml centrifuge tube or plated on Tryptic Soy Agar  at 37 °C of incubation unless otherwise indicated. All the plasmids and strains used for Tnp library construction were kindly provided by Timothy C. Meredith and supplemented glucose minimal medium (SGMM) was prepared as described previously23. TCA cycle mutants of sucA, sucB, sucD, sdhA, sdhB, acnA (NWMN-1263), citC and atpA were provided by bei Resources27. Ciprofloxacin, oxacillin, adenosine 5′-triphosphate disodium salt hydrate (ATP), carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other mentioned chemicals were from Sigma-Aldrich . All restriction endonucleases and T4 ligase were from NEB BioLabs .A list of bacterial strains, plasmids and primers used in this study can be found in Supplementary Table S. aureus Newman as reported previously24: (1) Tnp donor lysates were prepared by infecting TM43-45 with Ø11-FRT and the Tnp-plasmid packaging efficacy was determined by tittering these phage lysates on both RN4220 and TM19; (2) recipient strains were obtained by transforming plasmids pOrf5 Tnp+ or pOrf5 Tnp− into S. aureus Newman; (3) the Tnp donor lysates were gently mixed with recipient strains in SGMM and incubated at 22 °C overnight; (4) the cells were pelleted and incubated with TSB medium for 2 hours at 30 °C before plated on TSA plates containing erythromycin (5 µg/ml). Subsequently, the plates were left at room temperature until visible growth of individual Tnp insertion mutants could be observed.Transposon library was constructed in et al. by PCR amplification of the transposon/chromosome junction using primers Martn-F and Martn-ermR and an annealing temperature of 63 °C. After purification, the PCR products were sequenced with primer Martn-F43.The identification of the Tnp insertion site of library isolates was performed as reported by Bae 8 bacterial cells were harvested and re-suspended in 1 ml of pre-chilled 1x phosphate buffered saline (PBS) containing 3.7% formaldehyde. After 15 minutes of gentle mixing at room temperature, 500 µl of the fixed bacterial suspension was transferred to each well of a 24-well cell culture plate  lined with 12 mm diameter round coverslips. After centrifugation at 600 g for 5 minutes, coverslips were mounted onto slides with 3 µl fluorescence mounting medium .Approximately 10Image acquisition was performed in the confocal mode of an inverted Zeiss LSM 710 NLO microscope equipped with a spectral detector and employing Zeiss Plan-Apochromat 63x/1.40 oil DIC M27 objective . The following excitation wavelengths, laser sources and detection spectra were used for the promoter activity experiments, gpCerulean: 405 nm/diode laser/454–516 nm; gpVenus: 514 nm/argon laser/519–621 nm. Images were exported in the different channels or overlays as 16-bit tagged image files. The exported images were assembled with CorelDRAW X7 and adjusted for brightness and contrast.1 µl of pooled wild type or Tnp library was inoculated into 2 ml TSB medium for incubation at 37 °C with shaking at 200 rpm in 12-ml centrifuge tube, after 24 hours, 1 ml of the culture was taken out to be treated by 100 × MIC of ciprofloxacin (MIC = 0.5 µg/ml) at 37 °C for another 24 hours in 12-ml centrifuge tube. The culture was washed and re-suspended with 1 ml FK buffer (9 g/l sodium chloride), and then certain dilutions were plated on TSA plates to be incubated at 37 °C for 24 hours. The plates with 100–200 colonies were eluted with FK buffer and 2 µl of the suspensions were inoculated in 2 ml fresh TSB medium which then undergone the same treatment of ciprofloxacin. This procedure was repeated for four times in a row to enrich persister cells.ΔsucA mutant and parent strain cultures were taken at 1, 3, 5, 24 or 48 hours post the addition of ciprofloxacin and the dilutions were plated on TSA plate at 37 °C for 24 hours for CFU determination. The transductants of Tnp library isolates were also tested with 100 × MIC of oxacillin (MIC = 0.25 µg/ml) and the CFU/ml was determined over 7 days.2 µl of overnight culture of library isolates, TCA cycle mutants or wild type was inoculated into 2 ml TSB medium and incubated at 37 °C with shaking at 200 rpm for 24 hours in 12-ml centrifuge tube, then 1 ml of the culture was challenged by 100 × MIC of ciprofloxacin for another 24 hours still at 37 °C in 12-ml centrifuge tube. The persister frequency was determined by calculating the ratio between the CFU/ml counting of 24-hour post and right before the addition of ciprofloxacin by plating and incubating known dilutions of the samples on TSA plates at 37 °C for 24 hours. Aliquots of To evaluate the effect of medium acidification on persister generation, TSB medium with or without glucose  was used for the cultivation and the persister frequencies were compared between the cultures with these two media. CCCP dissolved in DMSO was added into bacterial culture right before the treatment of ciprofloxacin to test the effect of PMF on persister generation.TM Microbial Cell Viability Assay kit  according to manufacturer’s instruction. 100 µl suspension of bacterial culture (10-fold diluted with TSB medium) was carefully mixed with equal volume of BacTiter-GloTM Reagent and incubated at room temperature for 5 minutes before the luminescence was recorded with emission at 560 nm. The value of each sample was the average of three independent reads and there were 3 biological replicates for each individual sample.The ATP level of 24-hour cultures was tested with BacTiter-GloBacLightTM Bacterial Membrane Potential Kit  where 24-hour cultures were re-suspended with 0.5 ml filtered PBS, mixed with 10 µl of fluorescent membrane potential indicator dye, DiOC2(3) and incubated at room temperature for 30 minutes. The fluorescent signals were recorded by BD Biosciences Accuri C6 flow cytometer  counting 50,000 cells with the threshold of 16,000 at medium flow rate. The ratio between channel F3 (red fluorescence) and F1 (green fluorescence) was calculated with CFlow®  to indicate membrane potential.Membrane potential was assessed by The concentration of acetate in the supernatant of cultures from both early exponential (3-hour cultivation) and stationary phase (24-hour cultivation) was determined by Ultimate 3000 HPLC system  equipped with an Aminex HPX-87H column  and a Shodex RI-101 detector . The column oven temperature was set as 60 °C and the mobile phase consisted of sulfuric acid (5 mM) with a flow rate of 0.5 ml/minute.Results were represented as the mean of biological triplicates with standard deviation besides for the ATP measurements there were 3 independent luminescent reads for each biological replicate. Statistical significance was analyzed using one way ANOVA followed by Dunnett test with P < 0.05 in GraphPad Prism 7 .The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


Induced polarization (IP) mapping has gained increasing attention in the past decades, as electrical induced polarization has been shown to provide interesting signatures for detecting the presence of geological materials such as clay, ore, pyrite, and potentially, hydrocarbons. However, efforts to relate complex conductivities associated with IP to intrinsic physical properties of the corresponding materials have been largely empirical. Here we present a quantitative interpretation of induced polarization signatures from brine-filled rock formations with conductive inclusions and show that new opportunities in geophysical exploration and characterization could arise. Initially tested with model systems with solid conductive inclusions, this theory is then extended and experimentally tested with nanoporous conductors that are shown to have a distinctive spectral IP response. Several of the tests were conducted with nano-porous sulfides (pyrite) produced by sulfate-reducing bacteria grown in the lab in the presence of a hydrocarbon source, as well as with field samples from sapropel formations. Our discoveries and fundamental understanding of the electrode polarization mechanism with solid and porous conductive inclusions suggest a rigorous new approach in geophysical exploration for mineral deposits. Moreover, we show how induced polarization of biologically generated mineral deposits can yield a new paradigm for basin scale hydrocarbon exploration. These surveys supplement controlled-source electromagnetic2 and magnetotelluric5 surveys, which generate subsurface resistivity maps.As a key electromagnetic-based geophysical exploration methodωD) so ionic conduction dominates the electromagnetic response. There are two distinct, commonly recognized mechanisms that give rise to complex conductivity in this low-frequency regime4: (1) diffusive relaxation of neutral modes associated with ionic concentrations, usually called “membrane polarization”, and (2) capacitive charging of the electric double layer on the surfaces of non-ionic conductors, usually called “electrode polarization”. Regardless of the dominant mechanism, frequency dependence of complex conductivity is typically fitted to a Cole-Cole type model10 as the basis of various geophysical exploration methods18, where the effective complex conductivity of the porous media σeff has the form16:ω is the angular frequency of the applied electric field and σ∞ is the high frequency limit of σeff. The “chargeability” m quantifies the relative change in conductivity between the low- and high-frequency limits, whereas τ is a characteristic relaxation time and c is the Cole-Cole exponent. This model has originated from dielectric spectroscopy and has been successfully developed to understand metal-dielectric systems21, although its application to model observed IP response23 has been largely empirical.Frequencies of interest in these geophysical surveys are usually orders of magnitude below the Debye relaxation frequency , and ion-selective membranes in electrochemical cells . This type of polarization originates from inhomogeneities in the ionic transport properties, which drive ionic concentration gradients that ultimately relax through diffusion, therefore it leads to a relaxation time τ ∼ a2/D, where a is a characteristic length scale such as pore or grain size, and D is a characteristic diffusivity of dominant ions. In geophysical settings, this mechanism is more pronounced at lower pore fluid salinity . Chargeability, which directly relates to the magnitude of the signal in IP surveys, diminishes at the higher salinities that are typically encountered below freshwater aquifers and in offshore marine settings.Membrane polarization24 requires the presence of conductive grains with non-ionic charge carriers, for example electronic conductors or semiconductors, such as gold or sulfide ores like pyrite32. These grains act as short circuits in the high-frequency limit and insulators in the low-frequency limit, therefore the chargeability depends primarily on the effective volume fraction of the conductive grains, and does not diminish at higher salinities. Unlike membrane polarization, the electrode polarization relaxation time has strong dependence on the conductivity of the pore fluid32. There are several mechanistic and semi-empirical models for electrode polarization33 with broad agreement on chargeability but very different explanations for the relaxation time.In contrast, electrode polarizationIn this study, we show that capacitive charging of the Stern layer on the conductive grain is the primary mechanism for the observed electrode polarization relaxation time. This mechanism imparts a distinct and quantifiable spectral IP response linking directly to the intrinsic physical properties of the associated rocks, such as feature size, surface area, and electrical properties of the grains. In high salinity environments this mechanism dominates the observed IP signal and enables us to relate the response to the intrinsic physical properties of the conductive particulates. It also enables us to extend this mechanistic model to porous conducting grains in order to demonstrate the profound influence of nanoporous conductors on the characteristic relaxation time of the IP signal. We then discuss the geophysical implications of this insight in various geological settings. Treatment of IP response when membrane polarization effects are significant is beyond the scope of this study. Typically this occurs in low salinity environments.To isolate the fundamental mechanism for the electrode polarization relaxation time, we performed a series of frequency domain experiments with model systems. One of our model systems consists of natural cubic pyrite crystals (each side ~17 mm) embedded in a framework of glass beads (average particle diameter ~0.4 mm). The model systems are held in a custom-designed four-probe measurement cell as shown in Figs. 26 for salinities of 0.03 wt% NaCl or higher. With two cubic pyrite particles in the framework we observe significant frequency-dependent phase shifts in samples with different brine conductivities  in Fig. ϕc shows no discernible dependence on the brine conductivity σw  and relaxation time τ = aC0/2σm link directly to intrinsic properties of the porous media. Equation(1) the maximum phase shift 2) ϕc occurs at the characteristic frequency a and C0.Here the porous media (without conductive grains) is described in terms of its effective conductivity Equation  correspoϕc strongly agrees with the theoretical model Vcond, a, σm and a unit capacitance Cunit =1 μF/cm2. In principle, each rescaled spectrum should have a maximum phase shift C0 and is an invariant to a, σm, and Vcond. The solid symbols in Fig. To validate our theoretical predictions, we tested gold-coated glass spheres, silver grains, 304 and 316 stainless steels, and platinum-coated stainless steels of different sizes across different conditions  and stainless steel, due to the greater effective distance between the electronic charge and the ionic charge in the Stern layer34.The collapse of both gold-coated glass sphere and stainless steel sphere data onto the theoretically predicted frequency dependence indicate that our model is quantitative once the corresponding fc ∼ 1 Hz, attributed to presence of pyrites associated with hydrocarbon seepage above a known hydrocarbon reservoir7. Based on our quantitative model, generating this signature with solid (nonporous) pyrite would require the presence of meter-sized solid pyrite grains (with σm ∼ 0.2S/m and C0 ∼ 30 μF/cm2). This morphology is inconsistent with this geological setting7 and is only known to occur in massive ore deposits created by magmatic or hydrothermal processes. In this geological setting with sedimentary pyrite formation above a hydrocarbon reservoir, typical grain sizes tend to be in the tens to hundreds of micrometers. Here we propose that in such geological settings, one possible way of achieving dramatically increased relaxation times with this mechanism is to consider morphologies with very high surface-area-to-volume ratios, such as nanoporous conductive grains . For porous conductive inclusions, Eq. . To validate this modified scaling relation, we performed systematic experimental tests with a variety of nano-porous carbon beads as model materials. Carbon materials tested here are about a factor of 103 more conductive than the 3 wt% NaCl solution used, and thus behave like a conductive inclusion. Furthermore, the surface-area-per-mass (sm) of commercially available porous carbon are often calibrated and benchmarked46, and sf is a simple function of sm, ρ (density) and φ (porosity) as sf = smρ (1−φ). In Fig. sm ∼ 25 m2/g46, sieved into different size ranges. In this case with a constant sf, the characteristic frequency follows fc ∝ 1/a2 scaling almost exactly  and significantly different surface areas: Regal 660 (sm ∼ 90 m2/g)45 and Darco 12–20 (sm ∼ 600 m2/g)44 as in Fig. fc against C0 ∼ 3 μF/cm2 for porous carbon that is consistent with literature values48.Here tly Fig.  inset. Ttly Fig. . A more fc, porous conductors also distinguish themselves by an asymmetric spectral peak  with a nano-porous pyrite overgrowth due to the activity of sulfate reducing bacteria in the presence of hydrocarbons50. Understanding the IP response from these porous conductive materials could potentially lead to new methods to find hydrocarbon seeps or migration pathways. To illustrate the origin and electrical response of this nano-porous layer, we performed a biomimetic experiment with 1.6 mm carbon steel beads in the presence of sulfate reducing bacteria in an anaerobic environment (see Methods). Growing on the nominally flat surface of bare carbon steel beads  of the surfaces .Our improved fundamental understanding of the electrode polarization mechanism and the role of nanoporosity can now be applied to petroleum basins in a marine setting, where a specific type of porous conductive material, nano-porous pyrite, can be biologically formed in saline subsea environments by anaerobic sulfate reducing bacteriaads Fig. , the bacces Fig.  revealedces Fig. . The thices Fig.  and was 14, but in the absence of massive ore deposits associated with significant hydrothermal or volcanic activities54, we think that nano-porous pyrite is the only form of pyrite that would both correlate with hydrocarbon presence and would give rise to a distinguishable signature with the long relaxation times required for detection in conventional IP surveys. To form such specific nano-porous conductive structures, it is necessary to have sulfate reducing bacteria that produce H2S with the consumption of sulfate (xHy). The in-situ H2S production from anaerobic bacterial activity then reacts with different iron species and eventually forms nano-porous pyrite. In anoxic water columns, sulfate species diffuse downward from the seafloor, and are consumed by anaerobic bacteria in the sulfate reduction zone42. This process can also occur in euxinic water columns. In addition, mobile and constant hydrocarbon feed is necessary to form large clusters of framboidal pyrites with nano-porous overgrowths43 and aggregates42, as the conversion from localized organic carbon to pyrite is in some ways limited55 and the resulting euhedral and framboidal pyrites are relatively small.In subsea sediments, it is possible to detect IP features of different forms of pyrite7 observed in the geological setting with sedimentary pyrite formation above a hydrocarbon reservoir. For large solid euhedral pyrite(a ∼ 1 cm) or conventional framboidal pyrite  without any nano-porous pyrite overgrowth, we can use Eqs. (τ < 10 ms under representative conditions (σm ∼ 0.2 S/m and C0 ∼ 30 μF/cm2). Similar to the earlier results with euhedral pyrite in Fig. 43  would experience IP relaxation times τ ranging from ∼10 ms to ∼2 s assuming that their metallic nano-porous structures are inter-connected and filled with electrolytic conductive materials such as brine or clay. This mechanism based on nano-porous pyrite provides a possible explanation of the long IP relaxation time7 about 0.5–5 seconds observed in geological settings in the absence of massive ore deposits. It remains to be seen whether natural subsurface grains with the nanoporosity required to obtain large relaxation times form ubiquitously within hydrocarbon seeps, which would potentially allow IP surveys to help identify hydrocarbon charge, seep, or migration pathways7 by locating these nano-porous pyrite aggregates.The biomimetic experiment Fig.  along wiyritea ∼  cm or couse Eqs. , respectWhile most of our discussions so far are primarily focused on offshore marine environments, our understanding and interpretation of induced polarization can be readily applied to many other geophysical applications utilizing both frequency-domain and time-domain IP methods  in the subsurface. In such environments, salinity is typically high with σw > 1S/m, and as a result “membrane polarization” is less relevant leaving “electrode polarization” as the main signal for interpretation. For example, if the size distributions of the pyrite or other mineral deposits could be approximated from basin analysis, even just within an order of magnitude, it is possible to use Eq. (2) with the measured characteristic frequency and relaxation time scale to generate a subsurface map of surface-area-to-volume-ratio In offshore marine settings, either with a basin-scale controlled-source electromagnetic survey27 involving near surface fresh water, both “membrane polarization”27 from clay, sand, and other ion-selective materials and “electrode polarization” from conductive inclusions play a role in this low salinity environment. As a result meaningful interpretations of induced polarization signals would typically require additional information such as clay content distribution so that IP response from “electrode polarization” can be decoupled from “membrane polarization” with a correct mechanistic model. However, in near surface geological settings with a known type of massive mineral/ore deposit54, “electrode polarization” could potentially be the primary source of the measured IP signals. This electrode polarization response can easily be quantified when the mineral grain size distribution is relatively narrow. In this case the grain size can be estimated from σm and mineral information (C0). The abundance or volume fractions of the conducting grains can also be directly tied to the maximum phase shift of IP response as in In an electromagnetic-based land surveyσm is the effective conductivity of the porous media and μ is the magnetic permeability). Using this attenuation length we provide some perspectives to outline the detectability limitations of some specific materials  in different types of geophysical applications Like all electromagnetic-based geophysical surveys, the electromagnetic and IP signal strength will be limited by energy dissipation in conductive media, as electromagnetic waves in subsurface attenuate exponentially with the travelling distance. This attenuation length or skin depth is of the form of 42, the detectability from a basin-scale or borehole IP survey depends highly on their sedimentary environment and in particular the salinity. We have demonstrated that the characteristic frequency of their IP response is about 5000 Hz , the characteristic frequency of the same pyrites would be about 50 Hz leading to a skin depth of 700 meters due to both the frequency and conductivity changes. This large skin depth in the shallow freshwater condition could enable the acquisition and interpretation of meaningful IP response for identifying the abundance and potentially morphology of the shallow mineral species, assuming the minerals such as pyrite are relatively abundant in a local region.For conventional framboidal pyrite typically existing in subsea organic-rich layers SI-Text- or oil/g SI-Text- in high fc ∼ 1 Hz) resulting in a large skin depth over 1000 meters under representative conditions such as σm ∼ 0.2 S/m for brine saturated porous media. In addition, to our knowledge, the nanoporous layers demonstrated through natural samples43 and our biomimetic experiments . Given sufficient abundance, in principle such pyrite with nanoporous morphologies can be detected through offshore marine surveys and land surveys. Furthermore in borehole logging, along with resistivity, density, neutron porosity and other data, induced polarization data can potentially shed light on the nanoporous pyrite content and morphology and differentiate them from conventional framboidal pyrite from the unique frequency response.Unlike the conventional framboidal pyrites, pyrites with nanoporous morphologies are unique in the sense that even in marine environments with high salinity, these electronically conductive nanoporous structures can produce a uniquely low IP characteristic frequency  is a unique feature in sedimentary rocks containing nano-porous sulfides such as pyrite, which strongly correlates with the activity of sulfate reducing bacteria and hydrocarbon occurrence43. In addition to the broad geophysical applications, our discovery also leads to new strategies for surface area characterization of porous materials, super-capacitor design from biomimetic processes, and bio-inspired materials47.This study allows us to progress the interpretation of induced polarization measurements from the empirical and qualitative to the mechanistic and quantitative. We are now able to relate the measured spectral IP response to the abundance (volume fraction), morphology  and intrinsic material properties (specific capacitance) of the conducting grains. The broad agreements between theory and experiments, and the applicability to time-domain analysis  are used respectively for signal generation and data acquisition along with National Instruments chassis (PXIe-1078). Two current electrodes of the measurement cell are connected to two symmetric 1000 Glass beads are purchased through Sigma-Aldrich; Cubic pyrites are purchased through Ward’s science; 304 and 316 stainless steel spheres are purchased through McMaster Carr. Sodium Chloride is purchased through Sigma-Aldrich.50 nm of Gold and platinum coating on various sizes of spheres are achieved by plasma sputtering deposition (Denton sputter coater Hummer X) with gold and platinum targets (99.99% pure from Anatech USA). The uniformity of coating on spherical objects is achieved by a wireless controlled shaking device inside the vacuum chamber during the coating process. Our theoretical derivation suggests that the polarization effect of a hollow metal sphere would be the same as the solid spherical metal, since the solid shell behaves as a Faraday cage and the polarization charge is present only at the outer metal surface.sf  = 3/a and from Equation-2 we recover the earlier result with solid a conductive sphere.For solid spheres, a) composed of electronically-connected small conductive spheres (radius rp) with a porosity of φ, Cs ∼ a2C0 for a solid metal sphere, and therefore leads to a peak frequency For isotropic-shaped porous objects  on a solid metal bead (radius a), simple geometrical considerations suggest that the surface-area-to-volume ratio should be For a thin nano-porous conductive layer  and incubated at 30 °C overnight. After incubation, one set of carbon steel balls were transferred to 3 wt% NaCl solution for induced polarization measurement, while we process the other set with critical point drying and then use it for helium ion microscopy imaging.The wild-type Desulfovibrio vulgaris (D. vulgaris) was obtained from Judy Wall at the University of Missouri. Freezer stocks of D. vulgaris containing 10% glycerol were used to inoculate overnight cultures. The growth media contains 30 mM lactate, 30 mM sulfate, 8 mM MgClfc ∼ 3 Hz, h ∼ 5 μm, a = 0.8 mm, and C0 ∼ 30 μF/cm2 for pyrite), and by assuming a reasonable range of porosity (0.1 < φ < 0.5), we infer an average pore size < rp > ∼ 25–50 nm, demonstrating the consistency between induced polarization measurements and the helium ion images in Fig. From our earlier theoretical calculation Supplementary Information.