Il4r, Il10, Tgfbr2, colony-stimulating factor receptor (Csf1r), Itgam, suppressor of cytokine signaling 1 (Socs1), toll-like receptors 4 (Tlr4), P2rx7, P2ry12), and opposite responses were seen for others . In rat only, TGFβ1 affected microglial morphology and proliferation, but there was no apoptosis in either species. In both species, TGFβ1 dramatically increased Kv1.3 channel expression and current (no effects on Kir2.1). KCa3.1 showed opposite species responses: the current was low in unstimulated rat microglia and greatly increased by TGFβ1 but higher in control mouse cells and decreased by TGFβ1. Finally, we compared TGFβ1 and IL10 (often considered similar anti-inflammatory stimuli) and found many different responses in both species. Overall, the numerous species differences should be considered when characterizing neuroinflammation and microglial activation in vitro and in vivo, and when targeting potassium channels.The cytokine, transforming growth factor β1 (TGFβ1), is up-regulated after central nervous system (CNS) injuries or diseases involving microglial activation, and it has been proposed as a therapeutic agent for treating neuroinflammation. Microglia can produce and respond to TGFβ1. While rats and mice are commonly used for studying neuroinflammation, very few reports directly compare them. Such studies are important for improving pre-clinical studies and furthering translational progress in developing therapeutic interventions. After intracerebral hemorrhage (ICH) in the rat striatum, the TGFβ1 receptor was highly expressed on microglia/macrophages within the hematoma. We recently found species similarities and differences in response to either a pro-inflammatory  or anti-inflammatory interleukin-4 (IL-4) stimulus. Here, we assessed whether rat and mouse microglia differ in their responses to TGFβ1. Microglia were isolated from Sprague-Dawley rats and C57BL/6 mice and treated with TGFβ1. We quantified changes in expression of >50 genes, in their morphology, proliferation, apoptosis and in three potassium channels that are considered therapeutic targets. Many inflammatory mediators, immune receptors and modulators showed species similarities, but notable differences included that, for some genes, only one species responded (e.g.,  At the protein level, we also assessed the TGFβ1 receptor (TGFβR1), the interferon-γ (IFNγ) receptor (IFNGR1), the mannose receptor (CD206/MRC1) and the Ca2+-activated K+ channel, KCa3.1. While there were some species similarities in responses to TGFβ1, the observed differences reinforce the importance of analyzing both species in pre-clinical studies. Moreover, in comparing the TGFβ1 responses with our recently published analysis of IL-10 responses of primary rat and mouse microglia, substantial differences were seen between these two anti-inflammatory cytokines.There are surprisingly few studies comparing microglial responses in these rodent species. Our recent study showed many differences in responses of rat and mouse microglia to pro- and anti-inflammatory cytokines  and Ca2+-release-activated Ca2+ (CRAC) channels to regulate many microglial functions, including proliferation, migration, phagocytosis and cytokine production  for Kir2.1, Kv1.3, and KCa3.1. Again, despite some similar responses, there were species differences that should be considered for future translational studies.When considering microglial contributions to CNS damage and recovery, numerous recent studies using specific KAll procedures on animals adhered to the Canadian Council on Animal Care guidelines for humane animal use and were approved by the University Health Network Animal Care Committee .RRID:SCR_013553; Cat# MCA275G, RRID:AB_321301) and TGFβR1 . Secondary antibodies were Alexa-conjugated and used at 1:200 dilution  in phosphate buffered saline  with 0.2% Triton X-100  and 5% donkey serum . To label cell nuclei, 4’-6-diamidino-2-phenylindole  was applied for 10 min. Slides were mounted in Dako mounting medium  and stored in the dark at 4°C. Images were obtained with a confocal microscope  and processed using ImageJ software, version 1.47vRRID:SCR_003070).The methods for creating an ICH by injecting collagenase into the striatum of male Sprague-Dawley rats , and preparing the tissue and cryosections for immunohistochemistry were exactly as in our recent work  in cold Minimal Essential Medium . Mixed brain cell cultures were established by re-suspending the pellet in MEM, incubating in 75 cm2 flasks with 20 ml MEM supplemented with 10% fetal bovine serum  and 0.05 mg/ml gentamycin . The medium was changed after 2 days, and microglia (≥99% pure) were harvested a few days later by shaking the flasks  on an orbital shaker in the incubator, centrifuging the supernatant, and re-suspending the cells in fresh MEM . Microglia were seeded onto UV-irradiated 15 mm coverslips  at densities that are noted under each assay. Their initial state was similar for both species, as confirmed from the expression of numerous genes (see “Results” section).Microglia were isolated from Sprague-Dawley rat pups (P1–P2) and C57BL/6 mouse pups , as recently described , and centrifuged  to pellet the tissue. Following the manufacturer’s protocol for the Neural Dissociation Kit , the supernatant was removed, cells were dissociated using Pasteur pipettes. To trap myelin, cells were incubated with myelin removal beads  at 4°C for 15 min, washed in PBS containing 0.5% bovine serum albumin , centrifuged, resuspended in PBS/BSA buffer, and passed through LS columns . Eluted cells were centrifuged, resuspended in PBS/BSA buffer, and incubated at 4°C for 15 min with either human/mouse CD11b or rat CD11b/c microbeads . To collect eluted microglia, the cell suspension was washed, centrifuged, and resuspended in PBS/BSA buffer, and passed through MS columns . Microglia were centrifuged, resuspended in fresh MEM , seeded at 8 × 104 cells/coverslip, and grown for 1–2 days before treating with TGFβ1.Adult male Sprague-Dawley rats (300–350 g) or C57BL/6J mice  were deeply anesthetized with isoflurane and decapitated. After 400 mg of cerebral cortex was harvested, it was chopped into small pieces in 1 ml HBSS without CaRRID:SCR_006140; Cat# 7666-MB), and rat cells were treated with recombinant human TGFβ1  because a rat cytokine was not commercially available. rhTGFβ1 has often been used on rodent cells  or treated with TGFβ1, which was diluted from a stock solution in sterile PBS with 0.3% BSA that was stored at −20°C. The mature form of the cytokine binds to its receptor, and the amino acid sequences for mature rat, mouse and human TGFβ1 are 99%–100% identical. Therefore, either mouse or human recombinant TGFβ1 could be used. Mouse cells were treated with recombinant mouse TGFβ1  for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, washed, and labeled overnight with CD11b . The next day, cells were washed in PBS and incubated for 1 h with Alexa 594-conjugated secondary antibody . Cells were counterstained for 1 h with Acti-stain 488 phalloidin  to visualize filamentous (F-) actin, and with DAPI  to label nuclei. Coverslips were mounted on glass slides using Dako mounting medium and stored in the dark at 4°C until images were acquired with a confocal microscope  and processed in ImageJ. Unipolar cells (those with a lamellum and trailing uropod) were counted using ImageJ in three micrographs each (20× magnification) and expressed as a percentage of total cells counted (~120–200/culture).Microglial morphology and proliferation were examined, as before  or stimulated with TGFβ1 for 24 h. The detection dye was added to each well for 30 min , and the fluorescence intensity was measured using a multi-label plate reader , with excitation at 485 nm and emission at 535 nm. Duplicate readings were taken for 0.1 s at 3 mm from the bottom of the plate, averaged, and then the background was subtracted before normalizing to the control group.Proliferation was quantified using the CyQUANT™ NF assay , as before , treated with TGFβ1 for 24 h, and fixed in 4% PFA for 7 min, washed in PBS and post-fixed in methanol for 20 min. Apoptotic cells were detected using the TiterTACS™ Colorimetric Apoptosis Detection Kit , with positive controls (TACS-Nuclease™ added for 10–20 min to generate DNA breaks) and negative controls (omitting TdT enzyme). Absorbance was measured at 450 nm using a SPECTROstar Nano plate reader .Cells were seeded at 4 × 105 cells/coverslip in a 12-well culture plate, allowed to settle for 1–2 days , and then left untreated (control) or stimulated with TGFβ1 for 24 h. In a pilot study on primary rat microglia, some genes were also analyzed at 6 h after TGFβ1 treatment and the relevant results are mentioned in the text. The sample preparation and transcriptional profiling were conducted as before . Each transcript of interest was recognized by a capture probe and a reporter probe, and assay variability was minimized using negative reporter probes for background subtraction. Positive control reporter probes were included to assess the hybridization efficiency and detection range, and to calculate a scaling factor that was applied to all mRNA counts in each sample. Finally, a reference gene scaling factor was calculated in the same manner using the housekeeping genes, Hprt1 and Gusb, and used to adjust the counts for each sample .Data were analyzed with nSolver Analysis Software . Cells were incubated for 24 h with or without TGFβ1, and then briefly washed with PBS, lysed for 30 min in ice-cold RIPA lysis buffer with a mammalian protease inhibitor cocktail , and spun down to remove insoluble material. Protein concentration was determined with a Pierce™ BCA protein assay , and lysates were stored at −80°C until used. Proteins were denatured (100°C for 5 min in a dry-bath incubator) in NuPage LDS sample buffer  with 5% 2-β-mercaptoethanol. Samples were subjected to SDS-PAGE on 8% or 12% acrylamide gels at 10 μg protein/lane, and electrophoresed for 1.5–2 h at 80 mV (stacking gel) and 120 mV (resolving gel). Proteins were then transferred to a PVDF membrane and blocked (2–3 h) in 5% non-fat dry milk in Tris-Tween buffered saline (TTBS).Protein changes generally map well onto mRNA changes, which we previously validated for several molecules investigated here , anti-TGFβR1 , anti-CD206 , and anti-KCNN4 . After washing in 1% BSA-TTBS (4 × 10 min), membranes were incubated for 1 h at room temperature in horseradish peroxidase-labeled secondary anti-rabbit IgG antibodies  in 1% BSA-TTBS. After repeated washes, membranes were treated with GE Healthcare ECL™ Start Western Blotting Detection Reagent , and protein bands were visualized using the ChemiDoc™ XRS System (Bio-Rad).Protein levels were measured for IFNGR1, TGFβR1, CD206 and KCa3.1. Polyclonal rabbit primary antibodies were applied overnight (4°C) in TTBS with 1% BSA, as follows: anti-IFNGR1 , gel lanes and bands were user-delineated, background was subtracted using the software’s rolling disk algorithm, and signal intensities of identified bands were determined. Bands of interest were then normalized to the total Coomassie blue staining intensity of a given lane, and expressed as fold-changes relative to unstimulated (control) cells. Supplementary Figure S1 shows uncropped images of representative blots used for quantification.We used total protein normalization of Coomassie-stained immunoblots  at room temperature, and recorded in the whole-cell configuration. The bath solution consisted of (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 D-glucose, adjusted to pH 7.4 and 290–300 mOsm/kg H2O. Pipettes were pulled from thin wall borosilicate glass  on a Narishige puller  and fire polished with a microforge  from about 2 MΩ to 5–8 MΩ. The pipette  solution was 40 KCl, 100 K aspartate, 1 MgCl2, 10 HEPES, 2 MgATP , and with 0.5 CaCl2 and 1 EGTA to buffer internal free Ca2+ to ~120 nM. The same bath and pipette solutions were used to record all currents. Data were acquired using an Axopatch 200A amplifier, filtered at 5 Hz with a DigiDATA 1322A board, and analyzed with pCLAMP 10 software . Junction/offset potentials were calculated with the pCLAMP utility and used to correct each figure. Voltage protocols are indicated in the Figure Legends and the rationale briefly explained in the “Results” section text.Microglia were plated at 7–9 × 10RRID:SCR_003689; Cat# 4549) or Ba2+, and the blocker-sensitive Kir2.1 component was assessed, as before . To quantify the KCa3.1 current, it was evoked by the positive-gating modulator, 1 μM SKA-111  was added to the bath and the remaining current was subtracted from the total current to yield the Kv1.3 component. AgTx-2 was prepared from a stock solution in double-distilled water with 0.02% BSA. To quantify Kir2.1, the current was blocked with 20 μM ML133 . The p values for differences in treatment or species were adjusted using a 5% false discovery rate correction for multiple comparisons . Proliferation, apoptosis, Western blotting, and electrophysiological data were analyzed in GraphPad Prism using a 2-way ANOVA with Bonferroni or Tukey’s post hoc analysis.Quantified data are expressed as mean ± SEM for the number of biological replicates indicated in figure and table legends. For Nanostring mRNA analysis, a one-way analysis of variance (ANOVA) with Dunnett’s As mentioned in the “Introduction” section, TGFβ1 is often increased after acute CNS damage, and microglia are important responders to this cytokine. We previously reported an increase in TGFβ1 mRNA in the damaged rat striatum after ICH was induced by collagenase injection , and greater cell length in mouse (79.2 ± 3.9 μm) than rat . Consequently, the aspect ratio (lamellum width ÷ cell length) was 0.54 ± 0.02 for rat and 0.35 ± 0.01 for mouse (p < 0.0001). Moreover, only in rat cells was the lamellum ever wider than the cell length.To assess the general health of isolated rat and mouse microglia and their response to TGFβ1, we first examined morphology, proliferation and apoptosis in a pilot study using 5, 10 and 20 ng/ml of TGFβ1 Figures . As seen2). We measured cell capacitance during patch-clamp experiments (below) and showed no differences between species or with TGFβ1 treatment. For control microglia, the values (mean ± SEM) were 25.3 ± 1.8 pF (n = 32) for rat, and 24.2 ± 1.4 pF (n = 45) for mouse. For mouse microglia, 24 h treatment with TGFβ1 did not obviously affect the morphology or intensity of CD11b staining, and the capacitance was 22.1 ± 1.0 pF . Despite rat cells rounding up, their capacitance was not affected . Thus, the shape changes were due to rounding up, not to cell size changes.TGFβ1-treated rat cells showed increased CD11b staining and retracted their lamellae and uropods at all concentrations tested. However, visual inspection cannot determine whether the rounding up reflects a change in size or just a change in morphology at a constant size. The measured membrane capacitance can be used as a direct measure of total cell membrane area, regardless of 3-dimensional cell shape. For instance, capacitance accounts for retraction of cell processes, membrane ruffling and changes in cell height. This is because the bimolecular lipid membrane is a remarkably consistent dielectric, and specific membrane capacitance is, in essence, a biological constant  at 24 h in the number of rat microglia at all TGFβ1 concentrations; while mouse cell numbers were unaffected Figure . DespiteThe next goal was to compare responses of rat and mouse microglia to TGFβ1 by profiling transcript expression of >50 selected genes. We have organized the results into functional groupings, including microglial markers and immunomodulators, pro- and anti-inflammatory mediators and their receptors, and some molecules related to specific microglial physiological functions. The gene nomenclature is indicated for rat (Supplementary Table S1) and mouse (Supplementary Table S2), and where different, the rat names are used.First, we quantified expression of several genes that are often used to identify microglial “activation” after acute brain injury, as well as several immunomodulatory molecules (Table Itgam (CD11b), Cx3cr1 , toll-like receptors 2 and 4  and Nr3c1 , and high levels (>5000 counts) of Cd68 (ED1) and colony-stimulating factor receptor . The most striking species differences in control cells were higher Cd68 and much higher Aif1 (Iba1) in rat, as recently reported  and Socs3, and moderate levels of Nfkbia  and translocator protein (Tspo).Microglia of both species expressed moderate levels (>100 counts) of several well-known receptors on microglia: Cx3cr1, Itgam and Csf1r; mouse cells had reduced Nr3c1 and Tlr4 ; and rat cells had reduced Socs1. Several genes in this category were not significantly altered in either species: Aif1, Cd68, Nfkbia, Socs3, Tlr2 and Tspo (Supplementary Table S3). There were several similarities in a pilot study of 6 h TGFβ1 treatment of rat cells, including up-regulated Cx3cr1 and Itgam but no changes in Tlr2 or Tlr4 (not illustrated).Of genes in this category, 6/12 were altered in one or both species but there were several species differences in the magnitude of responses . Several genes associated with alternative activation were very low in both species: Arg1, Ccl22, Cd163 and Retnla (FIZZ1). Il1rn, Mrc1 (CD206), Myc, Pparg and Trem2 were moderately expressed in both species. The main species differences were that unstimulated rat cells had higher levels of Il1rn, Il4r, Il10ra, Il10rb, Pparg, Tgfb1, Tgfbr1 and Trem2.Microglia of both species had moderate resting mRNA levels of Mrc1 and Pparg, and increased Tgfbr1 . A pilot study of 6 h TGFβ1 treatment of rat cells showed several similarities: decreased Mrc1 and Pparg, and no change in Ccl22 (not illustrated).Changes were seen in 12/18 genes examined in one or both species. Both species had decreased r1 Table . Here, wNos2 (iNOS), Il6, Ptgs2 (COX-2), Ifng and Il1r1 . Nevertheless, cells of both species were poised to respond to the pro-inflammatory cytokines, IFNγ and TNFα, having moderate levels of the receptors, Ifngr1, Tnfrsf1a (TNFR1) and Tnfrsf1b , caspase-1 (Casp1), IL-1-converting enzyme (ICE), Ccl3 , and the protein kinase, Ptk2b . The main species differences were that rat showed much lower Ifngr2 and higher Ccr5, Ifngr1, Il1b, Tnfrsf1a, Tnfrsf1b and Trem1.As we previously found for rat microglia  and Trem1 was increased. There were some similarities in mouse  but a unique response was decreased Casp1, an enzyme that contributes to IL1β activation. Hallmark pro-inflammatory genes that did not change in either species included Ccr2, Ifng, Ifngr2, Il1r1, Il6, Nos2, Ptgs2, Ptk2b and Tnfrs1a (Supplementary Table S3). It appears that many transcriptional responses to TGFβ1 were rapid; i.e., in a pilot study of 6 h treatment of rat microglia (not illustrated), 17/25 genes showed qualitatively similar responses to those seen at 24 h; e.g., increases in Cx3cr1, Itgam, Ncf1 and P2rx7; decreases in Tnfa, Mrc1 and Pparg, and no changes in Nos2, Il4, Il4ra, Tlr4, Cd163 and Retnla.There were changes in 8/17 in one or both species, but again, there were species differences. 1 Figure  corroborCybb (NOX2), Ncf1, Fcgr3a; while IL-4 treatment reduced Cybb and increased Fcgr2b. Cybb is the main contributor to the respiratory burst in microglia  relocation to the plasma membrane , can promote NOX enzyme activity by allowing H+ efflux as charge compensation for NADPH-generated electrons , and Adora2a (adenosine A2A receptor). Both species had moderate to high levels of Hvcn1, Cybb, Ncf1, P2rx7, P2ry12, Itgb2 and the phagocytosis-related molecules, Fcgr1 (CD64), Fcgr2b (CD32), Fcgr3a (CD16) and Msr1  and Nox1 in both species. Species differences were decreased Fcgr3a and increased P2rx7  and P2ry12 in rat cells only. In both species, Adora1, Adora2a, Cybb, Fcgr1, Hvcn1, Msr1, Nox4 and P2ry2 were unchanged (Supplementary Table S3).Several genes were affected by TGFβ1 Table , includi2+ signaling (see “Introduction” and “Discussion” sections); thus, we asked whether TGFβ1 treatment affects expression of several ion channel genes that are linked to Ca2+ entry. These included the Ca2+-permeable channels, Trpm2, Trpm4 and Trpm7, the pore-forming subunits of CRAC channels , and the CRAC regulator, Stim1. We then assessed several K+ channels that potentially regulate the driving force for Ca2+ entry: Kcna2 (Kv1.2), Kcna3 (Kv1.3), Kcna5 (Kv1.5), Kcnj2 (Kir2.1), Kcnma1 (KCa1.1/BK/maxi K), Kcnn3 (KCa2.3/SK3) and Kcnn4 (KCa3.1/SK4). In addition, several important K+ channel regulators were monitored: Ptpn6 , calmodulin , RE1 silencing transcription factor (Rest), Phtp1 , and Nme2 . Note: for ease of comparison with the literature, results are presented using the protein names. While modest effects of TGFβ1 were seen in one or both species for Orai3, Rest, SHP-1 and Trpm7 . Those results are organized below under each channel type. For Kv1.3 and Kir2.1, adult microglia were also examined because some researchers have questioned whether their expression differs from neonatal microglia (see “Discussion” section).Many microglial functions depend on CaKcna2 (Kv1.2) 73 ± 47 in rat, 10 ± 6 in mouse ; Kcna3 (Kv1.3) 109 ± 18 in rat, 70 ± 14 in mouse (p = 0.013); Kcna5 (Kv1.5) 4 ± 2 in rat, 3 ± 2 in mouse. TGFβ1 treatment did not alter Kv1.2 mRNA, but significantly increased Kv1.3 mRNA: about 7.4-fold at 24 h in rat , and 5.6-fold in mouse . TGFβ1 increased the amplitudes of total Kv and Kv1.3 currents  and that, among cells with a detectable current, less was AgTx-2-sensitive compared to control rat microglia . TGFβ1 treatment did not significantly alter Kcnj2 mRNA expression in either species . In control microglia, there was a similar magnitude of Kir2.1 current in both species, and TGFβ1 treatment had no effect , and almost all the current was contributed by Kir2.1  6 ± 3 in rat vs. 30 ± 10 in mouse ; Kcnn3 (KCa2.3) 25 ± 14 in rat vs. 9 ± 3 in mouse (p = 0.08); Kcnn4 (KCa3.1) 52 ± 11 in rat vs. 644 ± 331 in mouse (p < 0.001). Calmodulin binds to KCa3.1 and KCa2-family channels and then, Ca2+ binding to calmodulin causes the channels to open  and was not affected by TGFβ1 in either species . Furthermore, the species difference in Kcnn4 mRNA corresponded with increased KCa3.1 protein in rat (but not mouse) microglia . In a pilot study on rat microglia (not illustrated), 500 nM NS309 failed to activate a current (3/3 cells) but after TGFβ1 treatment there was a robust current in 6/8 cells . Surprisingly, the opposite was seen for mouse cells. SKA-111 activated a large KCa3.1 current in control mouse microglia (17.9 ± 2.4 pA/pF) but after TGFβ1 treatment, almost no current was evoked .KCa3.1 is the only CaV Figure . Then, TCcl3, Ccr5, Csf1r, Itgb2, Kcnn3, Kcnn4, Orai3). . For ease of comparison, the bubble chart visually highlights both the magnitude and direction of changes. There were some intriguing differences in TGFβ1 and IL-10 responses, and we will highlight some examples under each category.As anti-inflammatory or resolving cytokines, TGFβ1 and IL-10 are often assumed to have similar effects (see “Discussion” section). In Figure Cx3cr1 expression; whereas, IL-10 decreased it. TGFβ1 decreased Socs1 only in rat. IL-10 increased Socs3 only in mouse. IL-10 increased Tlr4 in both species but TGFβ1 did not; indeed, Tlr4 decreased in mouse. Both cytokines increased Itgam in rat; only IL-10 increased it in mouse. TGFβ1 increased Csf1r in rat only; IL-10 did not affect either species. IL-10 increased Cd68 and Tspo in rat only. In mouse, TGFβ1 decreased Nr3c1 but IL-10 increased it.In both species, TGFβ1 increased Pparg and increased Tgfbr1; both were unaffected by IL-10. TGFβ1 decreased Mrc1 in both species; IL-10 increased it in mouse. TGFβ1 increased Tgfb1 and Myc in rat but decreased them in mouse. However, TGFβ1 decreased Il1rn in rat. In mouse only, TGFβ1 increased Il4 and Il10 and both cytokines increased Il4r and Il13ra1. Both cytokines increased Trem2 and Tgfbr2 in rat; whereas, IL-10 only elevated Tgfbr2 in mouse. IL-10 increased Il10rb only in rat.In both species, TGFβ1 decreased Trem1 but decreased Ccl3 and Ccr5. TGFβ1 decreased Tnfa and Tnfrsf1b only in rat and Casp1 only in mouse. Ifngr1 was oppositely affected by TGFβ1; increased in rat, decreased in mouse. IL-10 increased Il1b in mouse, while TGFβ1 decreased it in rat.In both species, TGFβ1 increased Fcgr2b, and only TGFβ1 increased Ncf1 and Nox1. In mouse, there were no other effects of TGFβ1; however, in rat, P2rx7, Itgb2 and P2ry12 were increased and Fcgr3a was decreased. In contrast, IL-10 increased Fcgr3a in both species. IL-10 also elevated P2ry2 in rat, and P2ry12 and Msr1 in mouse.In both species, both cytokines increased Kcna3, Kcna5 and Rest in both species; Kcnn4, Orai3 and Trpm7 in rat; and Kcnn3 in mouse. The only effect of IL-10 was reduced Kcnj2 in mouse. For the K+ currents, there were some notable differences. In both species, the Kv1.3 current was unaffected by IL-10  differ in transcriptional responses , and an important role in reducing microglial inflammatory cytokine and chemokine expression in vitro  up to 3 days after ICH, and then during the recovery period (days 7–14), they up-regulated Tgfb1, Tgfbr1 and Tgfbr2. Here, we found that 7 days after ICH in the rat, there was strong expression of the TGFβ1 receptor in microglia/activated macrophages within the hematoma.TGFβ1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties Colton, . TGFβ1 lIn vitro, microglia can be skewed to multiple anti-inflammatory phenotypes, which are usually assessed by molecular profiles and functional outcomes  did not affect proliferation 3 days later; but if proliferation was first stimulated by a mitogen , it was inhibited by as little as 0.01 ng/ml TGFβ1  and P2ry12. There were very few genes in common with the present study but qualitatively similar results following TGFβ1 treatment were seen for Cx3cr1, Myc, Tgfbr1 and Ccr5 in mouse microglia. Applying the blood product, thrombin, to cultured mouse microglia to model ICH, up-regulated Il6, Tnf and Ccl2 and reduced Tgfbr1, and these changes were reversed by adding TGFβ1 . TGFβ1 increased molecules involved in superoxide production , while IL-10 increased several phagocytosis-related molecules . The two cytokines evoked opposite changes in Cx3cr1. Differing responses were also seen in channel activity. Notably, in both species, TGFβ1 increased Kv1.3 currents (present study) but IL-10 had no effect  were moderately high in unstimulated rat and mouse microglia, and not reduced by either of the cytokine treatments  as well as anti-inflammatory receptors , suggesting an overall increased ability to respond to subsequent inflammatory stimuli.Differences in responsiveness to TGFβ1 and IL-10 were not accompanied by a deficiency or down-regulation of any of the cognate receptors in either species. Receptor transcript levels  and SOC channels  receptors. K+ channels affect the membrane potential , Cd68, Tlr2, Itgb2, and various Fc receptors) are part of the “sensome”, which is a large group of molecules and receptors at the cell surface involved in microglia sensing environmental perturbations, as in damage or disease (Hickman et al., Csf1r, Cx3cr1, P2ry12, Tgfbr1.Studies of disease mechanisms rely heavily on rodent models and translation to humans is a continuing challenge. Transcriptional profiling is increasingly used to investigate microglia in CNS damage and disease states, and an important question is whether differences reported in microglia responses Goals of molecular fingerprinting studies include: selecting and interpreting markers and molecules in animal models of CNS disease; identifying a reliable panel of human markers to help define disease progress and determine whether treatments are effective; and identifying molecular targets to control neuroinflammation. To this end, it is essential to compare human microglia with rodent models; however, there is very little published data comparing human responses to TGFβ1. A recent article raises concerns because a hallmark TGFβ1 response in rodents was not seen in human microglia (Smith et al., The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The project was mainly conceived and spearheaded by LCS, with important contributions from SL, DL and RW. SL carried out the analyses of ICH tissue, Nanostring, Western blotting and functional studies (morphology, proliferation, apoptosis; Tables 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. The reviewer RR and handling Editor declared their shared affiliation.


The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event—a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate. There is a pressing need for therapeutic agents against Zika virus (ZIKV). Here the authors present cryoEM structures of a neutralizing antibody (C10) complexed with ZIKV that show C10 preventing structural changes required for virus entry into the cell, suggesting it might be effective in treating Zika infections. Zika virus2The virus surface consists of 180 copies of E proteinAn E protein contains three domains—DI, DII and DIII6650=0.024 μg ml−1), as demonstrated recently in ZIKV infected cell culture11The recent explosion of the number of ZIKV cases, together with the association of ZIKV with the development of microcephaly in fetuses101110We solved the cryoEM structures of Fab C10 complexed with ZIKV at pH8.0, pH6.5 and pH5.0 mimicking the extracellular, early and late endosomal conditions, respectively, and compared them to the cryoEM maps of the uncomplexed ZIKV controls at pH8.0 ref. , pH6.5  and the Micrographs of the uncomplexed ZIKV control at pH8.0 sample show mostly smooth surfaced spherical particles . In the Micrographs of the ZIKV-C10 complexes at all pH conditions show spiky looking particles, due to the Fab molecules bound to virus surface . The 2D The cryoEM structures of ZIKV-C10 complex at pH8.0, pH6.5 and pH5.0 are determined to 4.0, 4.4 and 12 Å resolution, respectively . In eachIn the 4.0 Å resolution pH8.0 complex cryoEM map , the likThe ZIKV-C10 complex intra-dimer epitope is located on the DII (at and around the fusion loop) on one E protein and on DIII and DI of the other E protein in the dimer . A plot A comparison of the cryoEM maps of pH5.0 complex to the pH8.0 complex shows that the E protein layer has moved to a larger radius , whereasComparing the pH8.0 and the pH5.0 complex structures  shows thAlthough the E protein raft structure stays mostly intact in the ZIKV-C10 pH5.0 complex structure, the inter-raft interactions are disrupted , even thAll antibodies can cause ADE at some concentrations. HMAb C10, similar to other potent antibodies, causes ADE at a much narrower range of concentrations compared to the other weakly neutralizing antibodies20−1 were incubated with equal volumes of virus at 37 °C for 0.5 h. One hundred microlitres of each mixtures were then layered on BHK-21 cells in a 24-well plate and incubated at 37 °C for 1.5 h. The infected cells were washed with phosphate-buffered saline, overlaid with carboxyl-methyl cellulose and incubated at 37 °C for 5 days. Cells were fixed and stained, and the plaques were counted. Percentage neutralization was determined from the comparison of the number of plaques in specific antibody dilutions to the control (without antibody). PRNT50 is the concentration of the antibody that causes 50% reduction in plaque numbers.The neutralization activity of the HMAb C10 on Zika virus strain H/PF/2013 was determined by PRNT. Two-fold serially diluted HMAb C10 starting at 0.5 μg mlAedes albopictus C6/36 cells (ATCC) were grown in RPMI 1640 media supplemented with 2% fetal bovine serum at 29 °C. At about 80% confluency, the cells were inoculated with ZIKV strain H/PF/2013 at a multiplicity of infection of 0.5 and incubated at 29 °C for 4 days. The virus-containing media was clarified by centrifugation at 12,000g for 1 h. Virus was precipitated overnight from the supernatant using 8% (w/v) polyethylene glycol 8000 in NTE buffer  and the suspension was centrifuged at 14,334g for 1 h. The resulting pellet containing the virus was resuspended in NTE buffer and then purified through a 24% (w/v) sucrose cushion followed by a linear 10-30% w/v potassium tartrate gradient. The virus band, visualized by its light scattering ability, was extracted, buffer exchanged into NTE buffer and concentrated using a concentrator with 100-kDa molecular weight cut-off filter. All steps of the purification procedure were done at 4 °C. The concentrations and purity of the E protein were estimated with Coomassie blue-stained SDS-PAGE using different known concentrations of bovine serum albumin solution as standards.Monoclonal antibody (mAb) C10 was synthesized in transfected human cells from cloned plasmids . Briefly, previously published heavy and light chain variable region sequences−1) was incubated overnight with immobilized papain (Thermo scientific) at 37 °C. After digestion, the Fab fragment was purified with anion exchange chromatography  and gel filtration  on an AKTA purifier system.The Fab regions of C10 IgG were produced by papain digestion. Briefly, the whole IgG  for 10 s prior to adjusting the pH. The final pH of the virus was reached by addition of a volume ratio of 1.5 μl of 50 mM MES buffer at respective pH (pH5.0 or pH6.5) to every 1 μl of the virus-Fab mixture. The pH-adjusted samples were left on the grid for another 15 s. The grid was then blotted with filter paper and flash frozen in liquid ethane by using the Vitrobot Mark IV plunger . The corresponding controls (ZIKV without Fab) for each pH were prepared similarly.The images of the frozen ZIKV complexes were taken with the FEI Titan Krios electron microscope, equipped with 300 kV field emission gun, at nominal magnification of 47,000 for pH5.0 Fab C10 ZIKV complex, and 59,000 for pH6.5 and pH8.0 complex samples. A 4096 × 4096 FEI Falcon II direct electron detector was used to record the images.− Å−2 for pH8.0, total exposure of 1.05 s and total dose of 43 e− Å−2 for pH6.5 complex. The pH5.0 complex was collected at single image mode, with the dose of 20 e− Å−2. The frames from each ‘movie' were aligned using MotionCorr− Å−2 to use in 3D reconstruction. The images were taken at underfocus in 0.5∼2.5 μm range. The astigmatic defocus parameters were estimated with GctfLeginonThe pH8.0 and pH6.5 ZIKV-C10 structures were interpreted by fitting in the uncomplexed ZIKV (PDB ID 5IZ7) and Fab C10 (PDB ID 4UT9) first as rigid bodies in ChimeraElectrostatic potentials of protein surfaces were calculated using Adaptive Poisson-Boltzmann Solver(APBS)The cryoEM maps and the atomic models of the ZIKV-C10 complex at pH8.0, 6.5 and 5.0 have been deposited in the Electron Microscopy Data Bank (EMD) and the Protein Data Bank (PDB) under the accession codes EMD-9575, EMD-9573, EMD-9574 and 5H37, 5H30, 5H32, respectively. The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Zhang S, et al. Neutralization mechanism of a highly potent antibody against Zika virus. Nat. Commun.7, 13679 doi: 10.1038/ncomms13679 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables


Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass.pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2–BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification.In this study, we used a systematic genetic strategy based on the T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.These results demonstrate that genetically exploiting the potentials of  Depletion of fossil fuels and deterioration of ecological environments have attracted intensive attentions toward the utilization of renewable lignocellulosic biomass for production of biofuels, such as cellulosic ethanol , 2. An eIt is well-known that a multitude of enzymatic activities are required to degrade cellulose, the most abundant constituent of lignocellulosic biomass, into glucose as a fermentable sugar for ethanol production , 5. ThesTrichoderma reesei, which secretes all the core enzymes essential to the complete hydrolysis of lignocellulose [T. reesei, respectively [T. reesei cellulase system appear to be present in suboptimal ratios for lignocellulose degradation. Intensive research efforts with genetic engineering strategies have been done to increase single cellulase components in T. reesei for strain improvement [T. reesei have not been applied to optimizing the enzyme cocktail.Current commercial cellulase preparations are mainly derived from the filamentous fungus ellulose . The secellulose –19. CBH1ectively . Nonetheectively . Furtherectively . EG1 andectively . Effortsectively , 23. In ectively . In consrovement –28. HoweT. reesei exhibited a 17.1-fold increase in BGL activity and provided much better performance on the enzymatic saccharification efficiency [pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain. Individual overexpression of CBH2 or EG2 was firstly conducted and compared for cellulase production as well as saccharification efficiency. To assist multiple genetic manipulations, the marker pyrG was removed by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain to optimize the cellulase system. Differently pretreated corncob residues were finally used as substrates to assess the saccharification efficiency of the enzyme complexes.In our previous work, overexpression of BGL1 in ficiency . Here, wcbh2 (Gene ID: 72567) expression cassette, cbh2–pyrG, containing the cbh2 gene and a pyrG marker (pyrG+DR) was constructed by double-joint PCR [T. reesei uracil auxotrophic strain QP4 using the PEG-mediated method. The transformants were then screened on AMM plates containing Avicel as the sole carbon source. It was reported that growth rates of T. reesei CBH overexpression transformants on cellulose-containing plates correlated well with their CBH activities [cbh2 expression cassette in its chromosomal DNA  was performed to investigate the transcript abundance of T. reesei [egl1 and egl2 gene detected by qPCR analysis  at 30 °C for 7 days. The fermentation supernatants were collected at different time intervals. Then the activities of total cellulase , cellobiohydrolases, endoglucanases and the extracellular protein concentration were measured  and delignified corncob residues (DCR). In the saccharification of ACR, the glucose release (7.5 mg/mL corresponding to 21.4% cellulose conversion) using the QPC67 enzyme was comparable with that of QP4 (7.1 mg/mL corresponding to 20.0% cellulose conversion) after a total enzymatic reaction of 48 h  expression cassette, egl2–pyrG, was transformed into the strain QP4 using the method as described above for CBH2 overexpression  than that of the parental strain QP4. Particularly worth mentioning is that the expression levels of cellobiohydolase genes (cbh1 and cbh2) in QPE36 were also up-regulated with values of 2-fold in comparison to QP4 while the expression level of egl1 was not affected  was used as the recyclable marker that could be excised by homologous recombination. Figure pyrG marker. To reuse the pyrG marker in the egl2-overexpression strain, the conidio spores of QPE36 was plated on 5-FOA plates. The 5-FOA resistant colonies then were transferred to minimal medium plates containing uracil or not, which allowed the growth of strains in which the marker cassette has been looped out . Moreover, the abilities of these strains to hydrolyze cellulosic substrates were evaluated on the CMC-agar plates containing uracil  was overexpressed under control of a modified cbh1 promoter in T. reesei, leading to a 17.1-fold increase in BGL activity and a 65.0% increase in saccharification efficiency [bgl1 overexpression cassette was co-transformed with the pyrG+DR fragment into T. reesei QE51. One transformant QEB4, which exhibited the largest black zone around the colony on the esculin plates, was selected from 140 positive transformants according to the BGL activity-screening method [bgl1 gene using the primers Y1 and Y2  with the pyrG marker was used for strain improvement to increase the cellulase production as well as the biomass conversion efficiency  and also necessitate a relatively high proportion (5–10%) of EG2 [T. reesei provided an interesting basis for genetic improvement of cellulase producers. In this study, the transcriptional level of cbh2 in the overexpression strain QPC67 exhibited a 32-fold increase  resulted in a 4.3-fold increase in FPA, but they didn’t provide the experimental evidence of increased mRNA or protein level for CBH2 [Different types of optimized synthetic mixtures with purified component enzymes were recently designed for efficient deconstruction of lignocellulosic substrates to soluble sugars , 16–18. ) of EG2 . These r) of EG2 –44. Therfor CBH2 . Here, ifor CBH2 , it can T. reesei, EG2 is proposed to account for most of the endoglucanase activity, because its absence reduces the endoglucanase activity by as much as 55% [T. reesei and probably involved in the induction of cellulase synthesis by initially attacking cellulose to form oligosaccharides or cellobiose and thus finally become converted to the true inducers [T. reesei [T. reesei QP4. The transcript level of egl2 was increased by 46-fold and the secreted EG2 reached up to the protein level of CBH2, the second large amount of protein secreted by T. reesei  uracil when necessary. The CM was composed as follows: 2% microcrystalline-cellulose, 0.5% (NH4)2SO4, 0.5% KH2PO4, 0.06% MgSO4·7H2O, 0.1% CaCl2·2H2O and 2% corn steep liquor. To assay the transcript levels of cellulase genes, 108 spores were pre-cultured in 150 mL of glucose minimal medium (GMM) at 30 °C for 36 h, and subsequently 1 g of mycelia were transferred into 150 mL of Avicel minimal medium (AMM) supplemented with 0.1% (w/v) uracil when necessary. The GMM medium was composed of 2.0% glucose, 1.5% KH2PO4, 0.5% (NH4)2SO4, 0.06% MgSO4·7H2O, 0.06% CaCl2, 0.2% peptone, 0.001% FeSO4·7H2O, 0.00032% MnSO4·H2O, 0.00028% ZnSO4·7H2O, 0.0004% CoCl2, or supplemented with 0.1% (w/v) uracil when necessary. The AMM medium contained 1.0% Avicel substituted for 2.0% glucose as the sole carbon source and the remaining components in GMM.i QM9414 , was usecbh2–pyrG or egl2–pyrG was constructed with the double-joint PCR method [cbh2 gene containing its own promoter and terminator regions was generated from the genomic DNA of QM9414 using the primer pair CBH2-1183UF/CBH2-2817DR. The egl2 gene containing the native promoter and terminator regions was amplified from the genomic DNA of QM9414 using the primer pair EG2-1524UF/CBH2-1813DR. The pyrG+DR cassette containing the Aspergillus niger pyrG gene and the direct repeat (DR) region was constructed in three steps. Firstly, a 2.8-kb pyrG gene was amplified by the primer pair PyrG-S/PyrG-A used the plasmid pAB4-1 as template [pyrG by PCR using the primer pair DR-S/DR-A. Thirdly, the DR fragment was fused into the 5′ end of the pyrG gene. Subsequently, the cbh2 gene (or the egl2 gene) and the pyrG+DR cassette were further fused together by the primer pair CBH2-1179UF/pyrG-1172DR (EG2-1524UF/pyrG-1172DR) to generate the final expression cassette, cbh2–pyrG or egl2–pyrG . Synthesis of cDNA from total RNA was performed using PrimeScript RT reagent Kit  following the manufacturer’s description. LightCycler 480 System were used for qRT‑PCR . The 10 μL reaction mixtures containing 1× SYBR Premix Ex Taq™, 0.2 μmol/L forward primer, 0.2 μmol/L reverse primer, and 1 ul cDNA template (tenfold diluted) using the SYBR Premix Ex Taq™ (Tli RNaseH Plus) kit  were performed in triplicated. qRT‑PCR protocols were as following: initial denaturation of 1 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 20 s at 60 °C. Melting curve analysis from 65 to 95 °C was performed to confirm the specific of the applications. LightCycler480 software 1.5.0 was used for calculate Ct value. Transcript levels of target genes were normalized against the level of actin gene with ddCt method [For RNA extraction, 10t method .d-glucopyranoside (pNPG) as a substrate [T. reesei strains were measured by detecting the total intracellular protein amount extracted by 1 M NaOH [The total cellulase activity  was measured using Whatman No. 1 filter paper as substrate. The reaction mixtures contained 50 mg of filter paper, 1.5 mL of 50 mM citrate buffer (pH 4.8), and 500 µL of the suitably diluted enzyme fractions. These mixtures were then incubated at 50 °C for 60 min. EG activities were assayed with CMC–Na as substrate. The enzyme reactions were performed in 2 mL of 1% substrate citrate buffer (pH 4.8) at 50 °C for 30 min. The amount of reducing sugar released was determined using the DNS method . Cellobiubstrate . The dil1 M NaOH . ProteinThe corncob residues were kindly provided by LONGLIVE Co., Yucheng, Shandong province, China. Acid-pretreated (ACR) and delignified (DCR) corncob residues were used as substrates in the saccharification process and the components of these substrates had been described by Liu et al. . The cel


Streptomyces flavogriseus can produce both actinomycin D and holomycin. The actinomycete strain NJ-4 isolated from the soil of Nanjing Agricultural University was identified as S. flavogriseus. This S. flavogriseus strain was found for the first time to produce two antimicrobial compounds that were identified as actinomycin D and holomycin. GS medium, CS medium and GSS medium were used for the production experiments. All three media supported the production of actinomycin D, while holomycin was detected only in GS medium and was undetectable by HPLC in the CS and GSS media. The antimicrobial activity against B. pumilus, S. aureus, Escherichia coli, F. moniliforme, F. graminearum and A. niger was tested using the agar well diffusion method. Actinomycin D exhibited strong antagonistic activities against all the indicator strains. Holomycin exhibited strong antagonistic activities against B. pumilus, S. aureus and E. coli and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger. The cell viability was determined using an MTT assay. Holomycin exhibited cytotoxic activity against A549 lung cancer cells, BGC823 gastric cancer cells and HepG2 hepatocellular carcinoma cells. The yield of actinomycin D from S. flavogriseus NJ-4 was 960 mg/l. S. flavogriseus NJ-4 exhibits a distinct capability and has the industrial potential to produce considerable yields of actinomycin D under unoptimized conditions.This paper is the first public report that  Streptomyces remains a focus of systematic research and is especially of major pharmaceutical interest based on its commercial value as a rich source of numerous secondary metabolites.To date, several actinomycete genomes have been sequenced and annotated. Among actinomycetes, the genus Streptomyces species are soil bacteria, from which numerous antibiotic and antifungal compounds have been derived  and incubated at 28 °C for 7∼10 days. The isolate was purified and stored on GS agar medium.Actinomycete strain NJ-4 was isolated from the soil of Nanjing Agricultural University Campus in Nanjing, China. The method of isolating strain NJ-4 was as fllows: a soil sample (5 g) was suspended in 45 ml of 0.85% physiological saline solution. The soil suspension was incubated at 28 °C with shaking for 30 min and then allowed to settle. 100 µl of the suspension was coated evenly on Gause’s synthetic (GS) agar medium  consistiBacillus pumilus CMCC 63202 was obtained from the National Center For Medical Culture Collection; Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were obtained from the American Type Culture Collection; Fusarium moniliforme CGMCC 3.4017, Fusarium graminearum CGMCC 3.4598 and Aspergillus niger CGMCC 3.6478 were obtained from the China General Microbiological Culture Collection Center. Bacteria was cultured on nutrient agar medium  according to the methods described by the manufacturer. The 16S rDNA gene was amplified by PCR using the universal primers 27f (5′-AGAGTTTGATCTGCCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR programme consisted of initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, with a final extension for 10 min at 72 °C. The PCR product was purified using a PCR product purification kit , cloned into the pMD 19-T cloning vector , and then transformed into chemically competent oli DH5α . SequencStrain NJ-4 was grown on a Gause’s synthetic agar slant at 30 °C for seven days, and the mature spores were transferred into a 250-ml flask containing 50 ml of Gause’s synthetic medium. The seed culture was incubated at 30 °C with shaking at 180 rpm for 2 days. For holomycin and actinomycin D production, 5 ml of seed culture was transferred into a 250-ml flask containing 50 ml of Gause’s synthetic medium, which was incubated at 30 °C with shaking at 180 rpm for seven days.The cultured broth was centrifuged at 8,000× g for 15 min after seven days of cultivation, and the supernatant and mycelia were extracted three times with ethyl acetate at room temperature. The ethyl acetate fractions were combined, concentrated, and dried under reduced pressure to give a red-orange powder. The red-orange powder was then dissolved in methanol and loaded onto a Sephadex LH-20 column (2.6 × 120 cm). Elution was carried out with 80% methanol at a flow rate of 0.3 ml/min. Fractions were collected with a fraction collector, and antimicrobial activity was assessed in all fractions using the agar well diffusion method. Two fractions with antimicrobial activity were obtained. The two fractions were separately evaporated to dryness under vacuum and further purified by preparative RP-HPLC . The first fraction was suspended in methanol, and 100 μl of the concentrated first fraction was then loaded onto the column and separated using an isocratic gradient of 70% acetonitrile in water as the mobile phase at a flow rate of 4 ml/min for 20 min. The purified first fraction was collected automatically according to its absorbance at 443 nm. The second fraction was purified in a manner similar to that used for the first fraction but with an isocratic gradient of 25% acetonitrile in water as the mobile phase for 12 min and UV detection at 386 nm. The purified fractions were characterized using ultraviolet light (SHIMADZU UV-2600 UV-Vis spectrophotometer), infrared light (Thermo Mattson Fourier transform infrared spectrophotometer), electrospray ionization mass spectrometry  and nuclear magnetic resonance (NMR) spectroscopy (BRUKER).3 0.01%; (3) GSS medium consisting of soluble starch 2.0%, soybean flour 1.5%, KNO3 0.1%, K2HPO4⋅3H2O 0.05%, NaCl 0.05%, MgSO4⋅7H2O 0.05% and FeSO4⋅7H2O 0.001%. Every 24 h for seven days, 2 ml of culture was collected and prepared for HPLC analysis as follows: After centrifugation , the supernatants and mycelia were extracted twice with ethyl acetate. The two ethyl acetate fractions were combined and dried under reduced pressure. The crude extract was then re-dissolved in methanol, filtered through a 0.22- μm nylon membrane filter, and analysed with an RP-HPLC (DIONEX Ultimate 3000) instrument equipped with an Agilent Eclipse XDB-C18, 5- μm column (4.6 × 250 mm), with UV detection at 443 nm and 70% acetonitrile in water as the mobile phase for actinomycin D or with UV detection at 386 nm and 20% acetonitrile in water as the mobile phase for holomycin.Three types of fermentation media were used: (1) GS medium consisting of the same medium as in Gause’s synthetic agar medium except for agar (pH 7.0∼7.2); (2) CS medium consisting of corn flour 2.0%, soybean flour 1.5%, glucose 0.05%, yeast extract 0.025% and CaCOB. pumilus, S. aureus, E. coli, F. moniliforme, F. graminearum and A. niger was tested using the agar well diffusion method . The mycelia were washed three times with ice-cold PBS buffer. NAD+ and NADH were assayed using the NAD(+)H determination kit  according to the methods described by the manufacturer.All assays were done in triplicate and results were expressed as the mean ± standard deviation (SD). Results were statistically analyzed by ANOVA  by SAS 9.0 software.S. flavogriseus  in the phylogenetic tree according to a phylogenetic analysis  and –NH (3445.21 cm−1 and 3273.57 cm−1). There were bands at 2874.38 cm−1 and 2965.02 cm−1 because of the symmetrical and asymmetrical C–H stretching of the –CH2 group −1 and 16omycin D . The str+, which was the same as that observed with holomycin  were the same as the NMR spectrum of holomycin . Purified and commercial holomycin exhibited the same strong antagonistic activities against E. coli, B. pumilus and S. aureus and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger.As shown in The data obtained from the MTT assay showed that holomycin exhibited cytotoxic activity against all three human tumour cell lines. When the concentration of holomycin was 5 µM, the inhibition of all cancer cells and 293T normal human cells was weak. When the concentration of holomycin was 10 µM, the inhibitory effect on BGC-823 gastric cancer cells and HepG2 hepatoma cells became obvious, while the viability of all cancer cells was inhibited by over 50% at 20 µM holomycin .Research results indicate that holomycin not only has antitumour activity against A549 lung cancer cells, BGC-823 gastric cancer cells, and HepG2 hepatoma cells but also has cytotoxic activity against 293T normal human cells.Streptomyces strains, the Streptomyces genus has been widely used as an important biological tool for producing novel antibiotics  (S. parvulus (152 mg/l actinomycin D under optimized conditions) (S. sindenensis (850 mg/l actinomycin D)   , S. parvditions) , a mutanmycin D)  and S. anomycin) . Our S. S. flavogriseus NJ-4 was undetectable in CS medium and GSS medium. The biosynthesis of holomycin was probably strongly affected by the redox level of the cells  spectroscopy of the first active compound purification from strain NJ-4.Figure S4 The structure of actinomycin D.Figure S5HPLC analysis of the second active compound purification from strain NJ-4.Figure S6 UV analysis of the second active compound isolated from strain NJ-4.Figure S7 Fourier transform infrared (FTIR) spectroscopy of the second active compound purification from strain NJ-4.Figure S8 The structure of holomycin.Figure S9 Time course of redox potential of fermentation broth of strain NJ-4 cultured in GS(square) , GSS (circle) and CS (triangle) media.Figure S10 The ratio changes of NAD/NADH of strain NJ-4 cultured in GS (square), GSS (circle) and CS (triangle) media.Click here for additional data file.10.7717/peerj.3601/supp-2Data S1Figure 4 Time course production of actinomycin D of strain NJ-4 cultured in CS (triangle), GSS (square) and GS (circle) media.Figure 5Time course production of holomycin of strain NJ-4 cultured in CS (triangle), GSS (square) and GS (circle) media.Figure 6Proliferative inhibition of holomycin on 293T normal human cells, A549 lung cancer cells, BGC-823 gastric cancer cells, HepG2 hepatoma cells.Figure S9 Time course of redox potential of fermentation broth of strain NJ-4 cultured in GS (square), GSS (circle) and CS (triangle) media.Figure S10 The ratio changes of NAD/NADH of strain NJ-4 cultured in GS (square), GSS (circle) and CS (triangle) media.Click here for additional data file.


ZT by employing natural nano-wire-like electronic and thermal transport. The phonon-softening known as Kohn anomaly, and Peierls lattice distortion decrease phonon energy and increase phonon scattering, respectively, and, as a result, they lower thermal conductivity. The quasi-one-dimensional electrical transport from anisotropic band structure ensures high Seebeck coefficient in Indium Selenide. The routes for high ZT materials development of In4Se3−δ are discussed from quasi-one-dimensional property and electronic band structure calculation to materials synthesis, crystal growth, and their thermoelectric properties investigations. The thermoelectric properties of In4Se3−δ can be enhanced by electron doping, as suggested from the Boltzmann transport calculation. Regarding the enhancement of chemical potential, the chlorine doped In4Se3−δCl0.03 compound exhibits high ZT over a wide temperature range and shows state-of-the-art thermoelectric performance of ZT = 1.53 at 450 °C as an n-type material. It was proven that multiple elements doping can enhance chemical potential further. Here, we discuss the recent progress on the enhancement of thermoelectric properties in Indium Selenides by increasing chemical potential.Researchers have long been searching for the materials to enhance thermoelectric performance in terms of nano scale approach in order to realize phonon-glass-electron-crystal and quantum confinement effects. Peierls distortion can be a pathway to enhance thermoelectric figure-of-merit  Thermoelectric research is one of the efforts that promotes energy efficiency. Thermoelectric performance can be defined by the dimensionless figure-of-merit x1−EuxTe multiple quantum well structures 1− anionic layers  and In1+ cations with a weak van der Waals interaction along the a-axis between the layers and strong covalent-ionic interaction in the bc-planes within the layer 1− anionic staking layers along the a-axis 1− anionic layer of the In4Se3 structure. The vacant site of a dangling bonded Se (Se3 site) is more stable than other Se sites. The theoretical calculation proved that the Se vacancy strongly suppresses phonon propagation along the plane of charge density wave .L [0L = 2.45 × 10−8 W·Ω·K−2. The low Lorenz numbers of the compounds may be affected by the change of Fermi energy [ph. The total thermal conductivity κ  and lattice thermal conductivity κph  are presented in ph are increased with increasing Se-deficiency. It is consistent with the result of In4Sex3− for high Se-deficiency (x > 0.05) [4Sex3− (x > 0.05) is increased with increasing Se-deficient [L . The Lori energy . From th > 0.05) . The toteficient .4Se3 based compounds comes from the Peierls distortion due to quasi-onedimensional lattice and charge density wave instabilities [Basically, the low thermal conductivity of Inbilities . Recent bilities . In termS2σ of polycrystalline In4Pb0.01Sn0.03Sex3− (x < 0.5) and In4Se2.9 are presented in −1·K−2) have higher power factor than the ones of In4Pb0.01SnySe3 (0.1~0.19 mW·m−1·K−2) [4Se2.9, the Pb-/Sn-codoped and Se-deficient compounds have significantly enhanced values of the power factor. The high power factor over a wide temperature range (450 K ≤ T ≤ 725 K) is very important ingredient for practical thermoelectric applications.The temperature dependent power factors m−1·K−2)  near rooZT of In4Pb0.01Sn0.03Sex3− (x < 0.5) are increased with increasing temperature as shown in ZT value reaches up to 1.2 at 723 K for polycrystalline In4Pb0.01Sn0.03Sex3− (x = 0.1). Even though the ZT value of the x = 0.1 compound is lower than the one of In4Pb0.01Sn0.03Se3 (1.4 at 733 K) [opT range as the temperature range for ZT > 1.0, the operational temperature range of x = 0.1 Se-deficient and Pb-/Sn-codoped polycrystalline compound (opT > 600 K) is a little bit increased rather than those of In4Pb0.01Sn0.03Se3 compound (opT > 620 K). In addition, the ZT values of In4Sex3− significantly increased in the Pb-/Sn-codoped and Se-deficient polycrystalline compounds.The thermoelectric figure-of-merit t 733 K) , if we d4Se3 crystalline compounds In4Se3−xHy  is a good candidate to enhance chemical potential [4Sex3−H0.03 are significantly increased by halogen doping. It was found that the Cl-doping is the most effective for increase of Hall mobility. The polycrystalline In4Pb0.01Sn0.03Se2.9 compounds showed maximum ZT value of 1.2 at 723 K [4Pb0.01Sn0.03Se2.9 polycrystalline compound can increase furthermore because it is still far from the optimized chemical potential 0.8 eV from the Boltzmann transport calculation [4Se3 phase can be more increased by electron doping. The Cl-doping is believed to be a good candidate to increase thermoelectric performance in the multiple elements-doped In4Pb0.01Sn0.03Se2.9Clx compounds.The halogen elements doping in the Inotential . The Halat 723 K . Howeverculation ,29. The x = 0.02 and x = 0.04) based on the orthorthombic Pnnm space group, respectively. The XRD patterns show a single phase of In4Se3 with no noticeable impurity peaks except x = 0.06. Because of impurity phase of InSe , we cannot analyze the XRD pattern by Rietveld refinement for the x = 0.06 compound. This indicates that x = 0.06 is the solubility limit of chlorine which induces the phase separation from In4Se3 to InSe phase. The lattice parameter is calculated as shown in 4Pb0.01Sn0.03Sex3− , the lattice parameters are systemically changed [4Pb0.01Sn0.03Se2.9Clx do not show the systematic changes but show a decrease of lattice volume  polycrystalline compounds and x = 0.0  are preSe2.7Clx .xy and electrical resistivity ρ measurements, we obtain the effective carrier concentration Hn and Hall mobility μH, which are listed in 4Pb0.01Sn0.03Se2.9Clx (x = 0.02) compound shows the value of 1.58 times higher than the one of pristine In4Pb0.01Sn0.03Se2.9 polycrystalline compound. The carrier concentration does not increase with Cl doping for x = 0.04 and 0.06, implying the solubility limit of the compounds as in the case of the polycrystalline In4Se2.7Clx polycrystalline compounds (x = 0~0.05) [From the Hall resistivity ρ 0~0.05) .4Pb0.01Sn0.03Se2.9Clx polycrystalline compounds are decreased with increasing Cl-doping. In contrast with the result of In4Sex3−Cl0.03 single crystal [4Se2.7Clx [4Pb0.01Sn0.03Se2.9Clx decreased the Hall mobility by increasing the Cl-doping. It implies that the Hall mobility of polycrystalline In4Se3 is decreased by the excess Cl-impurity scattering at the grain boundary. However, the CuBr-doped In4Se2.5 polycrystal shows the increased Hall mobility [The Hall mobilities of the In crystal , the polSe2.7Clx  and In4Pmobility .4Sex3−Cl0.03 single crystal [The increase of Hall mobility in bulk crystalline materials is caused by the enhancement of crystallinity in chlorine doped In crystal . For exa crystal . This imS(T) of the polycrystalline In4Pb0.01Sn0.03Se2.9Clx  compounds are decreased with Cl-doping as compared with x = 0.0 as shown x = 0.02 sample is obviously increased as compared the one of x = 0.0, even though the Cl-doping dependence is not clear. The increased effective mass of carrier indicates less dispersive electron band than the non-Cl-doped sample. From the effective thermal band gap relation, g = 2eSEmaxTmax [x = 0.0) [x = 0.04). The Cl-doping on the In4Pb0.01Sn0.03Se2.9Clx compounds induces the less dispersive and decreased band gap than those of the In4Pb0.01Sn0.03Se2.9 compound.The temperature dependent Seebeck coefficient SmaxTmax , we can x = 0.0)  and 0.344Pb0.01Sn0.03Se2.9Clx  polycrystalline compounds are presented in x = 0.0, 0.02, 0.04, and 0.06) are decreased with increasing temperature. This conventional 1/T behavior is mainly caused by an acoustic phonon contribution for the thermal transport. There is no systematic change of the κ(T) with respect to Cl-doping concentration. In general, the total thermal conductivity κ is composed of electrical κel and lattice thermal conductivity κph. The electronic thermal conductivity κel can be calculated by the Wiedemann-Franz law κel = L0σT, where L0, σ, and T are the Lorenz number, electrical conductivity, and absolute temperature, respectively. In usual cases, the Lorenz number is written as:The total thermal conductivities of the polycrystalline Inr is the scattering parameter, η = F/kBTE is the reduced Fermi energy, and n(Fη) is the n-th order Fermi integral given by:However, the Lorenz number is incorrect in correlated metal and many degenerated semiconductors. In order to get a more reliable Lorenz number, we calculated the Lorenz number by using the following equation:r = For most cases, the scattering parameter for acoustic phonon scattering is 4Pb0.01Sn0.03Se2.9Clx  are shown in 0L = 2.45 × 10−8 W·Ω·K−2. The low Lorenz numbers indicate that the electrical contribution for thermal transport is lower than conventional metals. Using the calculated Lorenz number and electrical resistivity, we can obtain the lattice thermal conductivity by subtracting the electrical thermal conductivity from the total thermal conductivity as shown 4Pb0.01Sn0.03Se2.9Clx  polycrystals is not obvious. The similar thermal conductivity can be explained by the similarity of crystal structure and phonon dispersion relation between In4Se3 and InSe [By using the Fermi integral, the calculated temperature dependent Lorenz numbers of Inand InSe .S2σ of the polycrystalline In4Pb0.01Sn0.03Se2.9Clx  compounds. The decreased power factor with increasing Cl-doping is mainly caused by the decreased Hall mobility near room- and mid-temperature range. On the other hand, the slightly increased power factors of x = 0.02 and 0.04 are obtained near 723 K. The compound of x = 0.06 shows a decreased power factor than the other samples  implying that the InSe phase separation is not good for increasing power factor in the In4Se3 phase.ZT values of the polycrystalline In4Pb0.01Sn0.03Se2.9Clx  compounds are presented in ZT of x = 0.04 reaches up to 1.25 at 723 K, which is slightly increased value as compared with the one of pristine compound x = 0.0 (ZT = 1.2). The high ZT value over a wide temperature range in bulk crystals In4Sex3−Cl0.03 (1.53) is caused by the significant increase of Hall mobility, which is attributed from the enhancement of crystallinity by chlorine doping [4Se2.7Clx samples exhibit decreased ZT (0.67) value with increasing chlorine doping concentration, we believe that further enhancement of ZT can be possible in the compounds of In4Pb0.01Sn0.03Se2.9Clx for single crystalline form. Therefore, we should investigate the thermoelectric properties of the single crystalline compounds in further research projects.The e doping . Because4Se3−δ and In4Sex3−H0.03  were prepared by the Bridgman crystal growth method. Stoichiometric amounts of In, Se, and/or InH3  were loaded into a quartz tube under an argon atmosphere in order to keep the indium halides dry. The quartz tubes were vacuum sealed and heat treated at 860 K for 12 h in a rocking furnace for homogenization of the molten liquid. After the heat treatment of the compounds, the quartz tubes were loaded into a vertical Bridgman furnace. The compounds were melted at 860 K for 24 h and slowly pulled down at a constant rate of 2–5 mm/h.Bulk crystal ingots of In2, and so on were sealed in evacuated quartz tubes under high vacuum with respect to stoichiometric molar ratios. The quartz ampoules were heated at 1123 K for 24 h with following water quenching. The ingots were pulverized and sintered under argon atmosphere by hot-press at about 730 K for 1 h under uniaxial pressure of 70 MPa. In order to increase thermal stability, the pellets were annealed at 753 K for 5 days. The relative densities of annealed samples are above 95% (5.82~5.93 g/cm3) of the theoretical density.The polycrystalline compounds were synthesized by solid state reaction at 550 °C for 24 h and spark plasma sintering under uniaxial pressure of 70 MPa and 420 °C for 5 min. For cation doped materials, the samples were synthesized by melting, quenching, and annealing process. The elements of In, Pb, Sn, and Se, InClThe chemical inhomogeneity was examined by the inductively coupled plasma spectroscopy (ICP) and electron dispersive spectroscopy (EDS) measurements. The variations of chemical concentration of three different slices of one crystal ingot from top to bottom were about ±2 at.%. The EDS measurements at several different points of a slice of the sample showed the chemical homogeneity within an experimental error. The crystal structure and phase of annealed samples are identified by the powder X-ray diffraction (XRD) using Cu kα radiation . The Rietveld refinement was carried out using FullProf software.S and ρ), respectively. For anisotropic measurements, we cut the samples for two directions: along the growth direction and perpendicular to the growth direction. Typical sample sizes for thermal conductivity and electrical transport properties (S and ρ) measurements are of 10 mm diameter with 2 mm thickness and 10 mm long with (2~5) × (2~5) mm2 cross-sectional area, respectively.We prepared disk-shaped and bar-type samples to measure the thermal conductivity κ and electrical transport properties , and heat capacity pC ; κ = ρsλpC, where heat capacity pC was used the results from the Dulong-Petit fitting at high temperatures (T ≥ 300 K). The high temperature electrical resistivity ρ and Seebeck coefficient S were measured by the four-probe method . The orthorhombic bar was placed in the hot and cold side plate and the voltage leads were contacted at the sample surface. By applying the heat and electric pulse, the Seebeck coefficient S = ΔV/ΔT and electrical resistivity ρ = ΔV/ΔI were measured simultaneously. The Hall resistivity ρxy measurement was carried out by the five-contact AC -transport technique by the physical property measurement system . The Hall carrier concentration was calculated by the one-band model as following relation: H =n −1/(HeR), where Hall coefficient HR = ρxy/H and e = 1.602 × 10−19 C.High temperature thermal conductivity κ was obtained by the measurements of sample density ρk-point sampling is used for integration over the first Brillouin zone. The energy cutoff is chosen to be 240 eV and atomic positions are fully relaxed until all force components are smaller than 0.02 eV/Å. The unit cell is composed of 28 atoms with seven different sites (four sites for indiums and three sites for seleniums) and each site occupies four equivalent atomic positions. The calculated lattice parameters are of a = 15.429 Å, b = 12.442 Å and c = 34.142 Å, respectively, which are similar to the experimental values. For Se-deficient In4Se3−δ (δ = 0.25) crystal, single Se atom was eliminated from the twelve Se atoms that occupied three crystallographically nonequivalent sites in the unit cell. We found that the configuration with vacant Se1 site is lower in energy than other configurations with Se2 and Se3 sites by 0.14 eV and 0.19 eV per unit cell, respectively.The first-principles calculation was performed by the pseudopotential plane wave method using the Vienna Ab initio Simulation Package (VASP). We adopted the generalized gradient approximation (GGA) implemented by Perdew, Burke, and Ernzerhof (PBE) for the exchange correlation energy functional with the spin-orbit interaction. The 8 × 8 × 24 Monkhorst and Pack scheme of k-points in full Brilloun zone is used for the calculation. Exchange correlation energy functional is calculated using Engel-Vosko (EV) GGA for the Boltzmann transport calculation. Although PBE-GGA has been widely used for the self-consistent charge consistent calculation, it is known to underestimate the band gap. For a calculation of thermoelectric properties, a correct estimation of band gap is important. The EV-GGA has shown good agreement with experimental band gap. Indeed, PBE-GGA gives a band gap of 0.17 eV, but the EV-GGA gives a band gap of 0.57 eV, which is similar to the experimental value of 0.64 eV. We have used a rigid band approximation in the doping effect. In order to obtain the temperature dependent transport properties, we fix the chemical potential at μ = 0.22 eV, and use a constant time relaxation parameter τ = 2.2 × 10−14 s.The thermoelectric properties are calculated by using BoltzTraP program. Dense mesh of 19200 ZT on In4Se3−δ bulk crystals along the crystalline plane with the charge density wave were reported.The Peierls lattice distortion can be regarded as a new pathway to enhance thermoelectric performance. The inherent nature of quasi-one-dimensional charge transport with strong electron-phonon coupling breaks translational symmetry of crystalline lattices. The low dimensional electronic and thermal properties of Peierls system result in the intrinsic nanowire-like transport of electrons and phonons. The electronic transport with reduced dimensionality can give rise to high Seebeck coefficients if we control the electron-hole band asymmetry. The phonon softening driven by the strong electron-phonon interaction lowers phonon energy and the lattice distortion along the conducting plane significantly increases the phonon scattering in charge density wave system. The increased phonon scattering with lowered phonon energy has the effect of reducing thermal conductivity. Based on this concept, high 4Se3−δ shows anisotropic transport of carriers such as the significant electronic band dispersion along the c-axis, localized hole band along the b-axis, and the van der Waals energy gap along the a-axis. From the increase of the chemical potential by chlorine doping, the bulk crystalline In4Se3−δCl0.03 exhibited exceptionally high ZT over a wide temperature range. The thermoelectric properties of In4Se3−δCl0.03 are not optimized yet because the chemical potential is far from that of the highest power factor. If we increase chemical potential, the ZT value can be increased further in the In4Se3−δ based materials. The Peierls distortion can be manifested by band structure engineering on the quasi-one-dimensional chain structure and two-dimensional layered structure materials.The band structure of InT) and Seebeck coefficient S(T) of polycrystalline compounds of In4Pb0.01Sn0.03Sex3−  are decreased with increasing Se deficiency by increasing carrier concentration. The room temperature power factors are increased by introducing Se-deficiency for Pb-/Sn-codoped compound. The maximum ZT value is reached up to 1.2 (at 723 K) for In4Pb0.01Sn0.03Sex3− (x = 0.1) polycrystalline compound due to high Hall mobility 213.60 cm2·V−1·s−1 and increased carrier concentration.In order to utilize waste heat power generation for Indium Selenides, high thermoelectric performance in polycrystalline compounds are highly desirable. The electrical resistivity ρ compounds are synthesized well by the melting and solid state reaction with systemic change of lattice volume. Because of the chlorine impurity scattering, the electrical resistivities of the Cl-doped polycrystalline samples  are increased as compared with non-Cl-doped sample (x = 0.0) near room temperature which is mainly caused by the decrease of Hall mobility by chlorine doping. However, the electrical resistivities of x = 0.02 and 0.04 are decreased at high temperature accompanying with smearing out of broad shoulder near 670 K. Consequently, it exhibits relatively low thermal conductivity and high power factor over a wide temperature range. The power factor and ZT values of the Cl-doped polycrystalline compounds (x = 0.02 and 0.04) were increased as compared with the non Cl-doped compound (x = 0.0) near 700 K. The maximum ZT value of 1.25 is relatively high ZT for polycrystalline In4Se3 based compounds. Further enhancement of thermoelectric performance can be expected in single crystalline compounds by promoting crystallinity of the samples as well as enhancement by multiple elements doping in polycrystalline compounds.The Cl-doped polycrystalline In


Medicago truncatula suspension cultures overexpressing the MtTdp2α gene  and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli , the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights,  The nucleolus is a subnuclear entity with dynamic structure, where ribosomal RNA (rRNA) is synthesized and processed . This seThe study of the crosstalk between the nucleolus and the DNA damage response (DDR) has been recently recognized as a challenging issue . To dateTyrosyl-DNA phosphodiesterases (Tdps) are multifaceted DNA repair enzymes able to remove the 5′- and 3′-end blocking DNA lesions, among which are topo/DNA covalent adducts . Tdps reMedicago truncatula. Differently from animals, a small Tdp1 gene family is found in the plant kingdom, composed of Tdp1α and Tdp1β genes encoding distinct Tdp1 isoforms , rpL23 and rpL10  in relation to the Tdp1 function has been previously suggested by isoforms . Transgepartment . Accordind rpL10  that parbidopsis . To datebidopsis .M. truncatula plants, the overexpression of MtTdp2α gene, encoding the α isoform of Tdp2 enzyme, is associated with enhanced DNA repair ability and tolerance to osmotic and heavy metal stress , also known as TTRAP (TRAF and TNF receptor-associated protein), is not only required for repair of topo II-induced DSBs  but alsol stress . In a reDR genes . Based oI poison , were inM. truncatula Gaertn. (M9-10a genotype) cell suspension cultures used in the present work were derived from the MtTdp2α-overexpressing plant lines Tdp2α-13c and Tdp2α-28 previously obtained by Agrobacterium tumefaciens strain and the resulting engineered strain EHA105-pTdp2α was used to transform the M. truncatula leaf explants. A. tumefaciens-mediated transformation of M9-10a leaf explants and in vitro regeneration were performed according to in vitro on a modified (10 g l-1 sucrose) MS030A medium and propagated using stem nodal explants. Plantlets were maintained in a climate chamber at 22–24°C with a 16-h light/8-h dark cycle photoperiod and a photosynthetic photon flux of 65–70 μMol m-2 s-1 under a cool white fluorescence lamp. M. truncatula Gaertn. (M9-10a genotype) cell suspension cultures were subsequently obtained as described by in vitro grown plants of the MtTdp2α-overexpressing plant lines (Tdp2α-13c and Tdp2α-28) and control (CTRL) line  and maintained at 27°C in the dark for 2 weeks. The resulting calli were excised from explants and transferred to liquid MS medium (D (0.25 mg l-1) and kinetin . M. truncatula suspension cultures were routinely propagated in the same medium. Cells were subcultured every 8 days by transferring 5–45 ml of fresh medium and incubated at 80 rpm at 27°C in the dark. Treatments with etoposide  were carried out as described by 2.The RL) line  were traRL) line  supplemeS medium  containiM. truncatula cell suspension cultures (CTRL and MtTdp2α-overexpressing lines) were collected and fixed with 1.5% glutaraldehyde (Sigma-Aldrich) for 3 h at room temperature. Cells were rinsed in phosphate-buffered saline  overnight, post-fixed in 1% aqueous OsO4 (Sigma-Aldrich) and embedded in epoxy resin. Sections were stained with uranyl acetate and lead citrate (Sigma-Aldrich) and observed with a Zeiss EM900 electron microscope equipped with a 30 mm objective aperture and operating at 80 kV.Aliquots (2–3 ml) of 4-day-old For morphometric analysis, all specimens were observed with a Zeiss EM900 electron microscope equipped with a 30 mm objective aperture and operating at 80 kV. Images were acquired at a fixed magnification  and submitted to morphometric analyses using the software ImageJ. The surface area of 30 nucleoli per sample was measured. On the same samples, parameters such as the number of nucleolar vacuoles per nucleolus, number of nucleoli per cell, number of multinucleolate cells, and number of ring-shaped nucleoli, were determined. The results are expressed as mean values ± SD.M. truncatula cell suspension cultures at the final concentration of 100 mM. Incubation was carried out for 1 h. Cells were then rapidly rinsed in fresh medium and processed for TEM as described by -1. After rinsing with PBS containing 0.1% Tween and PBS, the grids were incubated with the specific secondary antibodies coupled with colloidal gold (1:20 in PBS) for 1 h at room temperature. Sections were finally stained with terbium citrate (Sigma Aldrich) 0.2 M pH 8.5 for 30 min at room temperature to detect RNA (Bromo-uridine (BrU) (Sigma-Aldrich) was dissolved in the culture medium and added to 4-day-old tect RNA . The celDNA diffusion assay was performed as originally reported by TM Total RNA Fatty and Fibrous Tissue Kit , according to manufacturer’s instructions. cDNAs were obtained with the High Capacity cDNA Reverse Transcription Kit  according to supplier’s suggestions. Gene-specific oligonucleotide primers for the M. truncatula MtTdp2α (Phytozome Database Accession No. Medtr8g146980) and MtTop2 (Phytozome Database Accession No. Medtr3g103270), coding sequences were designed using the Real-Time PCR Primer Design program from GenScript1  in a final volume of 10 μl according to supplier’s indications, and using a Rotor-Gene 6000 PCR apparatus . Amplification conditions included an initial denaturation step at 95°C for 10 min, and subsequently 95°C for 30 s, 59°C for 60 s, 72°C for 30 s (40 cycles). For each oligonucleotide set, a non-template control (water) was also included. The M. truncatula ELF1α (GenBank Accession No. EST317575) was used as a reference gene, based on previous work .For each treatment, two independent experiments were performed, using three replications. Asterisks indicate statistically significant differences determined using Student’s M. truncatula suspension cultures grown under physiological conditions. The proliferative state of M. truncatula cells was assessed by monitoring the expression profiles of the MtH4 (histone H4) gene , while the MtTdp2α-overexpressing cells showed expanded and decondensed nucleoli . Budding fibrillar structures (b) were observed at the nucleolar periphery in Tdp2α-13c and Tdp2α-28 lines . The appearance of such small bodies has been reported as an early sign of nucleolar decondensation (2. A significant increase was observed in MtTdp2α-overexpressing cells with an average nucleolar area of 1.04 ± 0.18 μm2 (Tdp2α-13c) and 0.91 ± 0.29 μm2 (Tdp2α-28), corresponding to 34 and 44% expansion of the nucleolar region, respectively (Table 1). In the nucleoli of MtTdp2α-overexpressing cells, TEM highlighted the presence of cavities  , another peculiarity of actively transcribing nucleoli , 2.3 ± 0.91 (Tdp2α-13c), and 3.2 ± 1.4 (Tdp2α-28) cavities per nucleolus (Table 1). Taken together, the reported data highlight changes in the nucleolus morphology and ultrastructure of MtTdp2α-overexpressing cells, compatible with conditions of enhanced nucleolar activity.The nucleolus ultrastructure was revealed by transmission electron microscopy (TEM) carried out on 4-day-old ensation . The estnucleoli . The numMtTdp2α-overexpressing lines compared to CTRL. Examples of mononucleolate and multinucleolate cells are shown in Figures 2A,B. The multinucleolate cells accounted for 8.3 ± 1.1% of the total population in the control suspension culture  while a significant increase  in the percentage of cells containing more than one nucleolus (up to three nucleoli in most cases) was observed in the Tdp2α-13c line, compared to CTRL . The percentage of multinucleolate cells was further enhanced in the Tdp2α-28 line, with an estimated value of 35.0 ± 4.8% (4.37-fold) compared to CTRL .Morphometric analysis showed that the percentage of multinucleolate cells was significantly higher in the M. truncatula cell suspensions collected at 4 days of culture, TEM revealed the occurrence of ring-shaped nucleoli characterized by a central vacuolization surrounded by a nucleolar shell . According to the current literature, ring-shaped nucleoli correspond to a reversible quiescent state generally induced in plant cells by stress conditions  whereas no ring-shaped nucleoli were present in the Tdp2α-28 line .In the nditions . MtTdp2αFigure 4). As for CTRL, the estimated transcript number in the nucleolus was 48.00 ± 5.07 particles per μm2. A significant increase was detected in Tdp2α-13c and Tdp2α-28 lines  . The quantitative evaluation in the region outside the nucleolus revealed as well an enhanced distribution of labeled transcripts in the MtTdp2α-overexpressing lines, compared to CTRL. The estimated values were 20.00 ± 3.10 particles per μm2 (CTRL), 31.00 ± 5.45 particles per μm2 (Tdp2α-13c), and 49.00 ± 3.97 particles per μm2 (Tdp2α-28) . Thus, MtTdp2α gene overexpression was associated with increased transcription levels. The overall transcriptional response within the nucleus corresponded to 68.00 ± 8.00 particles per μm2 (CTRL), 92.00 ± 13.4 particles per μm2 (Tdp2α-13c), and 127.00 ± 11.5 particles per μm2 (Tdp2α-28). The enhancement in transcription reported for the MtTdp2α-overexpressing cells well-correlates with the results from ultrastructural morphometric analysis, providing further evidence of increased nucleolar activity.Quantitative evaluation of BrU incorporation was performed in both nucleolar and extra-nucleolar regions  of the topo II inhibitor etoposide. The dose range was chosen based on the previous study by Figure 5). Following exposure to 75 μM etoposide, the CTRL showed only 50.0 ± 2.2% viable cells at 6 h whereas viability was significantly higher in both Tdp2α-13c and Tdp2α-28 lines . When 150 μM etoposide was provided, cytotoxic effects were evident at 0 h in the CTRL line, possibly due to the time required for sample processing. At 6 h, a drop in the percentage of viable cells (27.6 ± 2.2%) was observed in the CTRL line. PCD and necrosis frequencies accounted for 31.0 ± 3.0 and 41.4 ± 1.2%, respectively . The MtTdp2α-overexpressing lines showed a significantly higher the percentage of viable cells . PCD events occurred with frequency of 43.5 ± 2.5% (Tdp2α-13c) and 36.8 ± 2.1% (Tdp2α-28), while necrosis was observed at lower frequency . The highest dose (300 μM) resulted into 20.0 ± 2.2% viability in CTRL, with PCD and necrosis accounting for 36.0 ± 3.1 and 44.0 ± 2.9%, respectively .Four-day-old MtTdp2α-overexpressing lines, there was a progressive increase in the PCD/necrosis ratio with the 75 and 150 μM etoposide doses . In response to 75 μM etoposide, the level of MtTdp2α transcript was still significantly higher  at 6 h of treatment with the inhibitor. At the same timepoint, the MtTdp2α mRNA amount further decreased in cells treated with 150 μM etoposide, however the transcript levels remained significantly higher  compared to CTRL. Finally, in response to 300 μM etoposide, there was a drop in the expression level of MtTdp2α gene, in agreement with the low frequency of viable cells previously reported for this treatment . MtTdp2α-overexpressing cells showed significant up-regulation of the MtTop2 gene, encoding topo II, under physiological conditions , as previously described  at 6 h of treatment with the inhibitor, compared to CTRL. At the same timepoint, the MtTop2 mRNA amount did not significantly change in Tdp2α-13c cells treated with 150 μM etoposide whereas the transcript levels were decreased in Tdp2α-28, compared to CTRL. Finally, in response to 300 μM etoposide, there was a drop in the expression level of MtTop2 gene, in agreement with the cytotoxic effects of the treatment . The observed overexpression of MtTop2 gene might contribute to etoposide tolerance in Tdp2α-13c and Tdp2α-28 lines.Etoposide exerts its cytotoxic effects by stabilizing the transient topo II-DNA covalent complexes which result in increased levels of protein–DNA adducts subsequently processed into DSBs. In animal cells, Tdp2 is able to resolve the covalent link between topo II and DNA by directly excising these adducts . In the escribed . In the MtTdp2α-overexpressing cells, exposure to etoposide resulted in morphological features resembling the so-called nucleolar caps which in animal cells have been associated with the response to transcriptional stress and DNA repair . Other distinct morphological features were evident in the etoposide-treated nucleoli of M. truncatula cells and the corresponding nucleolar regions have been highlighted in Figure 7 using a colored line . The blue line surrounds the chromatin (ch) areas from which the nucleolus is stemming out. As shown in Figures 7A,B (blue line), the region involved in the release of nucleolar material into the surrounding nucleoplasm is visibly larger in the Tdp2α-28 nucleolus, compared to CTRL. This was a distinctive feature of MtTdp2α-overexpressing cells (data not shown). Another morphological change is evidenced by the red line which surrounds the dense fibrillar component (dfc) or the site of rRNA processing . Differences in the fragmentation and distribution of DFC areas were observed between CTRL and MtTdp2α-overexpressing cells. The finding that these rearrangements were more frequently detected in the MtTdp2α-overexpressing lines would further support the idea that transcription is maintained and enhanced under stress conditions in the Tdp2α-13c and Tdp2α-28 lines.Based on results of DNA diffusion assay, the treatment with 150 μM etoposide was then selected for ultrastructural analysis. In both CTRL and A repair . One of M. truncatula lines overexpressing the MtTdp2α gene: expansion of the nucleolar area, occurrence of ring-shaped nucleoli, presence of nucleolar vacuoles, increased nucleolar activity, and resistance to cytotoxicity.In the present work, several ultrastructural morphological hallmarks were identified in the M. truncatula lines overexpressing the MtTdp2α gene also showed an increased number of nucleoli per cell. To date, information concerning the mechanisms which determine the number of nucleoli per cell is still scanty (THAL (THALLO) gene in Arabidopsis. The THAL protein, member of the conserved SAS10 (SOMETHING ABOUT SILENCING 10)/C1D family with functions in pre-rRNA processing, has a peculiar role in the modulation of the nucleolar function, contributing to both rDNA transcription and pre-rRNA processing. According to these authors, hypertrophic nucleoli and multinucleolate cells might result from ectopic rDNA transcription and dispersal . Treatments with the drug caused changes in the nucleolar morphology, highlighting the presence of structures resembling the so-called nucleolar caps (M. truncatula cells exposed to etoposide have been described by Arabidopsis cells upon treatments with phosphorylation and transcription inhibitors (MtTdp2α-overexpressing cells, this might be an additional evidence of the response orchestrated by an extremely active nucleolus against cytotoxic and genotoxic stress.In animal cells, the Tdp1 enzyme also contributes to the repair of topo II-mediated DNA damage . As for lar caps . Structuhibitors . Since tMtTdp2α expression level was higher in Tdp2α-13c cells than Tdp2α-28, the observed phenotypic effects were always stronger in the Tdp2α-28 line. In this context, it should be considered the possibility that, as reported in animal cells, also in plant cells the Tdp2 gene might play a dual role, in the DNA repair processes as well as in the proliferation-related signaling pathways. At the moment, we cannot rule out any involvement of the MtTdp2α gene in plant signaling networks. This will be starting point for future investigations that will provide global profiles and additional information concerning the MtTdp2α gene function.Although the To date, many aspects of the plant response to genotoxic stress are still poorly investigated, despite their relevance in crop stress response within the scenario of global climate change. A useful way to investigate mechanisms involved in the plant response to genotoxic stress, and possibly disclose novel interactions, relies on the availability of cell and/or plant lines having altered expression of DDR genes as demonstrated in the present work for the Tdp2 function. This novel player in the stress response mediated by the plant nucleolus might be a useful tool to unravel the stress sensing mechanisms that act within the ‘nucleolar surveillance systems’ in plants.AB, AM, and SdSA conceived the work and wrote the manuscript. AM, MF, and MB contributed to the experimental data. MB performed TEM analyses. AB, AM, SdSA, MB, and DC contributed to the discussion of data. All authors read and agreed with 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. The reviewer TT and handling Editor declared their shared affiliation.