DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance. Cells are known to respond to external stimuli through changes to their biomechanical properties, such as cellular stiffness. Some studies have previously related chemotherapy treatments to changes in the stiffness of cells and tissues ,2. ChemoCisplatin, specifically, creates adducts within the double helix, which then lead to double-strand breaks (DSBs) in the DNA during replication, through replication-fork collapse .DSBs can result in large genomic aberrations and are, therefore, the most deleterious to the cell. In the event of a DNA break, the cell activates DNA damage response (DDR) pathways that allow detection and repair of this lesion. Failure to repair damage leads to cell death through apoptosis, or to the propagation of mutations that drive genomic instability and cancer development .It is well known that the ataxia-telangiectasia-mutated (ATM) kinase localizes to regions of damage, where it phosphorylates histone H2AX, producing γH2AX. This, in turn, promotes the recruitment of multiple repair factors to foci of damage ,8. HowevIt is established that chromatin and the lamina are major determinants of nuclear mechanics , and we Here, we investigated the relationship between DNA damage and nuclear mechanics. We used Atomic Force Microscopy (AFM) to probe mechanics of the nucleus of mammalian cells and monitor changes that occur after treatment with cisplatin. We found that, following DNA damage, large-scale mechanical alterations to the nucleus arise, caused by global decondensation of chromatin. This is dependent on DDR activation and increases molecular diffusion in the nucleus, potentially resulting in higher accessibility for repair factors. Surprisingly, we also found that mechanical relaxation of the nucleus, independently of chromatin, protects against genomic instability. Collectively, our data reveal how changes to chromatin architecture following DNA damage both suppress further genomic instability and provide an environment to promote repair. These findings may be harnessed to promote therapeutic approaches (e.g. resistance within a tumour environment).cis-diammineplatinum(II) dichloride] (Sigma) was resuspended in a 0.9% NaCl solution to a concentration of 3.3 mM, following manufacturer's instructions and used at a concentration of 25μM. Trichostatin A (TSA) (Sigma) was resuspended to 6.6. mM in DMSO and used at 400 nM in culturing medium, for 24 h. ATM inhibitor, KU55933 (Sigma), was resuspended in DMSO to 12.6 mM and used at a concentration of 20 μM, 30 min prior treatment and then for the whole duration of cisplatin treatment in culturing medium. Blebbistatin (Sigma) was resuspended to 50 mM in DMSO and used at a concentration of 50 μM for 1–2 h before AFM measurements and for 30 min, prior treatment and then the whole duration of cisplatin treatment for imaging. Latrunculin B (Sigma) was resuspended in DMSO to a concentration of 12.6 mM and used at 1 μM for 1 h before AFM measurements.Cisplatin [2 in MEM Alpha medium with GlutaMAX, supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). HeLa cells were transfected with 0.5 μg pSNAPf-C1 plasmid (Addgene 58186) using Lipofectamine 2000 (Invitrogen) and following manufacturer's instructions.HeLa cells (ECACC 93021013) were cultured at 37°C and 5% CO2, 25 mM KCl, 1 mM PMSF, 1 mM DTT and Protease Inhibitor Cocktail (Thermo Fisher Scientific). Cells were then re-suspended in cold hypotonic buffer N and incubated for 1 h on ice. Cells were homogenized on ice, using a glass Dounce homogenizer (Wheaton). Sucrose was added to the cell lysate to a final concentration of 220 mM and mixed well by inversion before centrifugation. The pellet, corresponding to isolated nuclei, was washed in cold Buffer N—10 mM Hepes pH 7.5, 2 mM MgCl2, 25 mM KCl, 250 mM sucrose, 1 mM PMSF, 1 mM DTT Protease Inhibitor Cocktail. The nuclei pellet was re-suspended in PBS and used immediately for AFM measurements.Nuclei were prepared based on protocols in ,18 HeLa 2 with 1μg ml−1 Hoechst 33342 in growth medium. Stained cells were fixed in 4% (m/V) paraformaldehyde (PFA). Residual PFA was quenched with 50 mM ammonium chloride for 15 min at room temperature. Cells were then permeabilized and blocked for 15 min with 0.1% (V/V) Triton X-100 and 2% (m/V) bovine serum albumin (BSA) in Tris-Buffered Saline (TBS). Antibodies were used as follows: mouse-Phospho-H2AX (Merck 05–636) at 1:500 dilution, rabbit-Lamin B1 (Abcam ab16048) at a dilution of 1:200, donkey anti-mouse Alexa Fluor 488-conjugated (Abcam Ab181289) at 1:500 and donkey anti-rabbit Alexa Fluor-488-conjugated  at 1:500 dilution. For actin staining, fixed and permeabilized cells were stained prior to immunofluorescence with 165 nM Rhodamine-Phalloidin (ThermoFisher) for 20 min. Coverslips were mounted on microscope slides with 10% (m/V) Mowiol, 25% (m/V) glycerol, 0.2 M Tris–HCl, pH 8.5, supplemented with 2.5% (m/V) of DABCO (Sigma).HeLa cells grown on glass coverslips were incubated for 10 min at 37°C and 5% COCells were visualized using Wide-field microscope Olympus IXT1, or Confocal microscope LSM 880. For confocal microscopy, a Plan-Apochromat 63 × 1.4 NA oil immersion lens  was used. Three laser lines: 405, 488 and 561 nm, were used to excite Hoechst, Alexa 488 and Alexa 647 fluorophores, respectively. Built-in dichroic mirrors  were used to reflect the excitation laser beams onto cell samples. For fluorescence collection, the used emission spectral bands were: 410–524 nm (Hoechst), 493–578 nm (Alexa 488) and 564–697 nm (Alexa 647). The green channel (Alexa 488) was imaged using a 1 gallium arsenide phosphide (GaAsP) detector, while the blue (Hoechst) and red (Alexa 647) channels were imaged using two multi-anode photomultiplier tubes (MA-PMTs). For imaging acquisition and rendering, ZEN software was used. Confocal Images were deconvolved using the Zeiss Zen2.3 Blue software, using the regularized inverse filter method.For Wide-field microscopy, a PlanApo 100xOTIRFM-SP 1.49 NA lens mounted on a PIFOC z-axis focus drive  was used, with an automated 300W Xenon light source  with appropriate filters . QuantEM (Photometrics) EMCCD camera, controlled by the Metamorph software (Molecular Devices) was used for image acquisition. All images were then analysed by ImageJ.2O: H2O2 : NH4OH , placed in a 70°C water bath. Cleaned coverslips were repeatedly washed in filtered water and then ethanol, dried and used for cell seeding. Transfected or non-transfected cells were incubated with 1μg ml−1 Hoechst 33342 in growth medium for 15 min at 37°C, 5%CO2. Following this, cells were washed with PBS and fixed in pre-warmed 4% (w/v) PFA in PBS and residual PFA was quenched for 15 min with 50 mM ammonium chloride in PBS. Immunofluorescence (IF) was performed in filtered sterilized PBS, unless when anti-phospho antibodies were used. Then, IF was performed in filtered sterilized TBS. Cells were permeabilized and simultaneously blocked for 30 min with 3% (w/v) BSA in PBS or TBS, supplemented with 0.1% (v/v) Triton X-100. Permeabilized cells were incubated for 1 h with the primary antibody and subsequently the appropriate fluorophore-conjugated secondary antibody, at the desired dilution in 3% (w/v) BSA, 0.1% (v/v) Triton X-100 in PBS or TBS. The antibody dilutions used were the same as for the normal IF protocol (see above), except from the secondary antibodies which were used at 1:250 dilution. Following incubation with both primary and secondary antibodies, cells were washed three times, for 10 min per wash, with 0.2% (w/v) BSA, 0.05% (v/v) Triton X-100 in PBS or TBS. Cells were further washed in PBS and fixed for a second time with pre-warmed 4% (w/v) PFA in PBS for 10 min. Cells were washed in PBS and stored at 4°C, in the dark, in 0.02% NaN3 in PBS, before proceeding to STORM imaging.Cells were seeded on pre-cleaned No 1.5, 25-mm round glass coverslips, placed in 6-well cell culture dishes. Glass coverslips were cleaned by incubating them for 3 h, in etch solution, made of 5:1:1 ratio of H® cell chambers (Invitrogen). Imaging was performed in freshly made STORM buffer consisting of 10% (w/v) glucose, 10 mM NaCl, 50 mM Tris–pH 8.0, supplemented with 0.1% (v/v) 2-mercaptoethanol and 0.1% (v/v) pre-made GLOX solution which was stored at 4 °C for up to a week (5.6% (w/v) glucose oxidase and 3.4 mg/ml catalase in 50 mM NaCl, 10 mM Tris–pH 8.0). All chemicals were purchased from Sigma.Before imaging, coverslips were assembled into the Attofluor2 and 7–12 kW/cm2, respectivelyImaging was undertaken using the Zeiss Elyra PS.1 system. Illumination was from a HR Diode 642 nm (150 mW) and HR Diode 488 nm (100 mW) lasers where power density on the sample was 7–14 kW/cmImaging was performed under highly inclined and laminated optical (HILO) illumination to reduce the background fluorescence with a 100× NA 1.46 oil immersion objective lens  with a BP 420–480/BP495–550/LP 650 filter. The final image was projected on an Andor iXon EMCCD camera with 25 msec exposure for 20000 frames for γH2AX and 60 msec for 60 000 frames for Hoechst imaging.Image processing was performed using the Zeiss Zen software. Where required, two channel images were aligned following a calibration using pre-mounted MultiSpec bead sample . The channel alignment was then performed in the Zeiss Zen software using the Affine method to account for lateral, tilting and stretching between the channels. The calibration was performed during each day of measurements.The images were then processed through our STORM analysis pipeline using the Zen software. Single molecule detection and localization was performed using a 9-pixel mask with a signal to noise ratio of 6 in the ‘Peak finder’ settings while applying the ‘Account for overlap’ function. This function allows multi-object fitting to localize molecules within a dense environment. Molecules were then localized by fitting to a 2D Gaussian.The render was then subjected to model-based cross-correlation drift correction. The final render was then generated at 10 nm/pixel and displayed in Gauss mode where each localization is presented as a 2D gaussian with a standard deviation based on its precision.2. For Blebbistatin pre-treated cells, 50 μM Blebbistatin was added prior to 4-h incubation with 50 μM Blebbistatin and 25 μM cisplatin. For cisplatin-treated cells, only cisplatin was added at 25 μM for 4 h. Cells were then incubated for 15 min with Hoechst dye, at 37°C, 5% CO2 and fixed using 4%(m/v) PFA. Cells were stained with 165 nM Rhodamine-Phalloidin (ThermoFisher) for 20 min and 1:500 dilution of mouse-Phospho-H2AX (Merck 05–636) antibody for 1 h, following the immunofluorescence protocol. High-content imaging was undertaken using a Cell Discoverer 7 (Zeiss), using a Plan-Apochromat 20 × 0.7 NA objective. Hoechst, Alexa 488 and Rhodamine fluorophores were excited using LED light at wavelengths 385, 470 and 567 nm, respectively. ZEN software was used for image acquisition and images were analysed using Zeiss Zen2.3 Blue software.HeLa cells were seeded in clear-bottom, black-walled 96-well plates at 10 000 cells/well in culturing medium and incubated overnight at 37°C, 5% COf-C1 (Addgene 58186) construct with Lipofectamine 2000 (Invitrogen), according to manufacturer's instructions. Following this, cells were treated with 25 μM cisplatin or 400 nM TSA in growth medium for 24 h.Cells were transfected for 24 h with 0.5 μg of pSNAPf-C1 construct were labelled for 15 min with 10 nM SNAP-tag-JF549 ligand, in cell culture medium at 37°C, 5% CO2. Cells were washed for three times with warm cell culture medium and then incubated for further 30 min at 37°C, 5% CO2. Cells were then washed three times in pre-warmed FluoroBrite Dulbecco's-modified Eagle's medium (ThermoFisher Scientific), before proceeding to imaging.Cells transiently expressing pSNAP2 at the back aperture of a Nikon 100 × 1.4 NA objective. Images were relayed through a custom optical system appended to the detection path of a Nikon Ti microscope with focus stabilization. The acMFM detection path includes a diffractive multifocal grating in a conjugate pupil plane, a chromatic correction grating to reverse the effects of spectral dispersion, and a nine-faceted prism, followed by a final imaging lens.Single molecule imaging was performed using an aberration-corrected multifocal microscope (acMFM), as described by Abrahamsson et al. . BrieflyThe acMFM produces nine simultaneous, separated images, each representing successive focal planes in the sample, with ca. 20 μm field of view and nominal axial separation of ca. 400 nm between them. The nine-image array is digitized via an electron multiplying charge coupled device (EMCCD) camera  at up to 32 ms temporal resolution, with typical durations of 30 s.3D+t images of single molecules were reconstructed via a calibration procedure, implemented in Matlab (MathWorks), that calculates and accounts for (i) the inter-plane spacing, (ii) affine transformation to correctly align each focal plane in the xy plane with respect to each other and (iii) slight variations in detection efficiency in each plane, typically less than ±5–15% from the mean.Reconstructed data were then subject to pre-processing, including background subtraction, mild deconvolution (3–5 Richardson-Lucy iterations) and/or Gaussian de-noising prior to 3D particle tracking using the MOSAIC software suite . Paramet−1) and therefore slightly stiffer than the nominal stiffness of 0.02 Nm−1. The nucleus was probed on several locations using the ForceMap macro with a maximum indentation force of 1.5 nN and the resulting force-indentation curves were analysed using a modified Hertz model within a self-written IGOR macro as described earlier  on top of an inverted fluorescence microscope . The combined instrument is mounted on a Halcyonics vibration isolation table  inside an acoustic enclosure (Asylum Research) to minimize noise. Force-indentation curves were executed in contact mode using the longer triangular cantilever of a TR 400 PB chip that has a pyramidal tip with an opening angle of 35°. The spring constant was determined with the built-in macro based on the thermal method and was in the range  using a 2° cone and plate geometry. To facilitate cell attachment, the gels were functionalized with rat tail Collagen type I  overnight at 4°C using the heterobifunctional crosslinker Sulfo-SANPAH  active with UV light (λ = 365 nm) for 10 min.Elastic polyacrylamide (PA) gels were prepared as described earlier ,24. In bCells attached to Aclar membrane (Agar Scientific) were fixed for 2 h in 2.5% glutaraldehyde (w/v) in 100 mM sodium cacodylate (CAB) buffer pH 7.2. Samples were washed twice for 10 min in 100 mM CAB and then post-fixed in 1% osmium tetroxide (w/v) in 100 mM CAB for 1 h before being dehydrated using an ethanol series of 50%, 70%, 90% (v/v) and three times with 100% ethanol for 10 min per step. The samples were then placed into propylene oxide, for (2×)10 min, and following this into a 1:1 mixture of propylene oxide and Agar LV resin (Agar Scientific) for 30 min. Following this, samples were embedded in freshly prepared Agar LV resin twice for 2 h before being placed in shallow aluminium moulds with the cells facing up and were polymerized at 60°C for 24 h before being examined with a dissecting microscope to identify areas confluent with cells. These areas were cut out with a jig saw and attached to polymerized resin blocks with superglue, and once attached, the Aclar membrane was peeled off, exposing a monolayer of cells in the block face. Sections of 70 nm were cut on a Leica EM UC7 ultramicrotome using a diamond knife (Diatome) and were collected on 400 mesh copper grids. Sections were counterstained in 4.5% uranyl acetate (w/v) in 1% acetic acid (v/v) for 45 min and in Reynolds’ lead citrate for 7 min. Samples were viewed in a Jeol 1230 transmission electron microscope and images were captured with a Gatan One View 16mp camera.Whilst the biochemical responses to DNA damage are well investigated ,25,26, tE. Figure Before AFM measurements, fully adhered cells on glass were treated with 25 μM cisplatin for 4 h, or 24 h, which induced DNA damage, recorded as fluorescent foci of γH2AX Figure . AFM meaWe found that long treatment with cisplatin (24 h), but not short treatment (4 h), significantly reduced cellular stiffness Figure . Young'sAlthough these measurements were taken at a central point above the nucleus, the complex actin cytoskeleton extends throughout the whole cell and exerts large amounts of force on the nucleus through compression, thus altering its physical properties and potentially masking mechanical changes. As a result, it is difficult to understand what the individual contributions of the nucleus and the cytoskeleton are to whole-cell mechanics.To understand if the mechanical changes observed were a result of alterations to the cytoskeleton, we labelled cells with phalloidin after cisplatin treatment . Our datTo understand the individual contribution of the nucleus to global mechanics, we decided to perform measurements on cells at initial adhesion stages, isolated nuclei Figure  and cellAt initial adhesion stages, the nucleus is the largest contributor to whole-cell mechanics. In this case, the cells are given enough time to adhere to the surface (2–3 h for HeLa cells) but the cytoskeleton and in particular stress fibres, are not fully established . As a reOur AFM data show that, as expected, fully-adhered cells (4.4 ± 0.7 kPa) are stiffer than Latrunculin B and Blebbistatin treated cells , initially adhered cells (2.9 ± 0.3 kPa) and isolated nuclei (2.6 ± 0.4 kPa), whilst there is no significant difference between these four latter conditions Figure .From these data we confirmed that nuclear measurements performed in initially-adhered cells are relatively free of cytoskeletal contributions and that the nucleus remains as the only large structural variable. Therefore, we decided to use this approach to investigate changes to nuclear mechanics, as this allows for the physical properties of nucleus to be probed whilst maintaining the organelle in its physiological environment and without the use of drugs that may have unknown effects in our study.P < 0.001) and 24 h  of cisplatin treatment , confirming these changes occur are intrinsic to the nucleus  by pre-treating cells with iATM for 30 min before 4-h treatment with 25 μM cisplatin . Immunofluorescence shows that treatment with iATM impairs the formation of γH2AX foci Figure . AFM meaThe nuclear lamina is a major structural component of the nucleus. We therefore tested if cisplatin treatment changed lamina integrity, thus changing nuclear mechanics. Our results show that for non-treated and cisplatin-treated cells there are no changes in thickness of the nuclear lamina . NegativTogether with the lamina, chromatin compaction is one of the other major contributors for nuclear mechanics, and previous studies have suggested that the state of chromatin condensation can change following DSBs ,34–36. ITo investigate this hypothesis, we used EM to image both non-treated and cisplatin-treated cells and compared it to TSA, a deacetylase inhibitor that leads to decondensation of chromatin. Dense regions of staining corresponding to condensed chromatin are clearly visible in the non-treated cells. This contrasts with both cisplatin and TSA-treated cells Figure . QuantifTo test if chromatin decondensation, following TSA treatment, had a similar effect to cisplatin on nuclear stiffness, we measured the Young's modulus in nuclei of initially adhered cells. Our AFM data show Figure  that nucTo complement EM data and confirm the observed effects of cisplatin on chromatin, we used super-resolution STORM imaging. Standard antibody staining was used to image γH2AX, whilst we took advantage of the photophysical properties of the Hoechst DNA dye to visualize chromatin Figure  and B. QA possible benefit that arises from chromatin decondensation is that it may allow for higher protein diffusion in the nucleus, thus enabling repair factors to reach areas of damage more easily. To test this, we used Multifocal Microscopy (MFM)  and 3D s2/s) is significantly higher than in non-treated cells (0.8 ± 0.03 μm2/s), but similar to TSA-treated (1.3 ± 0.04 μm2/s)  Figure . This suRecently, studies have linked the occurrence of DNA damage to external forces exerted on the nucleus ,40. SimiTo investigate how forces exerted on the nucleus influence DNA damage, we decided to pre-treat cells with Blebbistatin for 30 min before 4-h treatment with cisplatin . This allowed us to minimize cytoskeletal forces on the nucleus prior to the occurrence of damage. We used γH2AX as a marker for damage. High-content screening revealed a large decrease in the percentage of damaged cells when they pre-incubated with Blebbistatin. In non-treated cells, only 5.1 ± 0.2% of the population showed γH2AX signalling, as expected. 37.4 ± 0.9% of cisplatin-treated cells (short treatment) displayed signs of damage, whilst this value dropped to 21.2 ± 0.6% for cells that were pre-incubated with Blebbistatin Figure  and B. T2 to 456.0 ± 10.8 μm2 (Figure Matrix stiffness is tightly related to cell spread, adhesion formation and cytoskeletal force ,43,44. A2 Figure , in line2 Figure .If we expect lower force to have a protective effect, lower surface stiffness should decrease the amount of damage in cells, in a similar way to Blebbistatin treatment. As expected, our data reveal that cells treated in stiffer gels, 30 kPa, have 65% higher levels of γH2AX levels compared to 2 kPa gels and 30% higher than 11 kPa gels Figure . OverallOur work reveals a direct link between DNA damage, chromatin condensation and nuclear mechanics. It has been shown that treatment with cisplatin and other chemotherapy drugs changes whole-cell mechanics ,50. HoweMechanical changes to the nucleus do not arise spontaneously from breaks in the chromatin fibre, but are a result of downstream large-scale chromatin decondensation. The ATM kinase is activated by DNA damage, and its absence or inactivity can lead to a proportion of unrepaired DSBs in the cell ,52. Inteet al., which showed that nuclear softening protects against mechanically induced DNA damage (et al. (Interestingly, we found that alterations to biomechanics also appear to protect the cell from genomic instability. In addition to the intrinsic mechanical properties of the nucleus, the cytoskeleton also has a large impact on overall nuclear mechanics. Relaxation of the cytoskeleton results in reduced nuclear tension and, here, we show that this has a protective effect in DNA damage. It has been shown that reduction in matrix stiffness, a major determining factor for cytoskeletal organization, correlates with inhibition of replication . CisplatA damage . Moreove (et al. , which rDeformations to the nuclear envelope, as well as mechanical forces acting on the organelle are important for the regulation of cell-cycle progression  and tranin vitro cell lines and patient-derived primary samples, where the latter are more deformable. Our data fit with these scenarios, whereby decreased nuclear stiffness promotes repair and prevents further damage, which could subsequently drive drug resistance.There is growing evidence that tumorigenesis and resistance to chemotherapy agents correlate with changes in cellular and nuclear mechanics . DependiIn summary, here, we describe how nuclear mechanics change due to induced DNA damage through chromatin remodelling events, as part of DDR. Furthermore, we show that nuclear envelope relaxation protects against further damage. It will be of interest, in the future, to determine the molecular mechanisms through which nuclear mechanics directly influence DNA damage levels as these pathways may directly influence therapeutic resistance.The data supporting the findings of this study are available from the corresponding author on request.gkaa1202_Supplemental_FileClick here for additional data file.


This study investigated the activity of omadacycline against Legionella pneumophila strains isolated between 1995 and 2014 from nosocomial or community-acquired respiratory infections. Omadacycline exhibited extracellular activity similar to comparator antibiotics; intracellular penetrance was found by day 3 of omadacycline exposure.Omadacycline is an aminomethylcycline antibiotic with  in vitro activity against pathogens causing community-acquired bacterial pneumonia (CABP). This study investigated the activity of omadacycline against Legionella pneumophila strains isolated between 1995 and 2014 from nosocomial or community-acquired respiratory infections. Omadacycline exhibited extracellular activity similar to comparator antibiotics; intracellular penetrance was found by day 3 of omadacycline exposure. These results support the utility of omadacycline as an effective antibiotic for the treatment of CABP caused by L. pneumophila.Omadacycline is an aminomethylcycline antibiotic with  Legionella pneumophila, a leading cause of atypical community-acquired bacterial pneumonia   –3. L. pnive care –6. Betwelization , and morlization , 6, 8.L. pneumophila serogroups 1, 4, 5, and 6 are the primary causes of human disease; serogroup 1 is responsible for >80% of reported cases of legionellosis   . TherefoL. pneumophila isolates from 1995 to 2005 and 2006 to 2014. The minimum extracellular concentration (MIEC) inhibiting intracellular multiplication of L. pneumophila in human monocytes was determined for omadacycline and comparators against L. pneumophila strains.This study investigated the activity (MIC) of omadacycline and comparators against Antibiotic reference powders were provided by the following groups: Paratek Pharmaceuticals, Inc., King of Prussia, PA , Sigma Chemicals, Mississauga, ON , and Sanofi, Montreal, QC (telithromycin).L. pneumophila strains isolated during 1995 to 2005 and 50 strains isolated during 2006 to 2014 (serogroup 1 [n = 45] and serogroups 2 to 6 [n = 1 per serogroup]) were collected from mostly nosocomial or community-acquired respiratory tract sources. Strains were grown on buffered charcoal yeast extract (BCYE) agar. Five strains of L. pneumophila serogroup 1 were also used to assess intracellular activity. MICs were determined by broth microdilution methodology modified from Clinical and Laboratory Standards Institute (CLSI) guidelines (Fifty idelines .L. pneumophila (n = 100), MIC90 values for omadacycline (0.06 to 1 mg/liter) were either comparable to, or up to two dilutions lower than, those of azithromycin and erythromycin  was lower than the MIC90 values of doxycycline, azithromycin, and erythromycin and higher than the MIC90 values of telithromycin, levofloxacin, and moxifloxacin  than against L. pneumophila serogroup 1 . Against L. pneumophila serogroups 1 to 6, levofloxacin and moxifloxacin had the lowest MIC90 values observed, followed by telithromycin, omadacycline, azithromycin, doxycycline, and erythromycin.Against all serogroups of hromycin . Againstfloxacin . OmadacyL. pneumophila serogroup 1. The mononuclear cell method (6 to 2 × 106 cells/ml), and Legionella inoculum (104 to 105 CFU/ml) were used. After a 1-h exposure in a shaking incubator, 150 μl of infected cultures was maintained without shaking for 7 days at 37°C in 5% CO2 and 95% air. After 24 h (day 1), infected cultures were washed three times (300 μl). Antibiotics (150 μl of diluted antibiotic at 1× MIC) were added for a final volume of 300 μl, and cultures were incubated for 2 days. After 72 h (day 3), cultures were washed three times and split into two groups—one with the same antibiotic and one without antibiotic —for 4 days of incubation. Monocytes in a 20 μl sample taken at time zero and every 24 h until day 7 were diluted by 10-fold dilutions and lysed with distilled water. CFU/ml counts were determined in duplicate using BCYE agar at each time point.Intracellular activity of omadacycline was determined against five strains of l method  was perf10 CFU/ml or 99.9% of L. pneumophila serogroup 1 grown in macrophages was reached only with omadacycline and moxifloxacin after 3 days of antibiotic exposure .A reduction of 3 logexposure . CompareL. pneumophila serogroup 1. At days 1 and 3 of exposure, each strain was exposed to antibiotic concentrations of 1, 1/2, 1/4, 1/8, or 1/16 times the MIC required to determine the precise MIEC. Cultures were incubated with antibiotic for 4 days. CFU/ml counts were performed daily in duplicate using BCYE agar. MIEC was defined as the lowest MICs that produced intracellular reductions of ≥1 log10 (CFU/ml) of L. pneumophila and was calculated at days 3 and 5 of exposure.The MIECs of omadacycline and comparators  inhibiting intracellular human monocyte growth , 23 wereL. pneumophila growth in macrophages was detected at day 5 of omadacycline exposure, with an MIEC/MIC ratio of 0.24 (1/4× MIC) and MIEC of 0.06 mg/liter (L. pneumophila (Mean reduction of intracellular activity (≥92%) of mg/liter . At day umophila .0–24) value (17.23 mg · h/liter) and the observed mean alveolar cell (AC) AUC0–24 value (302.42 mg · h/l)  the area under the concentration-time curve from 0 to 24 h  was consistently lower than the MIEC/MIC ratio of moxifloxacin (0.5 or 1/2× MIC), doxycycline (1 or 1× MIC), and azithromycin (>1 or >1× MIC).in vitro activity against L. pneumophila serogroups 1 to 6. Based on the MIC90 values, omadacycline was 4-fold more potent by weight than doxycycline and erythromycin; omadacycline MIC90 values were 2-fold lower by weight than that of azithromycin. Omadacycline was 10-fold less potent by weight than telithromycin and fluoroquinolones tested. Noteworthy was the activity of omadacycline against L. pneumophila serogroup 1, the most common serotype isolated from nosocomial or community-acquired respiratory tract infections. Although L. pneumophila strains were isolated from patients across broad time frames, no change in MIC values was seen for omadacycline or comparators, indicating stable susceptibility across 20 years.Omadacycline demonstrated potent L. pneumophila is isolated as the cause of CAP in ∼2% to 5% of cases, but this incidence increases as much as 2-fold in hospitalized patients and the elderly (L. pneumophila is an intracellular pathogen, and understanding the intracellular activity, extracellular activity, and cellular penetration of an antibiotic is necessary to evaluate its potential utility. The current study results indicate that omadacycline demonstrates relative intracellular penetrance against L. pneumophila serogroup 1, comparable to other antibiotics used for CABP treatment. Findings also support those from a phase 3 study of CABP in which omadacycline was comparable to moxifloxacin, with a 87% early clinical success rate among 37 patients for whom L. pneumophila was identified as the causative pathogen (L. pneumophila, are suspected. elderly . L. pneupathogen . Thus, o


A receptor, also has antinociceptive properties. While progesterone and allopregnanolone are antinociceptive, their effect on estrogen-exacerbated TMD pain has not been determined. We hypothesized that removing the source of endogenous ovarian hormones would reduce inflammatory allodynia in the TMJ of rats and both progesterone and allopregnanolone would attenuate the estrogen-provoked return of allodynia. Baseline mechanical sensitivity was measured in female Sprague–Dawley rats (150–175 g) using the von Frey filament method followed by a unilateral injection of complete Freund’s adjuvant (CFA) into the TMJ. Mechanical allodynia was confirmed 24 h later; then rats were ovariectomized or received sham surgery. Two weeks later, allodynia was reassessed and rats received one of the following subcutaneous hormone treatments over 5 days: a daily pharmacological dose of estradiol benzoate , daily E2 and pharmacological to sub-physiological doses of progesterone , E2 daily and interrupted P4 given every other day, daily P4, or daily vehicle control. A separate group of animals received allopregnanolone (0.16 mg/kg) instead of P4. Allodynia was reassessed 1 h following injections. Here, we report that CFA-evoked mechanical allodynia was attenuated following ovariectomy and daily high E2 treatment triggered the return of allodynia, which was rapidly attenuated when P4 was also administered either daily or every other day. Allopregnanolone treatment, whether daily or every other day, also attenuated estrogen-exacerbated allodynia within 1 h of treatment, but only on the first treatment day. These data indicate that when gonadal hormone levels have diminished, treatment with a lower dose of progesterone may be effective at rapidly reducing the estrogen-evoked recurrence of inflammatory mechanical allodynia in the TMJ.Temporomandibular joint disorder (TMD) is associated with pain in the joint  and muscles involved in mastication. TMD pain dissipates following menopause but returns in some women undergoing estrogen replacement therapy. Progesterone has both anti-inflammatory and antinociceptive properties, while estrogen’s effects on nociception are variable and highly dependent on both natural hormone fluctuations and estrogen dosage during pharmacological treatments, with high doses increasing pain. Allopregnanolone, a progesterone metabolite and positive allosteric modulator of the GABA Women are genetically predisposed to experiencing certain chronic pain disorders. Some chronic pain conditions, such as endometriosis  nociception  were used in these experiments. Rats were housed two per cage in a colony room with a 12:12 h light:dark cycle (lights on at 8 a.m.). Food and water were available Stock solutions of estradiol benzoate (E2), progesterone (P4), and 5α-Pregnan-3α-ol-20-one  were dissolved in sesame seed oil , diluted prior to injection, and administered subcutaneously (s.c).Female rats were deeply anesthetized  with inhalation of gas  anesthesia. Topical lidocaine was applied before a single incision was made to the anterolateral abdominal area. The abdominal muscle was cut, ovaries were ligated with 3-0 Vicryl sutures, and excised. Abdominal muscle was sutured with 3-0 Vicryl sutures and the epidermal layer was stapled with an Autoclip Wound Closing System . Immediately before and 24 h following surgery, animals were administered the antibiotic Baytril (0.02 ml of a 22.7 mg/kg solution leading to approximately 4.4 mg/kg dose) intramuscularly (i.m.). The analgesic Rimadyl (0.03 ml of a 50 mg/kg solution leading to approximately 2.5 mg/kg dose) was administered subcutaneously (s.c.) immediately following surgery. Rats that received sham surgery received the same procedural manipulations and treatments except for removal of ovaries. Rats were allowed 2 weeks recovery from surgery and for the elimination of endogenous ovarian hormones.Complete Freund’s adjuvant  was dissolved 1:1 in saline solution and injected under brief isoflurane gas anesthesia into the intra-articular area of the TMJ. The TMJ area was palpated for the TMJ, confirmed by movement of the mandible, then the needle was directed to the joint and injected with CFA using a 30-gauge needle.Behavior was tested before CFA injections, 24 h after CFA injections, then 2 weeks after OVX. Von Frey filaments  were utilized to test the force to withdrawal threshold as a measure of mechanical allodynia at the cutaneous tissues surrounding the inflamed TMJ, as previously reported Ren, . For thiTo test the role of P4 after 5 days of hormone treatment, animals received one of the following hormone treatments s.c. every day for 5 days: (a) daily pharmacological dose of estradiol benzoate . One hour after hormone treatment, animals were rapidly decapitated under gas anesthesia  and trunk blood was collected in BD vacutainer® spray-coated K2EDTA collection tubes  on ice. Immediately after collection, blood was centrifuged at 3,000 rpm for 15 min. Serum was separated and stored at −20°C. After diethyl ether extraction, P4 levels were measured with a Progesterone Radioimmunoassay Kit  according to the manufacturer’s instructions.A separate group of 20 female rats were used to measure serum progesterone levels. Rats received CFA injections into the intra-articular area of the TMJ and were ovariectomized 24 h later. Two weeks post-OVX, animals received one of the following hormone treatments: (a) E2 (50 μg/kg); (b) E2 and P4 (16 mg/kg); (c) E2 and P4 (16 μg/kg); (d) E2 and P4  in ng/ml and analyzed by Mann–Whitney t-test and statistical significance was tested at p ≤ 0.05.Behavioral data were presented as the mean ± standard error of the mean of the force in grams (g) required to elicit a withdraw as a measure of the degree of mechanical allodynia. Data were analyzed by one-way analysis of variance (ANOVA) and two-way repeated measures ANOVA using GraphPad Prism 7 with time as the repeated measure and treatment as the independent factor. Tukey’s F = 20.6; p <0.05). CFA evoked a significant reduction in the force to withdrawal in all rats  compared to sham rats which remained allodynic (open bars). TMJ inflammation did not cause weight loss as animal weight 2 weeks post-surgery was significantly greater than animal weight prior to CFA injections  = 441.4; p <0.05; data not shown).Baseline mechanical threshold was measured before and after CFA injections then again 2 weeks after OVX or sham surgery  = 22.92; p <0.05). Ovariectomized rats that received daily E2 with daily P4 (mid gray bars) retained significantly higher mechanical thresholds (p <0.05), while rats that received only daily E2 (light gray bars) displayed similar mechanical allodynia to sham animals that received vehicle injections  = 42.8; p ≤ 0.05; p ≤ 0.05). In rats that were then ovariectomized, the force to withdraw returned to baseline levels  and was significantly greater than the sham surgery rats that remained allodynic . There was a significant effect of treatment  = 282.9; p ≤ 0.05), but not time  = 1.931; p > 0.05). On day 17 (1st day of hormone treatments), there was a significant effect of treatment 1 h following the first hormone treatment  = 83.40; p ≤ 0.05; p ≤ 0.05) and the sham rats that received vehicle treatment  when compared to all other groups. There was also a significant effect of treatment on day 19 1 h following the 3rd day of hormone injections  = 98.61; p ≤ 0.05; p ≤ 0.05) and the sham rats that received vehicle treatment  when compared to all other groups. We did not observe a decrease in mechanical threshold in ovariectomized rats treated with P4 (p > 0.05), ovariectomized rats treated with E2 and P4 (p > 0.05), or ovariectomized rats treated with E2 and P4 every other day (p > 0.05) compared to sham control (open bars) and E2-treated rats (light gray bars). There was also a significant effect of treatment observed 1 h after the last injection on day 21  = 161.6; p ≤ 0.05; p ≤ 0.05). There was no significant difference between sham controls (open bars) and ovariectomized females treated with E2 . No mechanical allodynia was observed in any group receiving P4 compared to sham or E2 injected rats (p > 0.05). On all 3 days, all rats receiving P4 treatment were similar to rats receiving vehicle treatment .We then repeated the experiment with an altered timeline to assess whether the observed effects of P4 on mechanical allodynia occurred prior to the last day of hormone treatments. Similar to F = 39.1; p ≤ 0.05), but not time  = 0.77; p > 0.05). As the treatment effect was again the same on each day, we compared the treatment groups within each day. There was a significant effect of treatment with 16 μg/kg P4 on day 17  = 18.65; p ≤ 0.05; F = 11.76; p ≤ 0.05; F = 26.13; p ≤ 0.05; p ≤ 0.05) and sham controls  compared to ovariectomized rats receiving vehicle (crossed bars) and all P4 treated rats with or without E2. There was no significant difference in CFA-evoked mechanical allodynia between sham controls (open bars) and ovariectomized rats receiving only E2 (light gray bars) on any treatment day tested (p > 0.05) and no mechanical allodynia was observed between any groups receiving P4 (p > 0.05).We next tested whether a lower dose of P4, 16 μg/kg, could also protect against the E2-elicited return of mechanical allodynia. Using the same treatment paradigm as F = 315.0; p ≤ 0.05), but not time  = 1.3; p > 0.05). As the treatment effect was again the same on each day, we compared the treatment groups within each day. There was a significant effect of treatment on day 17  = 162.2; p ≤ 0.05; F = 120.7; p ≤ 0.05; F = 268.2; p ≤ 0.05; p ≤ 0.05) and rats receiving daily P4 only (p ≤ 0.05). Significantly lower mechanical thresholds were also observed in ovariectomized rats treated with daily E2 (light gray bars) or in combination with daily or interrupted 16 ng/kg P4 compared to ovariectomized rats with vehicle treatments  and compared to ovariectomized rats with only P4 treatment (p ≤ 0.05).We then tested whether a sub-physiological dose of P4, 16 ng/kg, would have an effect on E2-elicited return of mechanical allodynia. There was a significant effect of treatment , females that received 16 μg/kg P4 (2.25 ± 0.40 (1.22–3.26) ng/ml; Mann–Whitney U = 0, n1 = 5, n2 = 5, p ≤ 0.05), and from females that received 16 ng/kg P4 (mean = 1.96 ± 0.41 (0.41–2.67) ng/ml; Mann–Whitney U = 0, n1 = 5, n2 = 5, p ≤ 0.05; data not shown). Also, plasma P4 levels following the 16 μg/kg dose were significantly different from ovariectomized control females , but did not differ from plasma P4 levels following the 16 ng/kg dose . Plasma P4 levels of females that received 16 ng/kg P4 did not differ from ovariectomized control females .Plasma P4 levels were measured following high E2 + P4  and in OVX females receiving the vehicle injection (sesame seed oil). The pharmacological 16 mg/kg dose of P4 resulted in plasma P4 levels significantly higher than OVX control females (40.02 ± 2.08 (37.11–48.04) ng/ml vs. 0.82 ± 0.16 (0.45–1.4) ng/ml; Mann–Whitney F = 39.1; p ≤ 0.05). There was a significant effect of treatment  = 295.6; p ≤ 0.01) and a significant effect of time  = 66.2; p ≤ 0.05). When comparing treatment groups within each day, there was a significant effect of treatment on the first day of hormone treatments  = 47.54; p ≤ 0.05; p ≤ 0.05) and the sham controls  when compared to ovariectomized females receiving vehicle (crossed bars), daily AP, daily E2 and AP, or daily E2 and AP every other day. No mechanical allodynia was observed in any group receiving AP compared to sham (open bars) or E2 injected rats . There was also a significant effect of treatment on day 19  = 1,762; p ≤ 0.05) and day 21  = 191.7; p ≤ 0.05). However, on day 19 (p ≤ 0.05). Similar to day 17, rats receiving daily AP did not display allodynia on day 19 or day 21 and were similar to vehicle treated rats (p > 0.05).The same behavior testing paradigm was used here, but AP was injected instead of P4 and mechanical allodynia was detected on day 17, day 19, and day 21 , could be linked to differences in the serum progesterone to estrogen ratio. Unfortunately, there appear to be no studies on the effect of altered progesterone to estrogen ratios in animal pain models for female-prevalent disorders linked to gonadal hormones. In the present study, all hormone-treated groups received the same pharmacological dose of estradiol (50 μg/kg) and while the plasma progesterone levels did not differ between the two lowest doses of progesterone, the resulting plasma progesterone levels from the 16 μg/kg, but not 16 ng/kg, were significantly greater than ovariectomized rats. We did not measure plasma estradiol levels, but this possibility warrants future studies to detect a potential role of ovarian hormonal ratios in nociceptive orofacial behavior.via inhibiting inflammatory mediators and preventing the estrogen-evoked upregulation of ion channel expression that triggers hypersensitivity. It is unclear where the site of action of gonadal hormones are in this system. Progesterone receptors are expressed throughout regions of the brain, such as thalamus, spinal cord, and medulla , while the latter converts 5α-DHP to allopregnanolone (Schumacher et al., Surprisingly, allopregnanolone treatment did not attenuate the return of mechanical allodynia on day 3 or 5 of hormone treatment. This is not in agreement with a study that reported allopregnanolone suppressed diabetes-induced thermal hyperalgesia up to 7 weeks (Afrazi and Esmaeili-Mahani, A receptor. This is supported by reports that allopregnanolone decreases pain in neuropathy models (Afrazi and Esmaeili-Mahani, A receptor subunits, resulting in a decrease in sensitivity to allopregnanolone (Zhu et al., A receptor in the trigeminal ganglia (Puri et al., A receptor in the trigeminal ganglia are needed to understand the mechanism underlying the protective role of progesterone in our treatment paradigm.Allopregnanolone may have acute antinociceptive effects through the GABAOverall, we show that removal of the endogenous source of ovarian hormones after orofacial inflammation relieves mechanical allodynia in the TMJ of female rats. We also showed rapid attenuation of the high estrogen-evoked return of mechanical allodynia by two different doses of progesterone and acute, rapid attenuation by allopregnanolone. These data suggest the allopregnanolone may provide short-term relief, whereas, progesterone may provide continual relief in the reemergence of TMD pain in post-menopausal women undergoing estrogen replacement therapy.All datasets generated for this study are included in the article.All studies were approved by Texas Woman’s University Institutional Animal Care and Use Committee. Experiments conformed to federal guidelines and the committee for Research and Ethical Issues of the International Association for the Study of Pain.RH contributed to experimental design, conducting experiments, data analysis and interpretation, and preparation of the manuscript. WB contributed to experimental design, conducting experiments, data analysis and interpretation, and approval of the manuscript. ST contributed to conducting experiments, data analysis and interpretation, and approval of the manuscript. LU and PK contributed to experimental design, data analysis and interpretation, and editing the manuscript. DA contributed to experimental design, data analysis and interpretation, and preparation 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.


Despite their contrasting redox chemistry, TU or H2O2 supplementation under NaCl [NaCl + TU (NT) or NaCl + H2O2 (NH)] generated a reducing redox environment in planta, which improved the plant growth compared with those of NaCl alone treatment. This was concomitant with better K+ retention and upregulated expression of NaCl defense related genes including HAK21, LEA1, TSPO and EN20 in both NT and NH treated seedlings. Under field conditions, foliar applications of TU and H2O2, at vegetative growth, pre-flowering and grain filling stages, increased growth and yield attributes under both control and NaCl stress conditions. Principal component analysis revealed glutathione reductase dependent reduced ROS accumulation in source (flag leaves) and sucrose synthase mediated sucrose catabolism in sink (developing inflorescence), as the key variables associated with NT and NH mediated effects, respectively. In addition, photosystem-II efficiency, K+ retention and source-sink relationship were also improved in TU and H2O2 treated plants. Taken together, our study highlights that reducing redox environment acts as a central regulator of plant’s tolerance responses to salt stress. In addition, TU and H2O2 are proposed as potential redox-based PBRs for boosting rice productivity under the realistic field conditions.Plant bioregulators (PBRs) represent low-cost chemicals for boosting plant defense, especially under stress conditions. In the present study, redox based PBRs such as thiourea  and hydrogen peroxide (H Salinity stress affects ~ 20% of world’s irrigated land and can go upto 50% of cultivable land by the middle of the twenty-first century2. Salinity decreases the soil water potential and thereby imposes an osmotic barrier for water uptake. Moreover, increased Na+ uptake also induces ionic toxicity which adversely affects overall plant metabolism3. Both osmotic and ionic disequilibrium results in increased production of reactive oxygen species (ROS), causing oxidative damage to various biomolecules like protein, lipid and DNA4. Plants possess various defense mechanisms including the activation of antioxidant system, maintenance of ionic homeostasis and activation of the defense genes. The non-enzymatic , and glutathione (GSH) and enzymatic  antioxidants functions to control the level of ROS5. Further the sequestration/redistribution of Na+ ions under salt stress maintains K+/Na+ homeostasis and thereby suppresses metabolic arrest and growth impairment6. Besides this, the activation of defense associated genes like late embryogenic abundant proteins (LEA)/dehydrin7, K+ transporters8, signalling mediators like phosphatidylinositol-4-phosphate 5 kinase9 (PIPK), translocator protein10 (TSPO) and early nodulin-2011 (EN20) are also reported to boost the ameliorative potential of plant towards salt stress.The rapid increase in the population together with shrinking of cultivable land is one of the major challenges towards ensuring global food security12. Various physiological processes like cell expansion, stomatal development, movement and conductance of leaf are negatively affected under salt stress13. In most plant systems, decrease in net photosynthetic rate14 was reported as one of the earliest quantifiable responses under salt stress. In plant photosynthesis, carbon dioxide (CO2) is fixed in chloroplasts via the Calvin cycle to yield triose phosphates (triose-P). Triose-P is transported to cytosol by triose-P/phosphate transporter for the synthesis of sucrose. In cytosol, aldolases catalyse the formation of fructose 1,6-bisphosphate , which is further metabolized to yield sucrose by the combined action of fructose 1,6-bisphosphatase and sucrose phosphate synthase (SPS). Sucrose as a final product of photo assimilation is translocated from its synthesis site (leaf) to various non-photosynthetic tissues15 (sink tissues) via phloem tissue. Sucrose translocated to sink is metabolized by cytosolic neutral invertase (NI), sucrose synthase (SuSy), and vacuolar acid invertase (AI), providing the hexose pool for the synthesis of structural and non-structural carbohydrates. The invertases (INV) catalyse irreversible hydrolyzation of sucrose into its hexose monomers (glucose and fructose), whereas SuSy catalyses reversible cleavage of sucrose using UDP to yield fructose and UDP-G. Salt stress negatively affects the sugar dynamics of the source leaf and developing sink tissue by inhibiting the synthesis, redistribution and utilisation of sucrose16.Both osmotic and ionic components of the salt stress negatively affect plant growth and yield associated traits−1 which leads to 50% yield loss17. Salt sensitivity also varies with age, from being moderately tolerant at seedling stage to highly susceptible at reproductive phase18. External application of chemicals and bio-molecules, referred to as plant bioregulators (PBRs), has been shown to minimize the salt-stress induced yield losses in multiple crops. Though PBRs may act differently, a unified redox/ROS dependent action has recently been proposed19. In the present study, two redox based PBRs such as thiourea20  and H2O222 , which chemically drive ROS levels in opposite direction, were assessed whether they will have an overlapping or independent effect(s). To this end, a short-term study was conducted at seedling stage to understand the impact of nutrient medium-supplemented TU and H2O2 on redox and ionic equilibrium. Further, the effects of foliar supplemented TU and H2O2 on field-grown plants were also studied in terms of source-sink relationship and yield attributes. The findings revealed that both TU and H2O2 maintain reduced redox status that could act as “core” regulator for improving K+ retention ability, photosynthetic efficiency and source-sink strength of the plants. These changes were ultimately reflected in the form of improved growth and yield under both control and NaCl stress conditions.Rice is the staple food crop which feeds more than half of the world population. Among the different abiotic stresses, soil salinity poses a major constraint for rice productivity. For rice, critical salinity level is estimated to be 6.9 dS mO. sativa) var. IR-64. The seeds were surface sterilized using 30% ethanol for 3 min followed by repeated washing with distilled water to remove traces of ethanol. The surface-sterilized seeds were germinated for 48 h and hydroponic cultures were established, as per the method described previously23. The ameliorative potential of TU and H2O2 towards NaCl stress was evaluated using two independent approaches. In the first approach, 14 days old hydroponically grown seedlings were subjected to different treatments including control (Yoshida medium), NaCl (50 mM), TU (7.5 µM), NaCl (50 mM) + TU (7.5 µM), H2O2 (1 µM) and NaCl (50 mM) + H2O2 (1 µM). Hereafter, NaCl + TU and NaCl + H2O2 treatments were denoted as NT and NH, respectively. A pre-treatment of 7.5 µM TU (TU and NT) and 1 µM H2O2 (H2O2 and NH) was also given for 24 h. The pre-treatment strategy has already been demonstrated to maximize the impact of TU-mediated amelioration of AsV in rice23. In shoots, activities of antioxidant enzyme and Na+ and K+ accumulation were quantified in a time-course manner ranging from 1, 4, 24 and 48 h post-stress. In addition, 6 h after the onset of treatments, expression levels of selected salt-responsive genes were analyzed. At 7 days post-stress, phenotypic parameters, both qualitative and quantitative along with antioxidant capacity were recorded. In the second approach, four healthy 30 days old hydroponically grown seedlings were transferred to plastic pots in six groups (5 pots/group), under the net-house experimental facility of Bhabha Atomic Research Centre, Mumbai (India). The fertigation and agronomic protocols were followed as previously described24. Group-1 plants were treated with NaCl (11 g/pot) dose twice, at 42- and 57-days post-transplantation. The NaCl dose  was calculated considering the total water holding capacity of 14 kg paddy soil (4.6 L) and the top-water (1.4 L). The group-2 and -3 plants were given foliar applications of TU (6.5 mM containing 0.01% Tween-20) and H2O2 (1 mM containing 0.01% Tween-20), respectively. A total of three foliar applications were given at vegetative, early anthesis and grain filling stages that corresponded to 40, 55 and 72 days post-transplantation, respectively. Group-4 and -5 plants were given the combined treatment of NaCl + TU (NT) and NaCl + H2O2 (NH) treatments, respectively. Group-6 plants were foliar-sprayed with water  and served as control. At 5 days post 3rd-foliar spray, various morphological traits  were recorded and biochemical attributes  were quantified from the flag leaves. Additionally, parameters of plant source-sink relationship and ion accumulation were quantified in three different tissues such as youngest flag leaf, old leaf from the bottom and developing inflorescence.The study was performed on Indian rice  using the pre-chilled buffer [(was extracted using 100 mM chilled potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 1% polyvinyl pyrrolidone (w/v)]. The samples were centrifuged at 15,000×g for 15 min at 4 °C. The supernatant was separated and used for the measurement of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities26. The protein content in the supernatant was quantified as per Bradford method27.The non-enzymatic antioxidant status of leaf tissues was analyzed according to the Oxygen Radical Absorbance Capacity (ORAC) method3, and finally dissolved using Milli-Q water. The Na+ and K+ contents were quantified using atomic absorption spectrometer and represented as % dry weight (DW).For the quantification of ions, leaf samples were dried at 70 °C till they achieve constant weight. The dried tissues (~ 100 mg) were acid digested using concentrated HNO2 and subjected to total RNA isolation using TRI-reagent (Sigma T-9494), as per manufacturer’s instructions. The quality assessment of the RNA sample and subsequent cDNA preparation were performed as described previously23. The details of gene-specific primers are given as Supplementary Table The leaf tissue samples from various treatments were harvested under liquid NThe various plant growth parameters viz shoot length, flag leaf length, leaf width and panicle length were quantified using a meter scale. The number of panicles per plant and 1000 seed weight were quantified manually. The leaf chlorophyll content was calculated as SPAD value using Chlorophyll Meter SPAD-502 plus-konica Minolta, representing an average value of five different points in the same leaf.−2 s−1 after optimization with a light curve. The photosynthetic efficiency was quantified using following parameters; cuvette air flow 750 mL min−1, chamber temperature (25 °C), Relative humidity (60%) and atmospheric CO2 concentration (400 ppm CO2). Net photosynthetic rate, stomatal conductance, and transpiration were recorded simultaneously. Water use efficiency (WUE) was calculated as the ratio between net photosynthesis and transpiration. Using the differential minimum (Fo) and maximum fluorescence (Fm) signals of from the open and close PS II centers, maximum quantum efficiency of PSII (Fv/Fm) was quantified. A 20 min prior dark adaptation is a prerequisite for the quantification of Fv/Fm, and can be defined as (Fm − Fo)/Fm. Similarly, maximum fluorescence of dark (Fm)and light adapted leaf (Fm’) were used for the quantification of Non photochemical quenching (NPQ); NPQ = Fm/F′m − 1. Further steady state chlorophyll fluorescence (Fs) was also quantified for the measurement of ETR and actual quantum efficiency of PSII (ΦPSII); ΦPSII = (F′m − Fs)/F′m28.The gas exchange of leaves was measured using an Infrared Gas Analyzer, GFS-3000 . The photosynthetic photon flux density (PPFD) was fixed at 1000 μmol m29. NBT stain intensity was quantified using image J software . The levels of ascorbate (ASA) and dehydroascorbate (DHA) contents were quantified using α-α′-bipyridyl-based colorimetric method30 and the results were presented as ASA/DHA ratio.Superoxide radicals were detected in situ using nitroblue tetrazolium (NBT) stainingg for 15 min at room temperature. The sucrose and starch were quantified in the supernatant and pellet respectively using sucrose quantification kit  and starch assay kit , respectively according to the manufacturer's protocol.Lyophilized leaf sample (10 mg) was extracted in 15 mL of 80% ethanol. The extract was boiled for 10 min and then subjected to centrifugation at 15,0002 ground plant material (~ 300 mg) using the pre-chilled buffer . The samples were centrifuged at 12,000×g for 15 min at 4 °C. The supernatant was separated and used for the measurement of SPS, SuSy31 and FPBase32. Additionally, Neutral invertase (NI) and acid soluble invertase (AI) activity in the plant samples were measured spectrophometrically following the extraction and assay methods31. The protein content in the sample was measured as per the Bradford method. All the enzyme activities were represented as units mg−1 protein which corresponds to μM of the product formed mg−1 protein min−1.For the quantification of enzymatic activities related to source sink homeostasis, total protein was extracted from liquid Np ≤ 0.05). Principal component analysis (PCA) was performed with datasets of the source leaf and developing sink, using Origin 2016 , and the first two components (PC1 and PC2) explaining the maximum variance in the datasets were used to make biplots.The experiments were conducted as randomized block design using three biological replicates. One-way analysis of variance (ANOVA) was performed with the whole dataset to confirm the variability of data and validity of results, and Duncan’s multiple range test (DMRT) was performed to determine the significant difference between treatments. Different letters indicate significantly different values  and compared across different treatments , K+ accumulation was increased by 147.32 and 105.47% under NT and NH treatments, respectively compared to NaCl treatment. Additionally, 33.42, 38.58% increase under TU, H2O2 was also observed at 48 h compared to control , LEA1 (late-embryogenic abundant 1), dehydrin and TSPO (translocator protein) which were upregulated by 279.42, 475, 113.41 and 162.78% under TU, and 285.92, 625, 94.72 and 362.58% under H2O2-treated leaves compared to control. Similarly, the expression of HAK21 , LEA1 , dehydrin  and TSPO  was found to be upregulated compared with those of NaCl-treated leaves , where it increased by 403.8%, followed by old leaves (OL) and developing inflorescences (DI), where it increased by 159.94 and 70.21%, respectively, compared with WS-treated plants , water use efficiency (WUE) and electron transport rate (ETR) which were decreased by 69.35, 61.90 and 53%, respectively compared with those of WS control. In contrast, significant photosynthetic recovery was noticed under both NT and NH treatments as these parameters were increased by 78.95, 62.5 and 101.23% in NT and 105.26, 75 and 72.84% in NH compared with that of NaCl treatment. In addition, quantum yield of photosystem II (PS-II yield) and non photochemical quenching (NPQ) were also increased specifically under NT (56.32%) and NH (31.25%), respectively compared with those of NaCl treatment  and sink  strength were also negatively impacted in NaCl-treated plants; except, for SuSy which was increased by 108.75%, in old leaves compared with WS control. The ameliorative effects were observed under both NT and NH treatments, at the level of source as well as sink. Besides, differential nature of NT and NH was also seen, especially in the source leaves. For instance, the SPS activity was increased by 93.47, 29.75 and 40.37% in NH-treated YL, ML and DI organs, respectively compared with those of NaCl treatment. However, under NT treatment, increased SPS activity was limited to YL (74.7%) and DI 22.36%) organ only. Unlike SPS, FBPase activity was significantly increased under both NT and NH, in all the three tested organs, compared with those of NaCl treatment. The sucrose degradation pathway was also activated under NT and NH treatment conditions. In source organs like YL and OL, the NI activity was increased 66.43 and 40.44% in NT and 59.05 and 120.97% in NH, respectively compared with those of NaCl treatment  which was associated with NT as well as NH treatments. The third category represented NaCl stress and this was not associated with any of the variables  signaling events36, maintained plant-water homeostasis37, enhanced antioxidant defense20 and improved sulphur metabolism23. Additionally, TU effectiveness has also been demonstrated under other types of abiotic stresses38, like drought, heat, UV radiation, metal stress. Similarly, H2O2 is another broad range effective PBR whose ameliorative potential has been demonstrated against various abiotic stresses39.Redox homeostasis is an essential constituent for sustained maintenance of plant growth and survival under stress conditions. The present study has evaluated widely used redox modulators like TU and H2O2  are expected to have a contrasting effect on the cellular redox state, which is described as an integrated ratio of reduced to oxidized form of all the redox couples present inside the cell. At whole plant level, the redox state is regulated by ROS-scavenging/producing enzymes and antioxidant metabolites40. Initially, the post-germination phenotyping was performed at seedling stage with variable doses of NaCl (ranging from 50 to 100 mM) and 50 mM was identified as the IC50 dose at which plant biomass is reduced by 48.15% compared with those of control . On the basis of biomass, 7.5 and 1.0 μM were identified as optimum doses of TU and H2O2 which were selected for all the medium supplementation studies  and H+ retention was also observed under both NT- and NH- treated plants  and stelar K+ outward rectifier (SKOR)47. The higher K+ retention ability of TU and H2O2 further substantiates our hypothesis that both these modulators have an overlapping capacity to generate reducing redox environment. This was also supported by TU and H2O2 dependent upregulated expression of HAK21  for foliar supplementation, which were also used previously in various other crops19. The significant reduction in PS-II efficiency under NaCl treatment was reflected by a drastic reduction in plant growth as well as yield  were equally improved; however, NPQ was identified as NH-specific parameter  is active under the reducing redox environment62. Thus, owing to the generation of reducing redox environment  and sink (developing inflorescence) strengths, respectivelyent Fig. C, the ovent Fig. A. GR is 2O2 alone treatments (Fig. 2O2 treated plants (Fig. 2O2 alone treatments (Fig. 2O2 under both control and NaCl stress conditions greatly increases their versatility to be applied under the realistic field scenario.A significant enhancement in growth and yield was also noticed under TU and Hnts Fig. ; Table 1nts Fig. , the hignts Fig. . Furthernts Fig. . Thus, t2O2 impart comparable level of NaCl stress tolerance in rice to a comparable extent. Both TU and H2O2 upregulated the expression of NaCl stress responsive genes in a constitutive manner, without showing any significant growth reduction under control conditions. In addition, reducing redox status was maintained along with better K+ retention and upregulated expression of NaCl stress tolerant genes like HAK21, LEA1, dehydrin, TSPO and EN10 under both NT and NH treated seedlings, resulting in improved growth. Under both control and NaCl stress conditions, foliar-supplemented TU and H2O2 improved the growth and yield attributes of the plants. The ameliorated phenotype under NT and NH was associated with reduced Na+ accumulation, improved photosynthesis efficiency and better source-sink relationship. Taken together, the results of this study suggest that the maintenance of reduced redox status acts as a “central” regulator for mitigating NaCl-induced damages. Besides, it also extends the concept of using redox modulators for improving rice crop productivity under the realistic field scenario.In conclusion, the study highlights that despite having contrasting redox chemistry, both TU and HSupplementary Information 1.Supplementary Information 2.


The obtained materials were utilized as nano-adsorbents for the elimination of methylene blue (MB), a hazardous water-soluble cationic dye, from aqueous solutions, and showed excellent regeneration capacity. The formation of the nanocomposites was followed by high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectrometry (EDX), Fourier-transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM), X-ray diffraction (XRD) and adsorption of N2 at −196 °C. The rate of adsorption was investigated varying several factors, namely contact time, pH, amount of adsorbent and MB concentration on the adsorption process. Studies dealing with equilibrium and kinetics were carried out in batch conditions. The obtained results indicated that the removal rate of MB was 99.53% in 90 min. Langmuir’s isotherm fitted better to the equilibrium data of MB. Fe3O4/AC/CD/Alg polymer beads shows amazing adsorption capacities in the elimination of cationic dyes (2.079 mg/g for polymer gel beads and 10.63 mg g−1 for dry powder beads), in comparison to other adsorbent materials. The obtained adsorbent is spherical with hydrophobic cross-linked surface properties that enable an easy recovery without any significant weight loss of in the adsorbent used.New magnetic iron oxide (Fe Balkizetal. [Ceibapentandra seeds. Natural polymeric materials are presently gaining more attraction, being used as adsorbents in wastewater treatment, as they were found to be non-toxic and biodegradable [Several contaminants with organic and inorganic nature end up often in water sources. Some of them are dyes, which have harmful effects on the environment, animals and humans. Even when present in small amounts, dye compounds discharged into water sources can affect the aquatic life. Synthetic dyes are often utilized for different purposes, like food coloring, textiles, paper, leather, cosmetics, medicines, inks for printing, paints, lacquers, soaps and lubricants . Therefokizetal.  used quakizetal.  used a pkizetal.  removed kizetal.  used comkizetal.  used phygradable . Activatgradable .β-Cyclodextrin (often named as β-CD) is an oligosaccharide with cyclic structure, containing seven glucopyranose units connected by α- linkages. β-CD has an inner cavity with hydrophobic nature, while the exterior is hydrophilic . Such stThe β-cyclodextrin can form complexes with several molecules, like aromatic compounds by means of intermolecular weak forces. Particularly, β-CD based polymers are quite effective in the elimination of reactive and disperse dyes from water solutions . Hence, 2+, Ca2+ and Fe3+) and is easily adsorbs heavy metals [Functionalized polymer nanocomposites attracted considerable interest due to their physical and chemical features, availability, easy separation and different reactive groups on the backbone chain ,31. Alg y metals , dyes [3y metals  and antiy metals ,35. At ty metals .In recent years, the polymer nanocomposites have been acquiring a great interest due to the not so high costs of production and good removal efficiency of some dyes ,38. Hydr3O4)/activated charcoal (AC)/β-cyclodextrin (CD)/sodium alginate (Alg) polymer composite gel beads. The sodium alginate-based material has high mechanical properties and high MB adsorption capacity, excellent regeneration ability and good separation properties [The present work aims to continue to explore the advantages of both polymeric and nanoparticle-based materials. In particular, our aim is to synthesize and characterize magnetic n , FeCl3·6H2O (ferric chloride), FeCl2·4H2O (ferrous chloride), CaCl2 , NH3 (ammonia) solution, NaOH (sodium hydroxide) and HCl (hydrochloric acid) were obtained from Merck . In the experiment, 0.1 M solutions of NaOH and HCl were used for pH adjustment. Activated charcoal, β-cyclodextrin, MB and ethanol were received from HIMEDIA . The structure of MB is illustrated in , dropwise, with strong stirring. A dark precipitate was immediately obtained, which was heated to 80 °C for 30 min. The formed black nanoparticles were removed with the help of an external magnetic field and three times washed with double distilled water and ethanol, heated at 250 °C in air for 2 h. The finely prepared 1.0 g Fe3O4 nanoparticles were dispersed in 10 mL double distilled water and then 1.0 g AC was added to the solution (1:1) and continuously stirred for 2 h. The liquid was removed from the resulting black slurry by decantation and filtration, and the solid was washed with double distilled water several times. The obtained precipitate was dried in an oven at 90 °C for 12 h and 500 °C for 2 h in a muffle furnace.Feterature , with sl3O4-AC nanoparticles were mixed with 10 mL double distilled water to obtain a colloidal solution that was mixed with 1.0 g β-CD under intense stirring at 40 °C. The formed nanoparticles were collected by applying an external magnetic field. The particles were washed with double distilled water several times and dried in an oven at 90 °C for 24 h.The obtained 1.0 g Fe3O4/AC/CD nanoparticles were well mixed with the solution. 2% CaCl2 solution was added to the gel beads with a syringe with non-stop stirring (300 rpm) and stored overnight to complete gelation and form stable beads. The latter were repeatedly washed with double distilled water and kept in an aqueous medium for later use. A scheme outlining all the steps involved in the synthesis of Fe3O4/AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg nanocomposites, from preparation to analysis, is shown in 2.0 g of powder sodium alginate were dissolved into 75 mL double distilled water with strong stirring until a viscous solution was obtained. Then 1.5 g of Fe2 adsorption and desorption analysis of Fe3O4/AC/CD/Alg polymer composite gel beads were carried out at −196 °C in a BELSORP-miniII, BEL, Japan apparatus. The samples were heated at 120 °C for 3 h in N2 atmosphere, to eliminate any water or gas adsorbed on the surface, on a pretreatment unit . The surface area was determined by the Brunauer–Emmett–Teller (BET) method with data from the N2 adsorption and desorption isotherms. The total pore volume (totV) was determined from the quantity of N2 adsorbed at a P/P0 = 0.99. The pore size distribution was obtained by the Barrett–Joyner–Halenda (BJH) method. The prepared Fe3O4-AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg polymer composite gel beads were characterized by X-ray diffraction (XRD) using a Philips PW3064/60 . Morphology was studied on a JEOL  analytical scanning electron microscope (SEM). A Fourier transform infrared spectrometer  was utilized to record the infrared spectra of Fe3O4-AC, Fe3O4/(AC)/β-(CD) and Fe3O4/AC/CD/Alg polymer composite gel beads in the range of 400–4000 cm−1, with KBr pellets. The microstructure of the prepared nanocomposite beads was analyzed by energy dispersive X-ray spectrometry . The magnetic properties were recorded for the Fe3O4/AC/CD/Alg polymer composite gel beads with a vibrating sample magnetometer . High resolution transmission electron microscopy (HR-TEM) images were obtained in a JEOL .N3O4/AC/CD/Alg polymer composite gel beads. A standard MB stock solution was used and diluted with deionized water in order to obtain different concentrations. The obtained MB solutions was kept in a flask with fixed volume (10 mL of 5 ppm) and the Fe3O4/AC/CD/Alg polymer gel beads were added (in different amounts) to the MB solutions. The flask was placed in a shaker (150 rpm) for 90 min, at pH 6, in an orbital incubator shaker, at room temperatures. The beads were removed with the help of an external magnetic field, when the experiment was over. The upper layer liquid was analyzed by UV-Vis , at a wavelength of 664 nm, after a certain time. The synthesis of Fe3O4/AC/CD/Alg polymer composite gel beads and image of gel beads being attracted by the external magnetic field are shown in R, %) was calculated from Equation (1) and adsorption capacity  from Equation (2), as follows:R, tq (kinetic adsorption capacity of MB dye) were calculated by Equations (1)–(3), respectively [oC and eC are the initial and equilibrium concentrations of MB (mg L−1), respectively, Ct is the concentration of MB at a given time t, m is the adsorbent mass (g) and V is the dye solution volume (mL). The equilibrium adsorptions of MB dye from aqueous solutions were investigated using the Langmuir and Freundlich models for isotherms. Furthermore, the kinetics of MB dye adsorption was examined using pseudo-first order and pseudo-second order kinetics. The Fe3O4/AC/CD/Alg beads were desorbed using 0.1 mol L−1HCl solutions after adsorption of MB until saturation. The flask containing 0.1 mol L−1 HCl and the saturated Fe3O4/AC/CD/Alg beads was placed in an orbital incubator shaker at room temperature, for 1 h, to full desorption. Subsequently, beads were filtered and three times washed until neutrality was reached before the following adsorption experiment.The MB adsorption experiments were conducted in batch conditions using the Feectively , where C3O4/AC/CD/Alg polymer gel beads (0.2 g) were placed in a conical flask containing MB solution  and the mixture was shaken in an orbital incubator shaker for 90 min at room temperature. The mixture was separated with a magnet and the final dye concentration was found. The Fe3O4/AC/CD/Alg polymer gel beads were recycled by washing with 0.1 N HCl and water under constant stirring for three times, respectively. Then the gel beads were added into another MB solution in order to begin a new adsorption. The recyclability of the material was followed up to five consecutive adsorption–desorption cycles.Fe3O4/AC/CD/Alg in more detail, and the results were shown in 3O4/AC/CD/Alg is a highly mesoporous material with particle sizes around 20 nm, and grain sizes around 2–15 nm. This decrease in size is mainly due to β-CD [High resolution Transmission Electron Microscopy (HR-TEM) was used to investigate the Fe to β-CD .3O4/AC/CD/Alg polymer beads before (A) and after adsorption (B), indicating that the surface of Fe3O4/AC/CD/Alg polymer beads had rough porous structure and large surface area. A 1000× magnification was used. 3O4/AC/CD/Alg showed irregular structure and large surface area, which improved the material adsorption capacity. The elemental analysis of the Fe3O4/AC/CD/Alg polymer beads are shown in 3O4/AC (A), Fe3O4/AC/CD (B) and Fe3O4/AC/CD/Alg (C) polymer nanocomposite beads, showing the presence of magnetic Fe3O4 particles, which can be utilized for magnetic separation D= nanocomposite were 11.04, 2.71 and 5.63 nm, respectively, as shown in In the present case, the mean crystallite size of the Fe3O4-AC, Fe3O4/AC/CD and Fe3O4/AC/CD/Alg polymer composite beads were analyzed by FTIR and results are in 3O4-AC, the bands at 3562.77, 3392.93, 3060.08, 2953.49, 1540.31, 1126.89, 632.92 and 563.79 cm−1 were observed in −1 were ascribed to Fe–O vibration modes, just like for Fe3O4/AC/CD and Fe3O4/AC/CD/Alg. The bands at 3562.77 and 3392.93 cm−1 were due to –OH and –NH2 stretching vibrations. The band at 3060.08 cm−1 was from the (=C–H) stretching vibration. The band at 2953.49 cm−1 was assigned to the antisymmetric stretching vibrations of the methylene group. The peaks for the C–N stretching vibration were found at 1126.89 cm−1. For Fe3O4/AC/CD, bands at 3415.26, 3021.36, 1713.47, 1563.97, 1140.92, 1032.28, 633.92 and 561.74 cm−1 were observed  stretching vibration. The peak at 1713.47 cm−1 was related with the stretching vibrations of the –C=O carbonyl groups and 1563.97 cm−1 was from the N–H bending of –NH2. Peaks at 1140.92, and 1032.28 cm−1  related to the glycosidic (C–O–C) antisymmetric vibrations and ʋ(C–C/C–O) coupled stretching vibration. Therefore, it could be concluded that Fe3O4/AC/CD was present [3O4/AC/CD/Alg (−1 were found. The bands at 570.97 cm−1 (characteristic peak of Fe3O4) and at 3427.52, 2923.88, 2856.61, 1625.96, 1411.60 and 1033.27 cm−1  were ascribed to –OH (stretching vibration), –CH2 (stretching) from CH3,–COO−(asymmetric stretching), –CH2 (bending) and C–O–C (stretching) vibrations [−1 were related to the carboxylic groups –C–O stretching vibrations. The absorption bands at 1320.90, 1247.16 and 808.28 cm−1 could be ascribed to the C–OH stretching vibrations, C–O–C stretching vibrations and C–H bending vibrations, respectively.The adsorption capacity of an adsorbent is related with its molecular structure and functional groups. For the identification of the functional groups, the Feobserved B. The ba polymer . The ban present . For Fe3C/CD/Alg C, bands brations , respect3O4/AC/CD/Alg nanocomposite, the magnetic-hysteresis (MH) loops were recorded at room temperature. Results can be found in 3O4/AC/CD/Alg nanocomposite exhibited hysteresis, which confirmed the usual ferromagnetic behavior of nanoparticles with negligible coercivities and moderate saturation magnetization. From MH loops, the saturation magnetization value (MS) of Fe3O4/AC/CD/Alg was found to be 0.12849 emu g−1. Since the composite was composed of a mixture of three non-magnetic materials, viz. activated charcoal, β-(CD) and sodium alginate, the MS obtained for Fe3O4/AC/CD/Alg had a lower value. The results were in conformity with those reported for the β-(CD) coated Fe3O4/carbon nanocomposite in literature, for which the MS was 1.2 emu g−1 [To determine the magnetic properties of the obtained Fe emu g−1 . The ads2 adsorption and desorption isotherms for Fe3O4/AC/CD/Alg at −196 °C. The Brunauer–Emmett–Teller (BET) analysis was utilized for determining the surface area of the material. The BET specific surface area of was 8 m2 g−1, and 3 g−1 with a mean pore diameter of 1.47 nm. This shows that the surface area of the material is larger than of other adsorbents [3O4/AC/CD/Alg nanocomposite are shown in sorbents . The tex3O4/AC/CD/Alg polymer beads = 0.2 g/10 mL dye solution, agitation speed = 150 rpm, contact time = 90 min, room temperature). These dye sorption behaviors could be explained the following way: at low pH, the lower dye adsorption onto Fe3O4/AC/CD/Alg can be linked to the large amount of H+ ions present, which compete with the dye positive groups for the adsorption sites of the beads. With the increase of the pH of the solution (above 3), the active sites of the surface of the Fe3O4/AC/CD/Alg onto the hybrid beads were deprotonated and the competition for the adsorption sites between the dye cations and H+ decreased, which increased the amount of dye adsorbed [3O4/AC/CD/Alg polymer beads and the positively charged dye ions. Similar results were observed for contact times of 60 min  and dry powder beads (0.06 g) were agitated for 90 min. The obtained results are shown in The adsorption capacity was determined as a function of time to evaluate the maximum time of contact during the adsorption process. −1, pH 6 and contact time of 90 min. As shown in To determine the effect of temperature on adsorption, tests were carried out at four different temperatures, i.e., 35 °C, 45 °C, 55 °C and 65 °C, at an initial dye concentration of 5 mg L3O4/AC/CD/Alg polymer gel beads and dry powder beads for removal of MB. The Langmuir model is applicable for monolayer adsorption and this isotherm assumes that the adsorbent surface contains active sites with uniform monolayer formation energy. The linearized Langmuir isotherm model is represented as:LK (L/mg) is the Langmuir adsorption constant, −1) is the equilibrium concentration and Adsorption isotherm models have been often used to explain the adsorption processes and mechanisms. They describe the interaction between adsorbate and adsorbent, under propitious conditions. The obtained equilibrium data were fitted to several theoretical models, as Langmuir  and FreuLR can be calculated by the equation below:−1) is the initial dye concentration. The LR = 1) or unfavorable (The main characteristics of the Langmuir adsorption can be expressed in terms of a constant (that is dimensionless), called separation factor or equilibrium parameter, used to predict if an adsorption system would be favorable or unfavorable. FK (mg g−1) is the adsorption capacity and n is an empirical parameter that varies with the intensity and heterogeneity of the adsorption. The values of 1/n and FK are calculated from the intercept and slope of the log logThe multilayer adsorption heterogeneous surface energy is described by the Freundlich isotherm that can be linearized as:The adsorption parameters derived from the isotherm models are in 2 = 0.992) model showed a better fitting than the Freundlich model (R2 = 0.971), proving the surface homogeneity of the adsorbent and showing that the monolayer adsorption capacity  is the rate constant, determined from the slope. t plot.The effect of contact time on MB removal by Fek2 (g/mg min−1) is the rate constant. The values of t/t shown in 3O4/AC/CD/Alg polymer beads.The pseudo second order kinetic model has this equation:The pseudo first and second order rate constants and correlation coefficients are listed in The high correlation coefficient obtained for pseudo second order kinetics indicated that the model nicely represented the experimental data.H°), entropy (ΔS°) and Gibbs free energy (ΔG°) change were calculated by the Equations (10)–(12), respectively [−1) is the distribution coefficient, ΔG° (kJ mol−1), ΔH°(kJ mol−1) and ΔS°(kJ mol−1) are the changes in Gibbs free energy, enthalpy and entropy, respectively, T is the temperature (in K). The adsorption capacity of the Fe3O4/AC/CD/Alg polymer composite beads increased with a temperature increase from 35 to 55 °C. Van’t Hoff Equation (11) related the distribution coefficient with S° at constant temperature (1/T). The values of entropy and enthalpy were calculated from the intercepts and slopes of T linear regression. ΔH° had a positive value suggesting the reaction to be endothermic, favoring the adsorption of the MB at higher temperature. The positive value of ΔS° indicated high randomness of adsorption, favoring the constancy of adsorption. The negative values of (ΔG°) confirmed the viability and spontaneity of the adsorption. The adsorption thermodynamic parameters, like enthalpy (Δectively .(10)ΔG°The reusability was tested up to five consecutive adsorption-desorption cycles and results are shown in 3O4/AC/CD/Alg polymer beads was considerable, compared to other biological adsorbents found in literature  or inorganic  layer.In this work, novel FeThe effects of several parameters on the adsorption process  were discussed. The versatility in surface modifications of magnetic nanoparticles offers advantages for diverse surface modifications. They could be easily functionalized using many different kinds of modification agents, including organic acids and polymers, based on their potential applications.Magnetic separation shows advantages, like easy separation from solutions and possibility to treat a large amount of wastewater within a short time. Moreover, enhanced performances were achieved, due to cooperative interactions between the constituents.Furthermore, a MB removal percentage of 99.53% at pH 6 was found, and the adsorbent could easily be regenerated and recycled, being reused up to five adsorption–desorption cycles, without any significant weight loss. The use of dry powder beads considerably enhanced the adsorption of MB, compared to gel beads, in terms of time and dosage.3O4/AC/CD/Alg polymer nanocomposite is a good adsorbent for large scale wastewater purification since it has high adsorption capacity and easy separation.In conclusion, this work opened a new path for effective elimination of cationic dyes from wastewater using an environmentally friendly biodegradable adsorbent. The Fe


In deionized water, the coating with the lowest corrosion current density (corri) of 7.865 × 10−8 A/cm2 was hard to erode. Meanwhile, it presented a lower friction coefficient and the lowest wear rate. In HCl solution, NiCrAlY coating gave the highest corrosion current density (corri) of 3.356 × 10−6 A/cm2 and a higher wear rate of 6.36 × 10−6 mm3/Nm. Meanwhile, the emergence of Al(OH)3 on the coating surface could reduce the direct contact between the counter ball and sample effectively, which was conducive to the lowest friction coefficient of 0.24.Atmospheric plasma spraying (APS) was taken to fabricate the NiCrAlY coating. The corrosion-wear properties of NiCrAlY coating was measured respectively under deionized water, artificial seawater, NaOH solution and HCl solution. Experimental results presented that the as-sprayed NiCrAlY coating consisted of Ni The results presented that the as-sprayed NiCrAlY/Al2O3 coating has the best cyclic oxidation resistance with an oxidation rate of 2.62 × 10−12 g2·cm−4·s−1 at high temperature and good adhesion during the cyclic oxidization treatment [In engineering fields, wear often occurs under different corrosive circumstances leading to the degradation rate of engineering parts . For insreatment . Currentreatment ,20,21,22reatment , so the reatment , and itsIn this work, the tribocorrosion properties of NiCrAlY coating were studied and the synergistic mechanisms between wear and corrosion in different corrosive environments were discussed in detail. The objective of this paper is to research how corrosive environments affect the tribological behavior of NiCrAlY coating and the interaction degree between corrosion and wear. This research would provide usable direction to the NiCrAlY coating application in corrosive environments.22Cr10Al1.0Y (wt.%) powder (53–106 µm) was bought from Sulzer Metco . The NiCrAlY coating was prepared by atmospheric plasma spraying (APS). The Inconel 718 alloy was sand-blasted, then ultrasonically cleaned with ethanol before spraying. The coating thickness was about 300 µm. The specific spraying parameters presented were: flow rate of Ar was 40 L/min; flow rate of H2 was 5 L/min; spraying angle was 90°; feed rate of the powder was 42 g/min; voltage was 60 V; the current was 500 A and spray distance was 110 mm.Gas atomized spherical Nia radiation, current 150 mA, potential 40 kV, Philips, Eindhoven, The Netherlands) was utilized to analyzed phase composition. The phase compositions on the worn surface were analyzed by Czemy-Tumer Labram HR800 Raman spectrometer .The micromorphologies of cross-section and worn surface of this coating were measured by field emission scanning electron microscopy . A Philips X’Pert-MRD X-ray diffractometer  and SEM were utilized to analyze the worn surface. The wear rate was got by W = V/LF, where W represented the wear rate (mm3/Nm), V represented the wear volume loss (mm3), L represented the sliding distance (m) and F represented the load (N).The tribocorrosion experiments were tested in deionized water (pH = 7), artificial seawater (pH = 8.2), 0.1 M NaOH solution (pH = 13) and 0.1 M HCl solution (pH = 1), with reciprocating ball-on-disk tribometer . The schematic diagram is shown in 3Al, NiAl and nickel-based solid solution and has high crystallinity , corrosion current density (corri), anodic and cathodic Tafel slopes (aβ and cβ) are obtained from pR) is calculated by Stern–Geary equation:corrE) of NiCrAlY coating under deionized water is the highest of −0.428 V (vs. SCE). However, the corrE of the coating in artificial seawater, HCl and NaOH shift to −0.516 V (vs. SCE), −0.559 V (vs. SCE) and −0.535 V (vs. SCE) respectively. Simultaneously, the corrosion current density (icorr) of this coating in deionized water shows the lowest of 7.865 × 10−8 A/cm2. Generally speaking, corrosion current density, whose rate is often used as corrosion rate, is a crucial reference to evaluate corrosion resistance [aβ, cβ and pR of 0.072 V/dec, 0.049 V/dec and 1.610 × 105 Ω respectively, which further illustrates that the coating in deionized water holds a good corrosion resistance.Results indicate that the corrosion potential 3 after sliding in HCl corrosive solution [To further research the influence of corrosive solution upon the corrosion-wear property of NiCrAlY coating, Raman analysis is tested. solution . The ressolution . The cor3 formation:3 is related to local pH changes in the hydrogen reduction region. Hence, the Al(OH)3 could be formed where the hydrogen evolution occurs. Furthermore, when the local pH rises to above 9, Al3+ ions will react with excessive OH− ions and forms aluminate anions [3 reaction :(8)2Al+22O3, which is well consistent with the micro-Raman results solution . Therefo(1)3Al, nickel-based solid solution, NiAl and Y2O3.The NiCrAlY coating is composed of Ni(2)−8 A/cm2 is hard to erode. Meanwhile, it presents a lower friction coefficient and the lowest wear rate.In deionized water, the NiCrAlY coating with the lowest corrosion current density of 7.865 × 10(3)corri) of 3.356 × 10−6 A/cm2 and a higher wear rate of 6.36 × 10−6 mm3/Nm.In HCl corrosive solution, the coating gives the highest corrosion current density ((4)3 on the coating surface could reduce the direct contact between the counter ball and sample effectively, which is conducive to the lowest friction coefficient of 0.24.In HCl corrosive solution, the emergence of Al(OH)In this work, the corrosion-wear properties of NiCrAlY coating were studied under deionized water, artificial seawater, 0.1 M HCl solution and 0.1 M NaOH. The main conclusions are given as follows:


Zinc oxide (ZnO) quantum dot (QD) is a promising inexpensive inorganic nanomaterials, of which potential toxic effects on biological systems and human health should be evaluated before biomedical application. In this study, the cytotoxicity of ZnO QDs was assessed using HeLa cervical cancer cell and HEK-293T human embryonic kidney cell lines. Cell viability was significantly decreased by treatment with 50 µg/ml ZnO QDs after only 6 h, and the cytotoxicity of ZnO QDs was higher in HEK-293T than in HeLa cells. ZnO QDs increased the level of reactive oxygen species and decreased the mitochondria membrane potential in a dose-dependent manner. Several gene expression involved in apoptosis was regulated by ZnO QDs, including bcl-2 gene and caspase. In HeLa cells, ZnO QDs significantly increased early and late apoptosis, but only late apoptosis was affected in HEK-293T cells. These findings will be helpful for future research and application of ZnO QDs in biomedicine. ZnO NPs could protect the cells against the ultraviolet (UV)-induced skin damage, and have been applied to cosmetics . ZnO NPsequences .Quantum dots (QDs) with a diameter of 1–10 nm, have unique optical properties, such as broad excitation, narrow emission spectra, and long fluorescence lifetimes . These aOur previous studies have demonstrated that ZnO QDs are located in the mitochondria, disrupt the activity of anti-oxidative enzymes, and induce oxidative stress after intravenous injection in mice . In thisFetal bovine serum was purchased from Hyclone . Dimethyl sulfoxide was purchased from Sigma–Aldrich . Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and trypsin were purchased from Corning . TRIzol reagent was purchased from Invitrogen . Cell Counting Kit-8 (CCK-8), ROS color reagent 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide (JC-1) were purchased from Beyotime . Deionized water was prepared from Millipore . Other reagents used were of analytical grade.2 in ethanol with stirring, and stirred for another 1 h. The obtained ZnO QDs was centrifuged and washed with absolute ethanol three times, then dried in vacuum. The characterization of ZnO QDs was described in The synthesis of ZnO QDs were as described previously . The rea2-controllable incubator  with 5% CO2. All the cell experiments were performed in a clean atmosphere.HeLa and HEK-293T cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin–streptomycin. The cell lines were maintained at 37°C in a humidity- and CO3 cells per well), and incubated at 37°C for 24 h. ZnO QDs in DMEM were added at final concentrations of 25, 50, 100, 200, and 400 µg/ml. At the end of the fixed incubation period , cells were washed with PBS to remove excess QDs. We added 200 µl fresh medium and 20 µl CCK-8 reagent. After 2 h incubation at 37°C, absorbance at 450 nm was recorded using a microplate reader. All experiments were performed in triplicate.Cell viability was determined by the CCK-8 assay. HeLa and HEK-293T cells were seeded into 96-well plates . Primers were designed following previous reports . Total RNA (1 μg) was reverse transcribed to synthesize complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit . Then, the messenger RNA (mRNA) expression levels of apoptotic markers including  reports , and the6 cells/ml. We added fluorescein isothiocyanate–annexin V and propidium iodide, and incubated the cells in the dark for 15 min at room temperature. Apoptotic rate was analyzed using flow cytometry.An Annexin V Apoptosis Detection Kit  was utilized to measure apoptosis of HeLa and HEK-293T cells. After incubation with control and 50 µg/ml ZnO QDs for 24 h, cells were washed, trypsinized, and resuspended in the binding buffer at a concentration of 10p < 0.05 was considered statistically significant.All the data are presented as mean ± SEM. Multiple group comparisons of the means were evaluated by one-way analysis of variance (ANOVA) followed Dunnett's test using SPSS version 16.0. The unpaired t-test was used to test the difference between the two groups. The average diameter of ZnO QDs was approximately 7.10 ± 0.30 nm according to transmission electron microscopy  and 0.76 ± 0.01  after incubation with ZnO QDs for 24 h (p < 0.001) and 0.10 ± 0.01  in HEK-293T cells  to 0.70 ± 0.02  for 6, 24, 48, and 72 h. The cytotoxicity was measured by CCK-8 assay. Cell viability was significantly reduced by 50 µg/ml ZnO-QDs after only 6 h treatment. This is contradicted with the result of Sudhagar et al. reports, that ZnO QDs were biocompatible and non-toxic even at 100 µg/ml for up to 48 h in MDA-MB-231 breast cancer cells . In our The release of ROS has been reported in cells exposed to ZnO nanoparticles, and involved in their toxic effects . Marfavivia the intrinsic pathway. Upon quantification by ﬂow cytometry, the total apoptosis ratio was significantly increased after exposure to 50 µg/ml ZnO QDs as compared to the control group. Persaud and Kim et al. reported that ZnO NPs induced significantly late apoptosis at the concentrations of 20 µg/ml , the National Natural Science Foundation of China , the Key Scientific Research Program for Universities of Henan Province (Grant No. 17A330001), the Key Science and Technology Program of Kaifeng City in China , and the Research Program for Young Talent of Henan University School of Medicine (Grant No. 2019018).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.