Lophophora williamsii (LW) extract can pass through the BBB and have antitumor properties. The aim of this study is to evaluate the cytotoxic potential of TMZ, PAN and LW extract against the glioma C6 cell line, and its role in the release of damage-associated molecular patterns (DAMPs), which is a hallmark of ICD. Our results indicate that all treatments induce cellular death in a time- and concentration-dependent manner, and that PAN and LW extract induce apoptosis, whereas TMZ induces apoptosis and necrosis. Also, that some of the treatments and their sequential administration induce the release of DAMPs. Furthermore, in a rat glioma model, we observed that all treatments decreased tumor volume, but the in vivo cell death mechanism was not ICD. Our findings indicate that TMZ, PAN, and LW combination have a cytotoxic effect against glioma cells but do not induce ICD. Glioblastoma multiforme is a malignant neoplasm of the brain with poor prognosis. The first-line drug against glioblastoma is the alkylating agent temozolamide (TMZ); unfortunately, treatment resistance and tumor re-incidence are common. In some cases, immunogenic cell death (ICD) inducers can decrease treatment resistance and tumor recurrence by stimulating an antitumor specific immune response. Not all ICD inducers, however, are suitable for glioma patients because of the low permeability of the blood-brain barrier (BBB). Panobinostat (PAN), a histone deacetylase inhibitor and  TMZ temozolamidePAN panobinostatLWLophophora williamsiiICD immunogenic cell deathBBB blood-brain barrierDAMPs damage-associated molecular patternsGlioblastoma multiforme (GBM) is malignant neoplasia of the brain that originates in the glial cells within the intracranial tissue. GBM infiltrates the surrounding tissue and decreases neurological function, resulting in a poor quality of life for the patients . CurrenThe alkylating agent temozolamide (TMZ) is a first-line drug for the treatment of glioblastoma; it acts by damaging tumor cells through DNA methylation, changing heterochromatin organization, and activating an oxidative stress response. However, tumor cells quickly become resistant to TMZ at pharmacotherapeutic concentrations .Some therapeutic drugs can induce immunogenic cell death (ICD) in cancer cells. ICD originates with endoplasmic reticulum stress in the target cell that results in an elevated production of reactive oxygen species and the release of damage-associated molecular patterns (DAMPs). The release of DAMPs includes the exposure of calreticulin (CRT) and the extracellular release of ATP, high-mobility group box 1 protein (HMGB1), and heat shock proteins (HSP70 and HSP90) , an epigenetic modulator that inhibits histone deacetylase activity and increases DNA-histone acetylation. This blocks multiple signals related to the development and progress of tumors, and induces apoptosis in the target cells  also known as “peyote”, is a spineless cactus known for the physical, visual, and perceptual changes it induces upon ingestion . LW extal., 2019). LW extract on a rat glioma model, and their capacity to induce ICD. The use of multiple treatments for cancer has been of great importance to improve therapeutic outcome. The present study was designed to determine the antitumor effect of TMZ, PAN, and Lophophora williamsii (LW) was obtained from a plant collection of the Laboratorio de Inmunología y Virología, Facultad de Ciencias Biológicas, UANL. IgG1 mouse HSP70 antibody (sc-32239), IgG1 mouse HMGB1 antibody (sc-56698), IgG2a mouse HSP90α/β (sc-13119), and IgGκ mouse HRP antibody (sc-516102) were purchased from Santa Cruz Biotechnology . Dulbecco´s Modified Eagle Medium (DMEM) was purchased from Sigma-Aldrich .Temozolomide (TMZ) was purchased from Schering-Plough , panobinostat (PAN) was purchased from Cellagen Technology , and the methanolic extract of Lophophora williamsii. The cacti were macerated and methanol extraction was performed for 48 h at 4 °C, after which the ethanolic phase was filtered. The methanol extract was lyophilized using a freeze-dryer . The resulting powder was dissolved in 1 mL of DMEM and the endotoxin levels were measured with the gel clot-based Limulus amoebocyte assay , which has a detection limit of 0.004 ng/mL.The cacti used in this study belonged to a plant collection of the Laboratorio de Inmunología y Virología, Facultad de Ciencias Biológicas, UANL, and had been previously identified as 2 atmosphere at 37 °C.The C6 murine brain glial cell line was purchased from the American Type Culture Collection . Cells were cultured in DMEM supplemented with bovine fetal serum (10 % v/v) in a 5 % CO3) were seeded into 96-well plates and cultured overnight in a 5 % CO2 atmosphere at 37 °C. Then, cells were treated with TMZ (2.15 - 43 mM), and LW extract (1.44 - 4.8 mg/mL) for 24 and 48 h, and with PAN (0.5 - 25 µM) for 24, 48 and 72 h. Also, the TMZ + LW extract + PAN treatment combination was evaluated using the IC30, and IC50 for 24 h. After the treatment, cells were washed with phosphate-buffered saline (PBS) 1X, and cellular metabolic activity was assessed using the resazurin assay. Cells were incubated with a resazurin solution (20 % v/v) for 30 min in a 5 % CO2 atmosphere at 37 °C and the fluorescence was measured using a microplate reader Synergy TM HT  at 535/590 nm, excitation/emission wavelength, respectively. The percentage of cell viability (%) was calculated according to the following equation [1]: C6 cells (5x10Relative viability (%) = treated cell fluorescence ÷ control cell fluorescence x 100 [1]3) were seeded into 96-well plates and cultured overnight in a 5 % CO2 atmosphere at 37 °C. Then cells were pre-exposed to TMZ (2.15 - 43 mM) for 24 h, followed by the treatments with LW extract  and PAN  for 24 h; pre-exposure to PAN (0.5 - 25 µM) for 24 h, followed by the treatments with LW extract  and TMZ  for 24; and pre-exposure to LW extract (1.44 - 4.8 mg/mL) for 24 h, followed by PAN  and TMZ  treatments for 24 h. Finally, cells were washed with PBS 1X and cell metabolic activity was assessed using the resazurin assay as previously described. C6 cells (5x103) were seeded into 96-well plates and cultured overnight in a 5 % CO2 atmosphere at 37 °C. After this, cells were treated with TMZ (2.15 - 43 mM), and LW extract (1.94 - 4.8 mg/mL) for 24 h, and with PAN (0.5 - 25 µM) for 24 and 72 h. The treatments were removed, and cells were washed twice with PBS 1X. Then DMEM supplemented with bovine fetal serum (10 % v/v) was added and cells were maintained at a 5 % CO2 and 37 °C atmosphere for 5 days. Finally, cells were washed with PBS 1X and cell metabolic activity was assessed using the resazurin assay. C6 cells (5x105) were seeded in 6 well plates and treated with TMZ (IC50 = 8.6 and IC100 = 43 mM), and LW extract (IC50 = 1.92 and IC100 = 4.8 mg/mL) for 24 h, and PAN (IC50 = 0.75 and IC100 = 25 µM) for 72 h. Also, cells pre-exposed to TMZ (8.6 mM) for 24 h followed by LW extract (1.92 mg/mL) and PAN (0.75 µM) 24 h treatments, were included. Briefly, cells were washed with PBS 1X and stained with 20 µL of AO/EB dye mix . Then wells were observed under a confocal fluorescence microscope (Olympus X70), at 250/605 nm  for EB and 502/525 nm  for AO. Viable cells were identified by bright green fluorescence, apoptotic cells by bright orange fluorescence, and necrotic cells by bright red fluorescence.Acridine orange/ethidium bromide (AO/EB) staining was used to determine viable and nonviable cells, based on disrupted cell membrane. Cells  were treated with the IC100 of TMZ (43 mM), LW extract (4.8 mg/mL), PAN (25 µM), or pre-exposed to TMZ (43 mM) for 24 h, followed by LW extract (4.8 mg/mL) and PAN (25 µM) treatments for 24 h. After the treatments, the cells were collected and centrifuged at 1,200 rpm for 10 min at room temperature. Then cells were washed with PBS 1X and homogenized using the SET 2X lysis buffer  supplemented with complete Halt protease inhibitor cocktail. Protein quantification was performed using the BCA kit . C6 cells  were treated with the IC100 of TMZ, LW extract, and PAN for 24 h, or a pre-exposure treatment with TMZ (43 mM) for 24 h, followed by LW extract (4.8 mg/mL) and PAN (25 µM) for 24 h. After this, the supernatants were collected and HMGB1, HSP70, and HSP90 were determined as described above.For the detection of DAMPs in the supernatant, C6 cells  of the Facultad de Ciencias Biológicas, UANL (2018-015).Data were obtained from 2- to 3-months-old female Wistar rats with a weight range of 240-260 g. The animals (n=25) were provided by the bioterium of the Facultad de Ciencias Biológicas, UANL. Rats were kept in 12 h light/dark cycles with 6 C6 cells were treated in vitro with TMZ (43 mM), LW extract (4.8 mg/mL), or PAN (25 µM) for 24 h, and a pre-exposure treatment with TMZ (8.6 mM) for 24 h, followed by LW extract (1.92 mg/mL) and PAN (0.75 µM) combination for 24 h. Next, the cells were centrifuged at 1,200 rpm for 10 min and washed twice with PBS 1X. Finally, cells were resuspended in 300 µL of PBS 1X and inoculated subcutaneously into the left flank of Wistar rats. The rats were randomly divided into five experimental groups: 1) Control group: without vaccination (n = 5), 2) TMZ group (n = 5), 3) LW extract group (n = 5), 4) PAN group (n = 5), and 5) pre-exposure treatment with TMZ followed by LW extract + PAN group (n = 5). After 7 days, rats were challenged with 5x106 viable C6 cells resuspended in 300 µL of PBS 1X via subcutaneous injection into the left flank. Tumor width and length were measured every third day for 21 days with a digital caliper and tumor volume was calculated according to the formula: V = (W^2 x L)/2, where V is tumor volume, W is tumor width and L is tumor length .The humane endpoint was used to avoid unnecessary suffering, and rats were sacrificed 21 days after inoculation.p-value ≤ 0.05. Statistical analyses were performed with the GraphPad Prism software version 6 .The experiments were performed in triplicate and statistical differences between groups were analyzed using ANOVA followed by the Tukey post hoc test. The data are presented as mean ± standard deviation (SD) and differences between groups were considered significant at a LW extract treatments significantly reduced the viability of C6 cells (p < 0.001) as compared to untreated cells in a time- and concentration-dependent manner. The IC50 value was 8.6 mM for TMZ, and 1.92 mg/mL for LW extract at 24 h  was observed after 48 and 72 h with all concentrations of PAN used  increased significantly (p < 0.0001) the cytotoxic effect of TMZ against C6 cells  and TMZ (8.6 mM) combination treatment  and TMZ (8.6 mM)  decreased cell recovery of C6 cells in a dose-dependent manner  decreased cell recovery in a time- and dose-dependent manner    difference in the HSP90 levels of LW treated cells and the control . The tumor volume was measured until day 13. The group vaccinated with the cell lysed by pre-exposition to TMZ and treated with LW extract and PAN presented the highest tumor volume, compared to the other groups (p = 0.001)  decreased C6 cell viability in a time- and concentration-dependent manner . To oural., 2019). The diLW extract treated cells were unable to proliferate even after a five-day recovery period, corroborating cell death.The recovery assay allowed us to discriminate cell death from a temporarily inactive metabolism. TMZ, PAN, and LW extract and PAN resulted in necrosis. The combination of cytotoxic drugs tends to cause antagonism, but this antagonism does not necessarily translate into clinical failure because of other properties, such as immune response modulation, which can also inhibit tumor growth . The usal., 2020; AsadzadLW extract and PAN.In this study, we did not observe a significant increase of HMGB1 and HSP90 in the supernatant of C6 cells treated with TMZ (8.6 mM) and PAN (20 µM) as compared to the control, but there was an increase of HSP70 in cells pre-exposed to TMZ, followed by in vivo. The treatments did not prevent the implantation of the C6 glioma, suggesting that immunogenic death was not the in vivo cell death mechanism. These results correlate with the recurrence of human glioma after a few months following completion of TMZ treatment , indicaal., 2019). If thiLW extract and its combinations do not induce immunogenic cell death, suggesting an explanation regarding the tumor recurrence in glioma patients after conventional treatment; furthermore, the use of LW extract should be considered for glioma treatment, alone or sequentially administrated with TMZ and PAN, but further in vivo experiments should be performed to determine its antitumor effect.In conclusion, this study contributes to the knowledge that TMZ, PAN, or “Fondo Sectorial de Investigación para la Educación”, grant A1-S-35951, CONACYT, México supported this work.We thank Alejandra Elizabeth Arreola Triana for article revision; and Universidad Autónoma de Nuevo León UANL, Facultad de Ciencias Biológicas, Laboratorio de Inmunología y Virología, P.O. Box 46 “F”, 66455 San Nicolás de los Garza, NL, México for facilities provided. The authors declare that there is no conflict of interest.


Here, we report that the reaction of fluorographene with azide anions enables the preparation of a material combining graphene-type sp2 layers with tetrahedral carbon–carbon bonds and nitrogen (pyridinic and pyrrolic) superdoping (16%). Theoretical investigations showed that the C–C bonds develop between carbon-centered radicals, which emerge in the vicinity of the nitrogen dopants. This material, with diamond-like bonds and an ultra-high mass density of 2.8 g cm−3, is an excellent host for the ions, delivering unprecedented energy densities of 200 W h L−1 at a power of 2.6 kW L−1 and 143 W h L−1 at 52 kW L−1. These findings open a route to materials whose properties may enable a transformative improvement in the performance of supercapacitor components.Supercapacitors have attracted great interest because of their fast, reversible operation and sustainability. However, their energy densities remain lower than those of batteries. In the last decade, supercapacitors with an energy content of ∼110 W h L via the radical chemistry of fluorographene, affording a symmetric supercapacitor with an energy density of 200 W h L−1 at a power of 2.6 kW L−1 and 143 W h L−1 at 52 kW L−1.A nitrogen-superdoped graphene with diamond-like interlayer bonds is obtained  Modern society relies on electricity. The demand is bound to grow due to the increasing electromobility, the number of mobile devices, and extending the networks for the internet of things. The depleting reserves of fossil-based energy resulted in efforts to support renewable resources, which are, however, intermittent in their production. These facts call for the development of electrochemical energy storage devices with improved performance, safety, eco-friendliness, and lower cost in order to contribute to the goal of the United Nations for affordable, reliable, and sustainable energy. Lithium-ion batteries have matured and currently dominate the field. Nevertheless, carbon-based supercapacitors offer independence of critical elements, alongside safety, long life-cycle, and ultrafast charging–discharging. Here, we present a nitrogen superdoped graphene material with diamond-like interlayer bonds that dramatically increases the energy content, which can be stored per volumetric unit of the electrode—the Achilles heel of contemporary supercapacitors. The electrode displays an ultrahigh mass density compared to porous carbons, keeping intact its ability to host the electrolyte ions—the energy carriers. Consequently, a supercapacitor device made from this electrode delivers energy density twice as high as that of top-rated materials and several-fold higher than commercial supercapacitor carbons, thus enhancing the performance of supercapacitor components.1.i.e. high power) and extralong cycle-life.1 Unfortunately, the energy density of the best existing supercapacitors (i.e. their ability to store charge/energy) is low. Commercial supercapacitors have cell-level specific energies (and energy densities) of 10 W h kg−1 (5–8 W h L−1),2,3 while lead–acid batteries offer 20–35 W h kg−1 (40–80 W h L−1)4 and state-of-the-art Li-ion batteries achieve ∼150 W h kg−1 (∼250 W h L−1).5,6 However, Li-ion batteries suffer from long charging times and, unlike supercapacitors, undergo irreversible processes during cycling that gradually reduce their energy density and thus their cycle-life. To exploit the benefits of supercapacitors in a broader range of applications, it will be necessary to identify electrode materials that have substantially improved energy densities combined with long life and high power. In addition, replacing metal atoms in electrode materials with non-metal and earth-abundant elements, such as carbon, would have significant environmental advantages, reducing our reliance on critical natural resources and increasing sustainability.Supercapacitors are energy storage devices with remarkable qualities including fast charging/discharging , carbon nanosheets9 (2500 m2 g−1), activated graphene10 (3100 m2 g−1), and carbon nanotubes11 (1300 m2 g−1). The specific energies of these materials range from ca. 10 to 90 W h kg−1, with the highest values being reported for activated graphene,10 carbon nanosheets,9 and nanotubes11 in ionic liquid (IL)-based electrolytes. Unfortunately, like most commercial electrodes,12 these carbon materials have very low mass densities (ca. 0.3–0.7 g cm−3).8,10 Consequently, the energy densities achieved with mesoporous carbon,8 activated graphene,10 and carbon nanosheets9  are only 22, 26, and 45 W h L−1, respectively.Due to the importance of the electrode material/electrolyte interface for charge storage,2–4,13–16 efforts have been made to increase the mass density of electrode materials. Compressing a graphene electrode increased its mass density from 0.34 to 0.75 g cm−3 and its energy density from 2610 to 48 W h L−1 .18 It was deduced that densification in the presence of the non-evaporating IL prevented the restacking of the graphene sheets and helped preserve the material's charge transport properties. Mechanical compression of a H2O2-treated reduced graphene oxide19 yielded a material with a density of 0.7 g cm−3 and a holey structure  that very effectively promoted three-dimensional ionic transport, delivering 85 W h L−1 at 1.75 kW L−1 (1 A g−1). In 2016, capillary drying was combined with a different pore-forming agent (ZnCl2), to afford a monolithic dense (0.9 g cm−3) graphene electrode14 exhibiting 60 W h L−1 at 0.4 kW L−1 (0.6 A g−1). Further attempts to increase the energy density by heteroatom tri-doping20 and densification21 were not more effective, resulting in energy densities of 40 and 65 W h L−1, respectively. Even high mass density inorganic phases such as 1T-MoS2 did not exceed 80 W h L−1 at 1.12 kW L−1 (0.5 A g−1).22 The highest energy density reported to date was obtained using electrodes consisting of interdigitated bilayers of exfoliated graphene-mediated hydrogen iodide-reduced graphene oxide (EGM-GO)23 with a mass density of ca. 1 g cm−3 and a capacitance of 203 F cm−3. These electrodes offered an energy density of 113 W h L−1 at 0.9 kW L−1 (1 A g−1) . Thus, over the last decade there have been small improvements in materials design for higher energy contents, and power densities have remained relatively low.To achieve higher energy densities, which is a key performance parameter,L−1 ref.  without −3 and an N2 sorption-based surface area of 128 m2 g−1 that can host ions even more efficiently than carbon materials with surface areas exceeding 2000 m2 g−1. GN3, which is prepared by reacting graphite fluoride with sodium azide, has tetrahedral (sp3) C–C bonds, which were identified by solid-state nuclear magnetic resonance in the same region as the C–C bonds in diamond. However, it retains a 2-D structure with a very high content of aromatic (and thus conductive) regions, together with nitrogen superdoping in the vacancies and holes of the aromatic lattice. The ultrahigh mass density of GN3 combined with its polar nitrogen moieties and vacancies facilitated an energy density of 200 W h L−1 at a power density of 2.6 kW L−1, corresponding to improvements of 74% and 190%, respectively, over the previous record.23Here we report a carbon-based electrode material, GN3, with an unprecedented density of 2.8 g cm2.24,25 in the synthesis of sp3-rich carbon materials,26–28 and by the high density of such materials,29 we hypothesized that fluorographene chemistry could produce carbon derivatives with high mass densities. This hypothesis was strengthened by the fact that in (C2F)n, whereby fluorine atoms occupy one side of every other carbon sheet in an FCCF manner, the carbon atoms in between adopt a diamond-like structure,30,31 ascribing high mass density.31 The formation of similar sp3-rich structures was also verified theoretically and experimentally for bilayer graphene.32,33 Despite their high mass density, fluorocarbons are large band-gap insulators34 and lack sites capable of interacting strongly with ionic species and facilitating their transport. However, because the defluorination and functionalization of fluorographene is known to occur via radical reactions propagated by fluorine elimination,35,36 these processes could potentially be exploited to drive sp3 C–C bond formation and create graphene-based materials with high mass density.Motivated by the importance of fluorine and radical chemistry36–38 Sonicated bulk graphite fluoride reacted very efficiently with NaN3 in dimethylformamide at 130 °C, resulting in nitrogen superdoping , (ii) non-functionalized sp3 carbons , and (iii) nitrogen bonded carbons . The other components in this spectral region were attributed to small amounts of residual fluorine and oxygen from the environment. The HR-XPS spectra of the N 1s envelope  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N moieties (pyridinic and pyrrolic),39,40 respectively. 19F → 13C CP MAS of the starting GF revealed peaks corresponding to CF2 (117 ppm) and CF (94 ppm) groups, typical for FG.41 Such non-functionalized tetrahedral carbons at 34 ppm are not detected in graphene oxide, reduced graphene oxide, or graphene.42,43 The reaction's progress was also verified by infra-red spectroscopy  were progressively replaced with bands at 1580 and 1210 cm−1 (characteristic of aromatic carbon rings44), indicating the formation of an sp2 network. Additional aromatic-ring vibrations, appearing at 1400 cm−1, could be ascribed to heteroatom substitution , as suggested by theoretical calculations.46,47 The Raman spectrum of GN3 featured broad D and G bands at 1300 and 1590 cm−1, respectively, and an ID/IG ratio of 1.3, which remained unchanged even after heating at 1000 °C in an argon atmosphere . Raman bands and X-ray diffraction peaks (XRD) deconvolution performed on GN3, and on the commercial porous carbon for comparison  of ca. 4 nm  calculations. The N3− anion initiated the reaction by nucleophilic attack on carbon radical defects, leading to N2 release and fluorine elimination  peaked at very high temperature (675 °C), at which CO2 emission also took place due to carbon lattice decomposition.Energy dispersive X-ray spectroscopy (EDXS) elemental mapping with high-angle annular dark-field scanning transmission electron microscopy  around the vacancies.48To better understand the formation of the tetrahedral C–C bonds, theoretical models of GN3 sheet fragments were studied using spin-polarized DFT , consistca. 10 μm thick film of GN3 to a thickness of 2 μm  and YP-80F Kuraray carbon (KC)  were formed by pasting slurries onto 15 μm-thick Al foils for density measurements and preparation of supercapacitor electrodes ESI, Methods. of 2 μm . From th of 2 μm , the den4) with 1,1,2,2-tetrafluoroethyl-2,2,3,3-tetrafluoropropyl ether (TTE) in a 9 : 1 ratio. The cyclic voltammograms . The efficiency improved to 100% at 20 A g−1 . At 1 A g−1, the GN3 cell achieved an ultrahigh energy density of 197.6 W h L−1 at a power of 2.6 kW L−1. At 20 A g−1, the energy density remained high at 143.5 W h L−1, while the power density jumped to 51.8 kW L−1  were evaluated under identical conditions. GN3 had a significantly better discharging time , which contained 50% exfoliated graphene.23For benchmarking, the carbons PC and KC . These comparisons highlight the excellent charge transport properties of GN3, which are also reflected in its electrochemical impedance spectroscopic features before and after cycling (Rs) of 3.9 Ω before cycling, which was only marginally increased to 4.4 Ω after 10 000 cycles.For comparative purposes, reported capacitance retention values for other high-performance graphene-based electrodes are  cycling . Based o cycling , the intRct) in the bulk material and at the interfaces.19,52 The almost vertical slope of the spectrum in the low frequency region (on the right of the x-axis) suggested a highly capacitive behaviour and effective ionic charge transport in the bulk of the electrode material.Furthermore, the total absence of semicircles in the high frequency region indicated a very low charge transfer resistivity , but this energy could be delivered at a power of 5.2 kW L−1 compared to 0.9 kW L−1. Importantly, the GN3 cell could also be operated at mass loadings of up to 10 mg cm−2, demonstrating almost identical capacitance (56–59 and 81% retention at the same testing conditions as the low mass-loading supercapacitor cell (see experimental part, ESIVolumetric performance is particularly important for devices in the modern portable energy storage landscape;GN3 cell  demonstracitance  at a tem3.17 capillary densification,18 and other methods.19,23 The new materials are prepared by leveraging the radical-based chemistry of fluorographene, which enables the fruitful combination of sp2 and sp3 carbon bonds in the same network, along with very high nitrogen doping and vacancies. This hybrid carbon achieves mass densities of 2.8 g cm−3, while retaining efficient charge transport, ion penetration, diffusion, and storage. Therefore, cells with electrodes made from these materials offer ground-breaking energy storage capability at very high charging/discharging rates. The discovery of this class of materials will spur intense research on other high-density conductive carbon materials with different functionalities, with the aim of further increasing the competitiveness of supercapacitors in the portable energy storage landscape.We have discovered a new class of carbon-based materials comprising nitrogen doped graphene with diamond-like tetrahedral bonds for high energy density supercapacitor electrodes that are significantly more dense than comparable materials prepared by mechanical compression,A European patent with the number EP 3907184 has been published.EE-015-D1EE02234B-s001


Genetic mapping of seed germination traits has been performed with many plant species. In tobacco, however, investigations are rare. In the present study, a bi-parental mapping population consisting of 118 doubled haploid lines and derived from a cross between ‘Beinhart-1000’ and ‘Hicks’ was investigated. Four germination-related traits, total germination (TG), normal germination (NG), time to reach 50% of total germination (T50), and the area under the curve after 200 h of germination (AUC) were considered by examining seeds either untreated or after a moderate controlled deterioration (CD). Quantitative trait loci were found for all traits distributed on 11 out of the 24 linkage groups. It was demonstrated that, as in many other species, germination-related traits are very complex and under polygenic control.The online version contains supplementary material available at 10.1007/s13353-021-00623-6. The speed of germination also plays an important role because, during the germination process, the plantlets are very sensitive to biotic and abiotic stresses. Quick germination can therefore be advantageous. Both germination and germination speed typically decline with an increase in seed storage time.A successful germination of seeds is the starting point of the growth cycle in higher plants. It is not only the germination N. tabacum L. and N. rustica L. stored at different temperatures. Reducing the temperature from 20 to 0 °C increased storability from about 10 to 30 years, and a further reduction to − 15/− 18 °C increased storability to more than 50 years. For materials stored at 20 °C, a significant effect of seed moisture content has been demonstrated.This behavior has been demonstrated for tobacco  after 200 h of germination.The mapping population was comprised of 118 doubled haploid (DH) lines developed from a cross between the cigar tobacco line ‘Beinhart-1000’ and the flue-cured tobacco cultivar ‘Hicks’  in Petri dishes placed in a climate chamber (Panasonic MLR-352-PE). The temperatures were 30 ± 2 °C and 20 ± 2 °C, during illumination (8 h) and darkness (16 h), respectively.In order to determine the germination speed, germinated seeds  were counted daily within a germination period of 10 days. Based on these counts, the time needed to reach 50% of TG and the AUC were determined using germination software GERMINATOR , to reach RH of 60% inside the boxes. The boxes were placed in aging chambers at a temperature of 45 °C for 30 days.For CD, seeds were placed into permeable paper bags and rehydrated in a climate chamber  to have identical initial moisture content before treatment. Afterwards, the bags with seeds were placed on plastic racks, which were positioned in airtight boxes containing 1 L of unsaturated LiCl solution (30 g LiCl/100 ml HIciMapping 4.2.53 (http://www.isbreeding.net/ (released in September 2019)), in order to detect putative additive QTLs, where the walking speed chosen for all QTL was 1.0 cM. A LOD score of > 2.0 ≤ 3 was applied to detect QTLs as significant and > 3.0 as highly significant. ICIM was chosen over composite interval mapping (CIM) implemented in other QTL mapping softwares like QTLCartographer as it avoids the possible increase of sampling variance and the complicated background marker selection process implemented in CIM  command implemented by The germplasm showed continuous variation for all the traits investigated Table , Fig. S1Additive QTLs were found for all four traits considered on eleven different linkage groups (LGs): 1, 4, 5, 6, 7, 12, 15, 18, 19, 22, and 24 Fig. . IndividThree QTLs were discovered for TG-C on LG 6, 22 and 24  whereas two QTLs were discovered for NS-C on LGs 7 and 24  where LG24 carried overlapped QTLs for TG-C and NS-C. Two QTLs were found on LGs 1 and 22 for TG-CD . Furthermore, another three LGs carried three QTLs linked with T50-CD  whereas only one QTL was discovered for AUC-CD  on LG 19. No QTL, however, could be detected for NG-CD.With regard to epistatic QTLs, a total of four and one pair was discovered for TG-C and TG-S, respectively Fig.  whereas In tobacco, there is only one previously published genetic study on seed germination , Rehman Arif and Börner , bi-parental mapping population QTL for seed longevity were detected on 7 linkage groups  by Rehman Arif and Börner ESM 2(XLSX 12 kb)ESM 3(XLSX 13 kb)ESM 4(XLSX 12 kb)


We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca Airway smooth muscle (ASM) is at the centre of AHR and is more sensitive to contractile stimuli in the diseased state. This may be exacerbated by airway inflammation and remodelling, which are extensively linked to tobacco smoking9.Tobacco smoking is the largest risk factor for developing COPD, which is associated with airway hyperresponsiveness (AHR), amongst other lung conditions such as emphysema, chronic bronchitis and infections10. Experimental analysis of CS utilises one or both of its two distinct phases: the gaseous phase and the particulate phase. The gaseous phase entails the smoke that passes through a glass fibre filter with 0.3 µm pores, while the particulate phase, also known as the tar phase, consists of the relatively larger aerosol particles trapped in these pores11. Delivery of CS can be performed by direct aerosolisation into a closed chamber, commonly used for animal smoking studies, but have also been applied in in vitro experiments. Another delivery model is known as CS extract (CSE), produced by bubbling CS into a buffer solution, which can then be applied to in vitro or ex vivo samples, or collected for chemical analysis. The two delivery models are often reported in the literature as though to be synonymous, but certain volatile constituents potentially lost through vigorous bubbling during the production of CSE are retained in the closed chamber in the CS model.Cigarette smoke (CS) is a complex chemical mixture, containing over 4000 identified compounds. The most prominent of these are nicotine, acrolein, N-nitrosamines, various volatile compounds and heavy metals18. However, CSE, or CS, may also have a direct effect on the intrinsic contractility of the ASM6. ASM contractility, and hence the tone and calibre of the airway, is tightly regulated by intracellular Ca2+ signalling. Chronic CSE exposure enhances agonist-induced Ca2+ release and SOCE in hASMC, which correlates with upregulation of several components of Ca2+ signalling, including Ca2+ permeable channels, potentially leading to long-term disruption of Ca2+ homeostasis19. In relation to ASM contractility, there are varying effects reported of chronic CS or CSE exposure. Chronic in vivo CS exposure sensitises the responsiveness of rat bronchial SM to acetylcholine ex vivo20. Interestingly, rat bronchial segments cultured with CSE chronically are found to be hyper-contractile21, but bovine tracheal strips and hASMC cultured with CSE exhibit attenuated contractile responses14. This makes it difficult to extrapolate to what may occur in these circumstances in hASMCs.In vitro experiments have shown that chronic exposure of human ASM cells (hASMC) to CSE augments ROS generation, chemokine secretion, collagen deposition, proliferation, migration, apoptosis and inhibits wound-healing, all of which are hallmarks of the inflammatory profile and airway remodelling in CS-related airway diseases12 reported that transient exposure to CSE increased [Ca2+]i in hASMC, but the mechanism of action was not explored. Acute CSE treatment also leads to ex vivo constriction of guinea pig bronchi, but rapid relaxation of mice lung slices23. Otherwise, the direct effects of acute gaseous CS or CSE exposure on hASMC Ca2+ signalling, and subsequent downstream effects, remain poorly understood. In the present study, we therefore explored the effects of acute exposure to CS, or CSE, on Ca2+ homeostasis in primary hASMC. Importantly, we applied both the aqueous extract model as well as direct exposure to gaseous CS in order to tease out potential differences between the two delivery methods. Using a variety of pharmacological and molecular interventions, we established that both CS and CSE stimulate Ca2+ influx in hASMC through the TRPA1 channel, and lead to downstream activation of contractile signalling mechanisms.On the other hand, Yoon, et al.2+ concentration in isolated primary hASMC, the cells were exposed to one whole Kentucky 3R4F reference cigarette as described above. Real-time changes in [Ca2+]i was reported by changes in fura-2 fluorescence ratio, and the amplitude (peak [Ca2+]i minus baseline [Ca2+]i) and rate (change in [Ca2+]i per minute fitted with linear regression) of the calcium response were calculated from each individual experiment. In the presence of 1 mM extracellular Ca2+, exposure to the gaseous phase of one whole cigarette led to a rise in [Ca2+]i that was significantly higher, in both amplitude and rate, than the change in [Ca2+]i elicited by room air puffed with the same volume/frequency . This Ca2+ response typically started to rise after 4–6 puffs, and reached an initial peak 4.5–8 min after the first puff. More importantly, the CS-induced [Ca2+]i increase required extracellular Ca2+, as the response was abolished when CS was administered in a nominally Ca2+-free solution . This points to Ca2+ influx from the extracellular space as the primary source of the CS-induced [Ca2+]i elevation, and that intracellular Ca2+ stores are not likely to contribute to this response.To investigate the effect of CS on cytosolic Ca2+]i was through a plasma membrane-bound Ca2+ channel, we tested the effect of gadolinium (Gd3+), a lanthanide ion that non-specifically blocks Ca2+-permeable pathways at the PM. Indeed, both the amplitude (52.6%) and rate (43.6%) of the CS-activated Ca2+ response were significantly inhibited by Gd3+ , suggesting that Ca2+ influx through PM-bound channels was the main source of this response.To ascertain if the CS-induced rise in [Ca2+]i, and this was concentration-dependent . This response typically reached a peak within 1–2.5 min of exposure to 50% CSE, and 3–6.5 min following exposure to 10% CSE. As for the CS-induced rise in [Ca2+]i, the CSE-induced change in [Ca2+]i was also abolished by the removal of extracellular Ca2+ . Since a 5-min exposure to CSE appeared to be sufficient to activate Ca2+ influx, we developed a calcium-addback protocol, whereby cells were first exposed to CSE in 0Ca2+ for 5 min to synchronise influx for the whole population of cells, then Ca2+ was added back in the absence of CSE. This reduced inter-experiment variability in response profiles.To directly compare the effect of CSE to that of gaseous CS, diluted CSE was delivered to hASMC through a perfusion system. Similarly to gaseous CS, exposure to diluted CSE also raised Ca+i, and t2+ addback protocol revealed that the continual presence of CSE was not required for maintained Ca2+ influx, and that the channels remained open following removing of extracellular Ca2+ (data not shown). More importantly, following an extended washout period (at least 25 min after removing CSE), both the amplitude and rate of the Ca2+ addback response was attenuated but not abolished (data not shown), suggesting that CSE’s activation of the Ca2+ influx pathway was likely to be reversible. Using the addback protocol, we examined the effect of Gd3+ on CSE-induced Ca2+ influx. Gd3+ also significantly attenuated both the amplitude (74.8%) and rate (76.0%) of CSE-activated Ca2+ influx in hASMC , providing further evidence that a PM-bound Ca2+ channel was responsible for this response.Experiments with this Ca2+ response elicited by the two tobacco product delivery methods were similar, the response profiles bore subtle differences. In the presence of 1 mM extracellular Ca2+, once cells were exposed to 10% and 50% CSE, [Ca2+]i started to increase within a minute; whereas in the CS model, there was a noticeable time lag of 2–3 min (4–6 puffs) before [Ca2+]i began to rise. Indeed, on average it took longer for CS-induced Ca2+ response to reach a peak than the CSE-induced response (4.5–8 min vs. 1–2.5 min for 50% CSE and 3–6.5 min for 10% CSE).Although the general Ca2+ influx pathways in hASMC, we considered the potential contribution of store-operated Ca2+ channels (SOCC) and the voltage-gated L-type Ca2+ channels (LTCC), both of which play an important role in refilling SR Ca2+ stores in ASMC24. The involvement of SOCC was studied using CPA (10 µM), a SERCA inhibitor, which depletes SR Ca2+ stores and activates SOCC. After store depletion and the subsequent SOC influx reaching a plateau, CS from one cigarette was puffed onto hASMC as previously described. CS was able to instigate an additional elevation in [Ca2+]i when SOCC were already active  did not significantly attenuate CS-induced Ca2+ influx  significantly inhibited the amplitude and rate of both CS- (71.2/94.7% inhibition) and CSE- (69.0/94.0% inhibition) induced Ca2+ influx in hASMC  against TRPA1. A shRNA sequence containing an empty vector (E) was used as a negative control. The relative expression of TRPA1 mRNA, normalised to two housekeeping genes, was significantly lower in cells transfected with the shRNA targeted towards TRPA1 than those transfected with the empty vector  was higher than reported in the initial screening studies (4.9–7.5 µM), albeit in different cell types and against different agonists40. We also used a genetic approach to ascertain the role of TRPA1, and used mRNA levels to quantify TRPA1 expression; studying TRPA1 protein levels, whenever a validated, specific, antibody emerges, could provide additional evidence for changes in TRPA1 expression in these cells. Our shRNA-driven knockdown of TRPA1 was not fully efficient (mean 2-ΔΔCT for TRPA1-shRNA transfected cells was 0.44–0.75) although we were able to detect a ~ 30–60% attenuation of CS/CSE-induced Ca2+ influx with this knockdown model. A complete knockout animal or cell model may provide more definitive evidence of this interaction. CSE and its components were reported to activate TRPA1-mediated Ca2+ influx in sensory neurons of WT, but not TRPA1-deficient, mice33; we hypothesise that ASMC from TRPA1-deficient rodents would not respond to CS/CSE challenge. Moreover, other Ca2+ channels may potentially be involved, since we observed that neither HC-030031 nor TRPA1 knockdown completely abolished CS/CSE-induced Ca2+ influx. For instance, although our data suggested that SOCC were unlikely to be the major facilitators of acute CS/CSE-induced Ca2+ influx, this interpretation assumed that CPA fully activated SOCE in hASMC, which may not be the case. Therefore, SOCC could play a minor role in mediating this influx, but several other candidates to consider include, TRPV141, TRPV442, TRPM843, and reverse-mode NCX44, as these proteins were reported to be activated by CSE or CSE constituents in other cell types. Furthermore, as CS-induced Ca2+ influx was blocked by Gd3+, the involvement of other Gd3+-sensitive channels, including stretch-activated cation channels45 and various TRPC, TRPM, and TRPV channels46, could be further explored.HC-030031 has been reported to be highly selective for TRPA1. At 10 µM, the compound did not inhibit other Ca2+ responses in hASMC  unless otherwise specified. HC-030031, felodipine, and CPA were purchased from Tocris . The primary antibody against p-MLC (3675S) and the secondary anti-mouse HRP-linked antibody (7076S) were purchased from Cell Signaling Technology , while the primary antibody against MLC (M4401) was from Sigma-Aldrich. 3R4F reference cigarettes were purchased from the Kentucky Tobacco Research & Development Center, University of Kentucky. Glycerol stocks of bacteria carrying the plasmid vector encoding shRNA targeted against TRPA1 (TRPA1-shRNA) or the empty control vector (EV) were obtained from Lenti-shRNA Core Facility, UNC Chapel Hill. PCR primers were purchased from Integrated DNA Technologies .56). These donors were all adults over the age of 18, and informed consent was obtained for procurement of lung tissues. All experiments presented in this study were performed in accordance with local and institutional guidelines and regulations.Human tracheal rings were procured from non-smoker excess donor lungs from Freeman Hospital, Newcastle upon Tyne and the Cystic Fibrosis Centre, UNC Chapel Hill. Ethical approval was obtained for the collection of non-smoking human donor lung tissues from Newcastle and North Tyneside Local Regional Ethics Committee (16/NE/0230) and the University of North Carolina Institutional Review Board , and cultured in DMEM/F-12 media . Media was replaced every 48–72 h. Cultured primary hASMC were grown in a humidified incubator at 37 °C with 5% CO2+ imaging experiments and shRNA transfection, hASMC were seeded onto 25 mm glass coverslips, and grown in 6-well plates until ~ 60–90% confluent (Ca2+ imaging) or ~ 40–60% confluent (transfection). For the MLC phosphorylation assay, hASMC were seeded directly onto 6-well plates until fully confluent. Cells were serum-starved for 24–48 h before Ca2+ imaging experiments or MLC phosphorylation assay.For CaCS generated from Kentucky 3R4F reference cigarettes was delivered through a Borgwaldt LM1 smoke engine , into a closed chamber in which hASMC were situated. A glass fibre Cambridge filter pad (0.3 µm pore size) was placed in the tubing to filter out the particulate tar phase. Exposure of hASMC to CS was carried out in 13 puffs with a 30 s interval in between; each puff was 35 ml, delivered over a duration of 2 s. CS persists in the closed chamber after delivery for the full duration of each experiment. Exposure to room air, with identical puff delivery conditions, was used as a control.2, 1 MgCl2, 10 NaHEPES, 10 D-glucose; for the nominally Ca2+-free solution, CaCl2 was omitted and replaced with 1 mM EGTA; solutions were adjusted to pH 7.4). The stock solution was made fresh at the beginning of each day of experiments, and kept on ice throughout the day. CSE was diluted into HEPES-buffered solution for use immediately prior to each experiment.The stock solution of 100% CSE was produced by bubbling one 3R4F reference cigarette, with the particulate phase filtered out, into 25 ml of HEPES-buffered solution  for 60 min at 37 °C, washed with HEPES-buffered solution, and left to de-esterify for 15 min at room temperature. Coverslips were mounted onto a Nikon epifluorescence microscope equipped with a 20 × lens and appropriate filters, and the 340/380 emission ratio (F340/380) of fura-2 fluorescence was recorded using a Princeton Instruments/Hamamatsu CCD camera. Single cells were circled as areas of interest , and images were collected every 5–15 s (5 s for the influx phase). For CS experiments, cells were left in a static bath (1 ml HEPES-buffered solution) and were covered with a customised chamber with tubing connected to the smoke machine for CS delivery. For CSE experiments, a perfusion system was assembled to allow for fluid exchange (2 ml/min). All experiments were performed at room temperature.Primary hASMC grown on glass coverslips were loaded with the cell-permeable i change.Summary data is presented as mean ± SEM. All experiments were performed using hASMC isolated from 2 to 4 donors . n denotes number of repeated independent experiments . Statistical analysis was performed using GraphPad Prism 8, with statistical significance indicated by an alpha value of p < 0.05. Non-parametric tests were performed for datasets that did not pass the D’Agostino & Pearson test. The type of analysis for each dataset is indicated in the respective figure legend. Linear regression was performed to estimate the rate of [CaSupplementary Figure S1.


Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5. Burkholderia glumae was first reported in Japan in the 1950s [B. glumae are bacteria pathogenic to rice plants, which cause a disease called bacterial panicle blight (BPB) in rice panicles [B. glumae has several virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems [B. glumae not only cause BPB in rice panicles, but also bacterial wilt in rice stems and various other economically important crops, such as tomatoes, eggplants, hot peppers, and sesame [B. glumae contribute to its resistance against the host immune system and cause damage the host. In particular, bacterial secretion systems have specialized, sophisticated, and highly developed strategies for the selective transport of toxic proteins (as effector proteins) across bacterial cell membranes. B. glumae BGR1 has four functionally distinct type VI secretion systems (T6SS): T6SS group_1, T6SS group_2, group_4, and T6SS group_5. T6SS group_1 of B. glumae BGR1 is involved in bacterial competition, whereas T6SS group_4 and group_5 contribute to bacterial virulence [B. glumae BGR1 have not clearly been identified.panicles ,3. B. gl systems ,7,8,9,10d sesame . The virOne of the bacterial secretion systems, the type VI secretion system (T6SS), is a contractile nanomachine which is prevalent in 25% of Gram-negative bacteria and contributes to interspecies interaction in a contact-dependent manner with adjacent bacteria or eukaryotic cells ,12,13. TA cell-envelope-associated membrane complex is assembled using TssM, TssL, and TssJ. The baseplate complex is assembled from TssE, TssF, and TssG, and the baseplate complex and membrane complex are connected by TssK. TssA is involved in baseplate assembly and TssB/TssC in sheath polymerization. The needle complex comprises TssD as an inner tube, a TssB/TssC sheath surrounding the inner tube, and a VgrG/PAAR spike complex at its end ,18. The Vibrio cholerae through its interactions with VgrG-1 and T6SS-dependent effectors (TseL) [V. cholerae are required for the secretion of TseL and TseL-mediated antibacterial activity [T6SS-dependent effectors are injected directly into adjacent target cells or with the aid of T6SS accessory proteins, such as T6SS adaptor/chaperones ,26. T6SSs (TseL) . DUF4123activity . HoweverB. glumae BGR1, was identified. The genes of bglu_2g07420 and bglu_2g07410, the functionally unidentified genes encoding these two proteins, are present in the gene cluster of T6SS group_5 in B glumae BGR1. Among them, bglu_2g07420 encodes the region of the DUF2169 domain, known as T6SS adapter/chaperones at its N-terminus and the region of pentapeptide repeats at its C-terminus. In addition, bglu_1g23320, which encodes only the DUF2169 domain in the genome of B. glumae BGR1, does not belong to the four T6SS gene clusters in B. glumae BGR1. Only loss of the DUF2169 domain of bglu_2g07420 belonging to the gene cluster of T6SS group_5 showed attenuated virulence in rice plants. In addition, the bacterial virulence of B. glumae BGR1 was found to be affected by the loss of the pentapeptide repeats region in bglu_2g07420 and bglu_2g07410. bglu_2g07410 encodes the pentapeptide repeats protein with only the pentapeptide repeats region. Our findings show that bglu_2g07420 and bglu_2g07410, belonging to the gene cluster of T6SS group_5 in B. glumae BGR1, encode the DUF2169 domain-containing protein and pentapeptide repeats protein, respectively, and contribute to the bacterial virulence to rice plants as the T6SS-associated proteins.In this study, DUF2169 domain-containing protein and pentapeptide repeats protein, which is involved in the bacterial virulence activity of A. tumefaciens; therefore, we focused on only the genes encoding the DUF2169 domain of the T6SS accessory protein [B. glumae BGR1, bglu_1g23320 and bglu_2g07420, were identified using the National Center for Biotechnology Information . In particular, bglu_2g07420 was present in the gene cluster of T6SS group_5 in B. glumae BGR1  composed of 362 amino acids, whereas bglu_2g07420 is a hypothetical protein (WP_012733555.1) composed of 878 amino acids. Functional analysis of the proteins encoded by the two genes was performed using the InterPro database  , encoding only the pentapeptide repeats region consisting of 356 amino acids  . The bglterminus . Additiono acids . We assubglu_1g23320 and bglu_2g07420 genes via homologous recombination. Single markerless deletion mutants, called Δu2g07420DUF2169 and Δu1g23320, were generated via the deletion of only the DUF2169 domain of bglu_2g07420 and bglu_1g23320, respectively , with a disease severity score of 4.38 ± 0.079 for wild-type BGR1  agar and in broth medium at 37 °C with shaking at 200 rpm. The growth curve of bacterial strains was determined every 2 h by measuring the optical density at 600 nm (OD600) using a UV-1800 spectrophotometer . When required, LB media were supplemented with antibiotics at concentrations of 100 μg/mL (rifampicin) and 50 μg/mL (kanamycin).All bacterial strains and plasmids used in this study are detailed in u1g23320, pK18u2g07410, pK18u2g07420DUF2169, and pk18u2g07420PPR, harboring portions of the target gene upstream (L fragment) and downstream (R fragment), were prepared. The L and R fragments were designed according to the Gibson assembly reaction. The L and R fragments were ligated by secondary PCR using LF and RR primer pairs. To construct pK18u1g23320, the ligated LR fragments and pK18mobsacB were digested with BamHI-HindIII . To construct pK18u2g07410, the ligated LR fragments and pK18mobsacB were digested with EcoRI and HindIII. To construct pK18u2g07420DUF2169, the ligated LR fragments and pK18mobsacB were digested with EcoRI and HindIII. To construct pK18u2g07420PPR, the ligated LR fragments and pK18mobsacB were digested with BamHI and HindIII. Competent E. coli DH5a cells were transformed with recombinant plasmids and selected in kanamycin-containing media. Recombinant plasmids were transformed into E. coli S17-1 cells. Recombinant plasmids were transformed into B. glumae BGR1 by conjugation through co-culture with E. coli S17-1 on LB agar plates. The first crossover was selected on media containing kanamycin (60 μg/mL) and rifampicin (100 μg/mL). The first crossover of colonies was confirmed by PCR using the primer pairs of genes, _UP_F and pK18_DOWN_R. The selected cells underwent a second crossover in LB broth containing kanamycin (60 μg/mL). The second crossover in the cells was selected on LB agar, containing 30% sucrose and rifampicin (100 μg/mL). Finally, single deletions of bglu_1g23320, bglu_2g07410, the DUF2169 domain region, and the pentapeptide repeats region of bglu_2g07420 gene were confirmed by PCR using the primer sets for gene_UP_F and gene_DOWN_R. The double deletion mutant, Δ2g07410-20, was constructed based on single Δ2g07410 deletion mutants using the above-mentioned methods.All primer pairs used for the construction of deletion mutants are shown in bglu_2g07410 and bglu_2g07420 genes was amplified by PCR using the primer pairs Cgene_F and Cgene_R. The amplified DNA fragments were cloned into the broad-host-range expression vector, pBBR1MCS2. The cloned vectors, pBu2g07410 and/or pBu2g07420, were conjugated into single and double deletion mutant strains by co-culturing with S17-1. Finally, complementation strains of single and double deletion mutants were selected on media containing rifampicin (100 μg/mL) and kanamycin (60 μg/mL) and were confirmed by PCR using primer pairs of pB_UP_F and pB_DOWN_R.For complementation, the entire open reading frame of B. glumae BGR1 at the vegetative and reproductive stages of rice plants, cultured bacterial cells in the mid-logarithmic phase were harvested by centrifugation, washed, and resuspended in distilled water. Thereafter, the optical density of the bacterial suspensions was adjusted to OD600nm = 0.8. The rice plants (Oryza sativa L. Saeilmi) used in this experiment were grown under greenhouse conditions . Subsequently, the stems of vegetative-stage rice plants were inoculated with bacterial suspensions using a syringe and grown for 8 days. Disease severity was observed in the inoculated area. Suspensions for each bacterial strain were prepared and adjusted to OD600nm = 0.5 to confirm bacterial panicle blight disease at the rice reproductive stage. At the flowering stage, the rice panicles were inoculated via dipping into 50 mL bacterial suspensions for 1 min. At 8 dpi, the disease severity of the rice panicles in individual rice plants was evaluated using the following scale: 0—healthy panicle; 1—0–20% diseased panicle; 2—20–40% diseased panicle; 3—40–60% diseased panicle; 4—40–80% diseased panicle; 5—80–100% diseased panicle. Disease severity was calculated using the following formula: disease severity = Σ /total number of panicles.To examine the bacterial virulence of putative T6SS accessory proteins in All experiments were conducted at least three times, with at least three replicates. Analyses of the disease severity scores were conducted with at least three replicates of individual rice plants. The mean disease severity scores between two groups were compared using the least significant difference test according to Tukey’s HSD assay.


Multisystem inflammatory syndrome (MIS) in adults associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is increasingly reported in published literature, although published reports remain sparse. In this report, we describe our first experience with a 31-year-old Caucasian male who developed severe MIS 31 days after a mild SARS‐CoV‐2 infection. The patient developed fever, elevated C-reactive protein (CRP), procalcitonin (PCT), reduced ejection fraction (EF), and shock. After extensive diagnostic work-up, nothing was found to justify his shock manifestation. A similar treatment to MIS in children (MIS-C) with immunoglobulins, corticosteroids, and anticoagulants led to a remarkable clinical improvement.MIS in adults (MIS-A) can be fatal. The early identification of MIS plays a crucial role in the prompt initiation of suitable treatment. Therefore, differential diagnosis and exclusion of other causes of illness are of priority. We believe that MIS in children treatment guidelines can be reformed in a way to include MIS in adults as well. An increasing number of studies in published literature reports multisystem inflammatory syndrome (MIS), in children (MIS-C), associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections. Reports from Europe and North America have described instances with symptoms resembling Kawasaki syndrome, toxic shock, and MIS manifestations  requirinBefore October 2021, the case definition for MIS in adults (MIS-A) was not available. According to the recently published CDC (Centers for Disease Control) definition for MIS-A, the patient must be over 21 years of age, hospitalized for ≥24 hours, or with an illness resulting in death, which meets specific clinical and laboratory criteria, and in the absence of alternative diagnosis for the illness such as bacterial sepsis or exacerbation of a chronic medical condition . DespiteFrom December 31, 2019, to late November 2021, 129,158 cases of SARS‐CoV‐2 have been reported in the Republic of Cyprus , includiThe case refers to a 31-year-old Caucasian male with a COVID-19 infection confirmed by reverse transcription-polymerase chain reaction (RT-PCR) of a specimen collected with a nasopharyngeal swab on February 12, 2021. His initial symptoms included mild fever and malaise. After an uneventful initial recovery, 31 days after his initial positive PCR test, the patient developed a sudden high fever, malaise, and difficulty breathing, which led him to seek medical attention.The patient visited a private hospital with tachycardia of 140 beats per minute, pyrexia of 39.6°C, oxygen saturation of 90% (on ambient air), and a blood pressure of 70 over 45 mmHg. He was given 2 g ceftriaxone IV and then was immediately transferred to the Limassol General Hospital, Limassol, Cyprus, where he was admitted to the ICU.2/min, arterial blood gases revealed a PO2 of 62 mmHg, PCO2 of 32 mmHg, pH of 7.39, and lactate levels less than 2 mmol/L. His chest x-rays were unremarkable in terms of infiltrates . On a non-rebreathing mask at 15 litOPhysical examination revealed conjunctivitis, lip cheilitis, and diffuse rash on his palms and lower extremities Figure .3/μL (1-3.4), white blood cells (WBC) of 14.6 x 103/μL (3.6-9.2), a troponin of 0.75 ng/mL (0 and 0.04), creatinine of 2.44 mg/dL (0.67-1.17), urea of 111 mg/dL (17-43), and procalcitonin (PCT) of 10 µg/L (<2) (Table The results of laboratory tests upon admission showed an elevated C-reactive protein (CRP) of 372.46 mg/L (<5), a lymphocyte count of 0.78 x 102/FiO2 ratio of 68 on non-rebreather mask [NRM] 100%/15 litO2). The patient was intubated and mechanically ventilated due to severe persistent tachypnoea and hypoxia (PO2/FiO2 ratio of 114 on FiO2 1.00). That day’s laboratory tests revealed ferritin of 3420 ng/ml (peak on day 4: 8060 ng/ml) and an elevation of WBC to 22.11 x 103/μL (peak on day 3: 22.62 x 103/μL)  specimens. Given that the patient's symptoms were similar to MIS associated with COVID-19, the patient was started on intravenous immunoglobulin (IVIG) 0.4 g/kg/day, methylprednisolone 62.5 mg three times daily, low molecular heparin 0.6 IU, and aspirin 75 mg daily Table . DespiteOutcomeThe patient's condition improved with the treatment provided. He remained on mechanical ventilation for six days, and oxygenation returned to near normal by the seventh day. The exanthem disappeared on the fifth day, and the fever settled on the sixth day Figures , 4. RepeMIS has been linked with COVID-19. It is likely underdiagnosed, and the pathogenesis remains undefined , but theBastug et al. illustrated that hypotension, tachycardia, fever, conjunctivitis, diffuse exanthem, dyspnea, and cough are among the most prevalent MIS clinical presentation as well as gastrointestinal symptoms and reduced left ventricular EF. More than 40% of the patients may require vasopressors for hypotension, and almost 20% may require mechanical ventilation . A recenThe recently published CDC case definition criteria for MIS-A  is a steAccording to recently published criteria on the syndrome , the patHe was characterized as a severe case of MIS-A since respiratory and circulatory support was needed. Management of the patient was guided by the available reports on MIS-A, similar to what has been reported for MIS-C ,4,12,13.Healthy young adult patients may present with severe multiorgan dysfunction several weeks after a mild infection with SARS-Cov2 requiring respiratory and cardiac ICU support. Since early MIS-A identification plays a significant role in prompt pharmacological treatment initiation and patient recovery, rapid exclusion of other causes is of great importance.MIS-A identification relies mainly on clinical symptoms; therefore, clinicians should consider MIS-A as a possible cause for patients with SARS-Cov2 infection history. Local authorities should promote awareness and reporting. Treatment guideline for MIS-A is currently not available; but given the rapid response in our patient, we believe that the MIS-C treatment guideline can be adapted for MIS-A.


Staphylococcus aureus. Thin-layer chromatography demonstrated the entrapment and accumulation of rhodomyrtone within the bacterial cell wall and cell membrane. The incorporation of radiolabelled precursors revealed that rhodomyrtone inhibited the synthesis of macromolecules including DNA, RNA, proteins, the cell wall, and lipids. Following the treatment with rhodomyrtone at MIC (0.5–1 µg/mL), the synthesis of all macromolecules was significantly inhibited (p ≤ 0.05) after 4 h. Inhibition of macromolecule synthesis was demonstrated after 30 min at a higher concentration of rhodomyrtone (4× MIC), comparable to standard inhibitor compounds. In contrast, rhodomyrtone did not affect lipase activity in staphylococci—both epidemic methicillin-resistant S. aureus and S. aureus ATCC 29213. Interfering with the synthesis of multiple macromolecules is thought to be one of the antibacterial mechanisms of rhodomyrtone.As the burden of antibacterial resistance worsens and treatment options become narrower, rhodomyrtone—a novel natural antibiotic agent with a new antibacterial mechanism—could replace existing antibiotics for the treatment of infections caused by multi-drug resistant Gram-positive bacteria. In this study, rhodomyrtone was detected within the cell by means of an easy an inexpensive method. The antibacterial effects of rhodomyrtone were investigated on epidemic methicillin-resistant  Staphylococcus aureus is a significant cause of morbidity and mortality worldwide in both hospital- and community-acquired infections uridine, >97%, 250 µCi (9.25 MBq); L-leucine, >97%, 250 µCi (9.25 MBq); N-acetyl-d-[1-3H]glucosamine (NAG), 250 µCi (9.25 MBq); acetic acid, sodium salt, [3H]-specific activity: 2–5 Ci (74.0–185 GBq)/mMole, 5 mCi (185 MBq). All standard chemicals were of analytical grade. Purified rhodomyrtone was isolated as previously described  thymidine, [3H]uridine, [3H]leucine, [3H]NAG, and [3H]sodium acetate, respectively. The cultures were added to universal test tubes containing rhodomyrtone at 4× MIC, MIC, and 1/2× MIC. The radiolabelled precursors were added at a final concentration of 1 µCi/mL. Cultures were incubated at 37 °C with constant shaking (200 rpm). Samples (350 µL) were withdrawn at intervals and the reaction was terminated by adding 350 μL ice-cold 10% TCA. The mixtures were then centrifuged at 20,000 rpm at 4 °C for 15 min. Supernatants were discarded and pellets were washed twice with 500 µL ice-cold 5% TCA. The pellets were dissolved in 200 µL distilled water, transferred to scintillation vials, and then added up to 4 mL scintillation fluid. The samples were read with a scintillation counter. ELB21 (DNA), rifampicin (RNA), chloramphenicol (protein), and vancomycin  were used as positive control agents for macromolecular synthesis inhibition assays. For lipid synthesis, the bacterial cells in the mid-exponential growth phase were added to falcon tubes containing rhodomyrtone at 4× MIC, MIC, and 1/2× MIC. [3H] sodium acetate at 0.2 µCi/mL was added to the mixtures, and the reactions were incubated with shaking at 37 °C. Samples (1000 µL) were withdrawn at intervals, and 3750 µL of chloroform/methanol (1:2) was added to stop the reaction. The mixture was vortexed for 10 min, chloroform (1250 µL) was added to the mixture, vortexed for 2 min, followed by addition of 1250 µL distilled water and vortexed for 1 min. The mixtures were centrifuged at 13,000 rpm for 10 min, and 500 µL of organic phase was transferred to a scintillation vial and allowed to evaporate to dryness in a fume hood. The samples were then counted using a scintillation counter. A negative control was used with 1% DMSO. All experiments were carried out in triplicate.Macromolecular biosynthesis assays were performed on cultures of EMRSA-16 and ribed by  with somS. aureus ATCC 29213 was investigated as previously reported with some modifications [The effect of rhodomyrtone on the lipase activity of ications . Lipase p < 0.05).Each experiment was performed in triplicate, and experimental values were expressed as means ± SD. The statistical significance of the differences between the control (DMSO-treated) and test (rhodomyrtone- and standard inhibitor-treated) cells were analyzed using one-way analysis of variance (ANOVA). The Statistical Package for the Social Sciences 20  was used for the analysis at a 95% confidence interval (Scheffe test at Rhodomyrtone, a potent plant-derived antibacterial compound, has demonstrated membrane-active effects against several pathogenic Gram-positive bacterial isolates. Although several studies have demonstrated the effectiveness of rhodomyrtone in the inactivation of microbial cells, its mode of action is still not conclusively understood. In this study, we investigated the mechanism of action of rhodomyrtone by evaluating for rhodomyrtone accumulation in bacterial cell walls and the cell membrane and assessing its effects on the synthesis of cellular macromolecules. This study demonstrated that rhodomyrtone accumulates in the bacterial cell wall and the cell membrane and inhibits the synthesis of multiple cellular macromolecules, including nucleic acid, proteins, the cell wall, and lipids. The results revealed that rhodomyrtone targets multiple cellular components in MRSA and thus might be an alternative antibacterial agent, effective for the management of methicillin and other drug-resistant Gram-positive bacteria.


Caleyachetty R, Barber TM, Mohammed NI, et al. Ethnicity-specific BMI cutoffs for obesity based on type 2 diabetes risk in England: a population-based cohort study. Lancet Diabetes Endocrinol 9: 419–262021; —In figure 4 of this Article, the horizontal line representing type 2 diabetes incidence at a BMI of 25·0 kg/m2 in White populations was incorrectly placed. This correction has been made to the online version as of June 16, 2021, and printed version is correct.


We found that ALOS4 promoted healthy weight gain in A375-engrafted nude mice and reduced melanoma tumor mass and volume. Despite these positive changes, examination of the tumor tissue did not indicate any significant effects on proliferation, mitotic index, tissue vascularization, or reduction of αSMA or Ki-67 tumor markers. Modulation in overall expression of critical downstream αvβ3 integrin factors, such as FAK and Src, as well as reductions in gene expression of c-Fos and c-Jun transcription factors, indirectly confirmed our suspicions that ALOS4 is likely acting through an integrin-mediated pathway. Further, we found no overt formulation issues with ALOS4 regarding interaction with standard inert laboratory materials  or with pH and temperature stability under prolonged storage. Collectively, ALOS4 appears to be safe, chemically stable, and produces anti-cancer effects in a human xenograft model of melanoma. We believe these results suggest a role for ALOS4 in an integrin-mediated pathway in exerting its anti-cancer effects possibly through immune response modulation.We examined the effects of ALOS4, a cyclic peptide discovered previously by phage library selection against integrin α Among the approaches [vβ3 with monoclonal antibodies [vβ3 inhibitors such as the cyclic peptide Cilengitide [vβ3 antibodies such as Abegrin [Integrin αgression , metastagression , invasiogression , and anggression . Structusequence  shared wsequence . Due to sequence  endothelc target  and is cc target  that corc target ,11 and ic target , prostatc target ,14, ostec target , breast c target ,15, and c target . Melanomc target ,18. Currc target . The seaanocytes  mediatesanocytes , which hproaches  used to tibodies , cyclic tibodies , or otheengitide  and func Abegrin  have fai2-CSSAGSLFC-COOH (MW = 871.98)) was previously discovered using a phage–display technique targeted to integrin αvβ3 binding [vβ3. In addition, we also characterized the physiochemical aspects of ALOS4 formulated stability and toxicity issues such as alterations in mouse behavior, blood cell profile, and blood chemistry in healthy  mice. Our findings suggest that ALOS4 is stable in chemical formulation and poses no overt toxicity risks, yet is effective in melanoma tumor reduction by an αvβ3-related mechanism and perhaps other mechanisms.ALOS4, a synthetic 9-amino acid cyclic non-RGD peptide , which differentiated between responder and non-responder individuals, excluding two animals from analysis (p = 0.0239).In our previous research, we have shown that ALOS4 treatment leads to tumor growth inhibition and increased survival of C57BL/6J mice inoculated with murine B16F10 melanoma cells. In this study, we used a xenograft model to further confirm ALOS4 anti-cancer properties using immunodeficient nude mice, which were SC inoculated with human A375 melanoma cells followed by administration with 0.3, 3, or 30 mg/kg ALOS4. We found that 3 and 30 mg/kg of ALOS4 preserved normal weight gain of nude mice compared with untreated control animals, whose weight was significantly decreased during tumor development . We similarly conducted an ROC analysis of the results to distinguish responders from non-responders . Youden’s index values for ROC analysis of each ALOS4 dose were as follows: 231.6, p = 0.0056  in all examined concentrations showing maximal two-fold changes in growth inhibition with 30 mg/kg on day 17  and all examined tissue sections, regardless of treatment, had evidence of vascular invasion of the tumor mass . Mitotic indices were also similar between controls and ALOS4 treatments .Analysis of the effect of ALOS4 on the expression of alpha smooth muscle actin (αSMA), a marker of vascular smooth muscle cells, was used to assess the number of blood vessels in the tissue sections to indicate the vascular invasion A–D. Contp = 0.089). Thus, ALOS4 0.3 mg/kg dose and saline-treated controls both appeared to have a higher score in Ki-67-positive cells within the neoplastic cell population , while not affecting levels of extracellular signal-regulated kinase (ERK) and pERK .A375 cells were treated with concentrations of 0.01, 0.1, or 1.0 µM for 48 h and protein extracts were prepared for Western blots. We observed that 1.0 µM of ALOS4 significantly upregulated focal adhesion kinase (FAK), as well as proto-oncogene tyrosine protein kinase (Src) and pSrc levels . A similar phenomenon was observed in c-Jun transcription levels, which showed significant decrease after 24 h at higher doses (p = 0.0005), whereas c-Jun was increased by 0.01 µM ALOS4 at 48 h (p = 0.0172).We also examined the expression of the immediate early genes We chose to perform a series of chemical stability and recoverability tests on ALOS4 to determine its practical applicability as a drug in formulation. To ensure that ALOS4 was stable and did not adhere to standard laboratory materials, we incubated ALOS4 formulated in 0.9% NaCl solution at a range of concentrations from 1–100 µM in polypropylene microtubes for 60 min at room temperature, then transferred solutions to either polypropylene or borosilicate glass liquid chromatography (LC) vials. LC-MS analysis showed that ALOS4 recoverability was near 100% in both borosilicate LC glass vials and standard polypropylene LC vials A.p < 0.0001).Solubilizing agents are commonly used to stabilize peptides in solution and to reduce inert substrate interaction. Therefore, we compared two solvent options for ALOS4, standard saline solution (0.9% NaCl) and saline solution containing 0.1% Tween-80 (polysorbate). Solutions of ALOS4 ranging from 0.5–5000 pmol/µL formulated in both solvents were incubated for 60 min at room temperature in standard polypropylene tubes. LC-MS analysis showed that ALOS4 formulated in the saline solution containing Tween-80 had significantly reduced peptide stability by 1.2-fold in comparison with ALOS4 formulated in 0.9% NaCl only  stability at different ranges of acid/base and temperature conditions was conducted in saline after 1 or 24 h in different pH solutions above and below the physiological pH (7.4): pH 3.4, 4.4, 5.4, 6.4, 7.4, and 9.4. Stability analysis was also performed under three regimes: at 4 °C (storage temperature), 25 °C (room temperature), and 37 °C (body temperature). LC-MS analysis indicated that ALOS4 was highly stable (90–100% recoverability) at all measured temperatures and all analyzed acid/basic conditions at both time points C.To evaluate ALOS4 safety and toxicity, uninoculated  ICR mice were IV administrated ALOS4 and monitored for clinical signs of toxicity including body weight changes, body temperature, alopecia, nasal bleeding, and mortality. ICR mice were injected with doses of ALOS4 every other day over 21 days with 10, 30, 90, 180, or 360 mg/kg . Mouse weights were taken daily and rectal temperatures were recorded 30 min following ALOS4 injection. We observed no alterations in weight-gain D or bodyTo evaluate potential effect of ALOS4 on mouse locomotory activity and anxiety-like behaviors, we used two standard behavioral paradigms: the open-field ambulation and elevated plus maze (EPM) tests.p = 0.4441) with only an exception for total traveled distance at the dose of 180 mg/kg , and did not produce any anxiety-like behaviors .We found that an IV acute single dose of ALOS4 of 30, 90, or 180 mg/kg administered to ICR mice did not affect locomotor activity in general  and basic blood chemistry profile. Analysis of blood compared with the normal rage of ICR mice blood scores [vβ3 integrin is an important cell adhesion receptor involved in various biological activities [vβ3 in many cancers [vβ3 inhibition targeting the Arginine-Glycine-Aspartate (RGD) motif. Despite their promising potential, these peptides failed in late clinical trials [vβ3 integrin signaling pathway. This peptide was discovered in our laboratory using a phage display technique and previously demonstrated an anti-cancer efficacy in a murine melanoma model [αtivities ,31 actintivities . Due to  cancers , this in cancers ,32 were l trials , possibll trials . In thisma model . The potUsing a subcutaneous model of A375 human melanoma, we demonstrated high efficacy of ALOS4 in tumor growth inhibition during an 18-day trial. Since there were limitations regarding the number of mice permitted for study, thus also restricting the number of dosing groups, we applied receiver operating characteristic (ROC) analyses  and calcvβ3 integrin-targeted molecule, it is likely that observed anti-cancer effects were achieved through modulation of αvβ3 integrin signaling. To confirm this suggestion, we analyzed changes in expression of selected candidates from the αvβ3 integrin signaling pathway, including extracellular signal-regulated kinases  initiated by activation of focal adhesion kinases (FAK) and Src kinases, which in complex or individually further activate downstream ERK signaling [c-Fos and c-Jun, were both affected by ALOS4 treatment in vitro. ALOS4 significantly reduced c-Fos mRNA levels at all doses, while downregulation of the c-Jun gene was significant at 0.1 µM dosage after 24 h and showed a non-significant tendency to decrease after 48 h. We speculate that the differences in levels of significance in c-Fos expression at 24 and 48 h of treatment may indicate the attempt of the cancer cells to stabilize expression of this oncogene, whereas its downregulation by ALOS4 remains to be explained. These results indirectly confirm that ALOS4 is able to modulate αvβ3 integrin signaling and differences in the effect of ALOS4 on c-Fos and c-Jun expression may be explained by additional ALOS4-independent processes involved in c-Fos and c-Jun transcription. The c-Fos results are not without precedent considering the actions of other peptide-based integrin antagonists (flavoridin) in melanoma cell lines, which increase activation of downstream integrin pathway elements (such as increased FAK phosphorylation) while also effecting downregulation c-Fos expression [νβ3 integrin and its ability to bind ανβ3 leading to metastatic arrest was previously demonstrated [Since ALOS4 was developed as an αignaling ,45. We fignaling , we suggpression . Thus, snstrated , we belinstrated  or integnstrated , is presWe also examined ALOS4 safety and stability as a potential drug candidate. Due to a known tendency of peptides to adhere to standard inert laboratory materials , we demoBlood chemistry was also examined during toxicity studies and revealed reduction of white blood cell counts (40%) with ALOS4 treatment at 180 mg/kg. This reduction could indicate higher levels of tumor-infiltrating lymphocytes (TILs), suggesting that the immune system may be involved in the ALOS4 activity. Unfortunately, potential effects and mechanistic outcomes of immune interactions of ALOS4 could not be determined in this study due to the immunodeficient nature of the mice required for the xenograft model. Nevertheless, cumulative results from this work and prior studies suggest an interaction of ALOS4 with immune system elements, which needs to be further evaluated.In summary, ALOS4 appears to be completely non-toxic, remarkably prolongs lifespan, and increases weight of treated mice. The latter feature makes ALOS4 beneficial to counteract cachexia experienced by cancer patients during the process of disease progression. We believe that demonstrating the anti-cancer activity through modulation of components of integrin signaling together with its safety profile suggests that ALOS4 peptide is a promising patient-tolerable prospective anti-cancer drug candidate.vβ3 binding using phage display technology. This synthetic cyclic peptide is composed of the following nine-amino-acid sequence: H-cycl(Cys-Ser-Ser-Ala-Gly-Ser-Leu-Phe-Cys)-OH. ALOS4 was custom-synthesized by Shanghai Hanhong Scientific Co. . Stock solutions of ALOS4 at 10 mM, were prepared in sterile physiological saline solution  with the addition of 0.02% BSA  and maintained at either −20 °C for short term use, or −80 °C for long-term storage. For each experiment, ALOS4 was thawed and freshly diluted to working concentrations in physiological saline.ALOS4 was developed based on α2 at 370 °C.A375 human melanoma cells  were grown in Dulbecco’s Modified Eagle Medium  with 4.5 g/L glucose and L-glutamine, supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin . Cells were maintained on uncoated dishes in atmosphere of 5% COAdhesiveness to inert materials was measured for ALOS4 0.9% NaCl(aq) solution in concentrations of 1, 3 10, 30, or 100 µM and incubated in standard laboratory polypropylene microtubes for 60 min at room temperature. Solutions were transferred to either polypropylene LC vials or borosilicate glass LC vials. Optimal formulation stability of ALOS4 was analyzed in either saline solution (0.9% NaCl) or saline solution containing 0.1% tween 80  at concentrations of 0.5, 5, 50, 500, or 5000 pmol/µL. Solutions were incubated in standard polypropylene tubes for 60 min at room temperature. Acid/base stability was measured for ALOS4 formulated in saline at a concentration of 10 µM and incubated for 1 or 24 h at 3.4, 4.4, 5.4, 6.4, 7.4, or 9.4 pH at temperatures of 4, 25, or 37 °C. Recovery of ALOS4 following these materials assays was assessed by LC-MS.To investigate the effect of ALOS4 on human melanoma cancer cells, nude Fox nu/nu mice were used for SC- or IV-injected inoculations. Additionally, uninoculated  ICR mice were used for safety and toxicity studies. Mice were obtained from Envigo, Israel, and arrived at the age of 4–5 weeks old. Upon arrival, mice were habituated to vivarium conditions for one week before initiation of experiments. All mice were maintained under a 12:12 light–dark cycle and provided Purina rodent chow  and water ad libitum. Animals were housed five to a cage in a room maintained at 22 ± 0.5 °C (nude mice cages were held in a laminar-flow cabinet).To evaluate the effect of ALOS4 on mouse locomotor activity, we used the open field (OF) behavioral test ,60. ThisTo evaluate the effect of ALOS4 on mouse anxiety-like behaviors, we used the elevated plus maze test (EPM) . The EPMAcute single or repeated doses of ALOS4 at 10, 30, 90, 180, or 360 mg/kg were administered intravenously to uninoculated  ICR mice. Body weight, rectal temperature, and survival were evaluated for 14 days following injections. Mice were also evaluated for locomotory and anxiety-like behaviors (open-field and EPM) on treatment day 14.6 cells in 100 μL in serum-free DMEM medium/mouse. Following inoculation, all mice were randomly divided into experimental groups, then treated IP with either ALOS4 or saline (negative control) at day one post-inoculation. Mouse body weights were monitored during the course of the experiment. The base-weight of mice was determined by the weight on the second day to account for acclimation-related changes, then mice were weighed twice a week until tumor appearance and thereafter daily until the experiment was terminated. Termination resulted from mouse death, when tumor diameter reached or exceeded 1500 mm3, or when 30 days of treatment had elapsed, whereupon mice were CO2 euthanized. Mice were IP-injected with ALOS4  or saline (control) at identical fixed volumes. SC inoculation of cells usually formed a palpable tumor in 7–14 days. Tumor volumes were estimated by digital caliper and calculated with the following equation: V = π/6 × width × length × height. Survival rate of mice was documented at the end point of experiments. Tumor growth rates were calculated by the following formula: TGI% = (relative tumor volume ALOS4-treated)/.We used a subcutaneous (SC) melanoma model to study the effect of ALOS4 on localized solid tumor growth. Nude mice (Fox nu/nu) were SC injected with A375 cells at 2 × 106 cells in 100 μL normal saline/mouse) and treated for 18 days with ALOS4  injected IP with daily monitoring for clinical signs. Mice were euthanized by CO2 asphyxiation when the first mouse reached the ethical protocol limit of 1500 mm3 tumor size. Tumors were harvested, weighed, and fixed in 4% formalin. After 24 h fixation, samples were rinsed with PBS and transferred to 70% ethanol for transport to the pathology laboratory. Embedding, 5 μm sectioning, and slide preparation were performed for the 5 tumors from each experimental group  according to routine procedure.Nude mice (Fox nu/nu) were SC-inoculated with A375 human melanoma cells  using the following scales when examined at 40× magnification: Pleomorphism , mitotic index (mitotic indicators were counted in 10 different 40× fields and averaged), degree of vascular invasion . Prepared tumor tissue slides were also evaluated for the presence of tumor-related-markers by monoclonal antibody staining for the α-smooth muscle actin (αSMA), the marker of vascular smooth muscle cells, as well as the nuclear protein cell proliferation marker Ki-67 using the following rating scales: αSMA , Ki-67 .c-Fos and c-Jun was performed using 2× PCR SYBR Green Master Mix , with a 100 nM mixture of forward and reverse primers , 4 µg of cDNA and RNase/DNase free water. Samples were placed in Real-Time PCR  and reactions were performed in a thermocycler: 180 s at 95 °C, followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C.RNA from A375 cells 24 and 48 h after the treatment was purified from cells using a quick RNA miniprep kit . DNase treatment was performed using on-column DNase digestion. RNA concentration was measured at 260 nm using NanoDrop spectrophotometer  and 260/280 ratio method was used to verify that the samples met proper purification standards around 2. A total of 1 μg of total RNA was reverse-transcribed using a reverse transcription system . The master mix for cDNA synthesis insisted of 10× Reverse Transcription buffer, dNTP mix, oligo (dT) (18T) primers, and AMV enzyme. The reverse transcription reaction was performed in a thermocycler  using a two-step program: 42 °C for 60 min followed by heating to 70 °C for 15 min to terminate the reaction, and maintained at 4 °C. The quantitative RT-PCR for 3VO4; Sigma-Aldrich, Cat#S6508, Darmstadt, Germany), 5 mM sodium fluoride , protease inhibitor cocktail , and phosphatase inhibitor . Protein concentrations were assessed using a Bradford assay. Proteins were separated by gel electrophoresis in an 8% polyacrylamide gel. Blots were incubated with blocking solution  for 1 h with gentle shaking at room temperature. After blocking, separate membranes were each probed with one of target-specific antibodies , then hybridized with horseradish peroxidase-conjugated streptavidin secondary antibodies  and developed using ECL solution  according to the manufacturer protocol. After the antibody of a specific protein on each membrane was evaluated, we performed a GAPDH  re-probe for all of membranes to quantify the target proteins. FAK and Src were performed after stripping on the same membrane and that is why they share their common GAPDH. Blots were visualized using ChemiDoc™ Imaging System  apparatus and densitometry analysis was performed using Image Lab Software . The grouped data sets representing phosphorylated and total proteins demonstrates percentage of each treated group normalized to untreated control.Proteins from A375 cells were extracted in RIPA lysis buffer solution  with freshly added 1 mM sodium orthovanadate  and blood samples were collected at 24 h post-injection to EDTA and serum tubes. Complete blood cell count (CBC) and blood chemistry analyses were performed for saline and ALOS4 treatment groups at a certified animal laboratory .All data are expressed as means ± SE (±SD in a few measures). Threshold for significance was set to α = 0.05. Multiple treatments were Bonferroni-corrected and compared by unmatched one-way ANOVA for single time point results or by two-way ANOVA for multiple treatment outcomes over time. ANOVA tests were followed with a Bonferroni means separation test to identify specific differences between treatments. For ordinal or nominal data of tumor immunohistochemistry scoring, groups were compared by Kruskal–Wallis ANOVA followed by a Dunn’s post-test for intergroup comparisons . To diffPinhasov A. has an ALOS4 patent .