Mutations in isocitrate dehydrogenase (IDH) are frequently found in low-grade gliomas, secondary glioblastoma, chondrosarcoma, acute myeloid leukemias, and intrahepatic cholangiocarcinoma. However, the molecular mechanisms of how IDH2 mutations induce carcinogenesis remain unclear. Using overlapping PCR, transfection, immunoblotting, immunoprecipitation, measurements of enzyme activity, glucose, lactic acid, ATP, and reactive oxygen species (ROS), cell viability, protein degradation assays post-inhibition of the 26S proteasome (bortezomib) or HSP90 (17-AAG), and a homology model, we demonstrated that the properties of ten cancer-associated IDH2 variants (R140G/Q/W and R172S/K/M/W/G/C/P) arising from point mutations are closely related to their structure and stability. Compared with wild-type IDH2, the R172 and R140 point mutations resulted in a decrease in IDH2 activity, ROS, and lactate levels and an increase in glucose and ATP levels under normal and hypoxic conditions, indicating that mutant IDH2 increases cell dependency on mitochondrial oxidative phosphorylation, and reduces glycolysis under hypoxia. Overexpression of most of IDH2 point mutants showed anti-proliferative effects in the 293T and BV2 cell lines by inhibition of PI3K/AKT signaling and cyclin D1 expression and/or induced the expression of TNF-α and IL-6. Furthermore, bortezomib treatment resulted in dramatic degradation of IDH2 mutants, including R140G, R140Q, R140W, R172S and R172K, whereas it had little impact on the expression of WT and other mutants . In addition, targeting HSP90 minimally affected the expression of mutated IDH2 due to a lack of interaction between HSP90 and IDH2. The homology model further revealed that changes in conformation and IDH2 protein stability appeared to be associated with these point mutations. Taken together, our findings provide information important for understanding the molecular mechanisms of IDH2 mutations in tumors. IDH codes for three isoforms . IDH2 catalyzes the oxidative decarboxylation of isocitrate to form α-ketoglutarate (α-KG) and NADPH using NADP+ as a cofactor in mitochondria1. To date, the IDH crystal structures of wild-type (WT) or mutants from prokaryotes and humans complexed with various ligands/inhibitors5 or from porcine heart mitochondrial IDH  with a 97% homologous sequence to human IDH26 have been resolved, such as IDH1-R132H2, IDH2-R140Q3, and IDH2-R172K4.Isocitrate dehydrogenase (IDH) is an important enzyme for cellular metabolism that is part of the Krebs cycle. Homo sapiens IDH2 and other point mutants associated with cancers remains unclear. Numerous studies have shown that IDH1/IDH2 mutations frequently occur in low-grade gliomas7, secondary glioblastoma8, chondrosarcoma9, acute myeloid leukemia (AML)10, and intrahepatic cholangiocarcinoma11. Point mutations are mainly found in the arginine residues within the isocitrate binding sites of IDH active sites, including IDH1/R132, IDH2/R140, and IDH2/R172.However, the structure of 12. Specifically, due to the similar structure between 2-HG and α-KG, 2-HG competitively blocks the binding of α-KG to histone demethylases, such as lysine-specific demethylase (KDM), and then it increases histone methylation13. 2-HG also inhibits the activity of ten-eleven translocation hydroxylases, which are important for global DNA methylation14. In addition, the accumulated 2-HG results in inhibition of prolyl-hydroxylase (PHD) activity and induction of pyruvate dehydrogenase kinase (PDK), which hydroxylates hypoxia-inducible factor 1α subunit (HIF-1α) at specific proline residues and increases HIF-1α expression. Furthermore, the high levels of 2-HG because of IDH mutations stabilize HIF1α by decreasing levels of the HIF-1α antagonist endostatin, which results in upregulation of HIF1α-dependent genes, including vascular endothelial growth factor (VEGF)16.Although their mechanisms of tumorigenesis have not been fully demonstrated, IDH mutations have been found to convert α-KG to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases11. R140Q (17.9%) and R172K (20.2%) are more common point mutations in AML17. R140 and R172 mutant proteins possess neomorphic enzymatic activity resulting in 2-HG accumulation, indicating that both mutations are associated with cancer progression.Recently studies have found that the point mutation frequency of IDH2 is relative lower than that of IDH1, for example, in glioma (1.6–26.8%), intrahepatic cholangiocarcinoma (5.7–23.1%), and chondrosarcoma (10.7–19.4%), IDH2 R172 mutants include R172K (39%), R172S (29%), R172W (12%), R172G (10%), R172M (5%), and R172T (4%)Mutated IDH2 has been found to be an important therapeutic target in above-mentioned cancers. However, it is still unclear for the molecular mechanism of pathogenesis, specially, the conformational changes and stability of the mutated IDH2 protein associated with function. In the present study, the effects of ten very common IDH2 point mutations (R140G/Q/W and R172S/K/M/W/G/C/P) on the function, structure, and stability of protein were evaluated by the measurement of enzyme activity, glucose, lactate, ATP, and ROS levels, degradation analysis post-treatment with 26S proteasome inhibitor (bortezomib) or HSP90 inhibitor (17-AAG), and bioinformatics homology modeling. The current data demonstrated that IDH2 point mutations reduced their enzymatic activity and protein stability due to conformation changes. These data provide important information for understanding the molecular mechanisms of IDH2 mutations in tumorigenesis.Phospho-AKT, polyclonal antibodies against AKT and TNF-α, and the monoclonal antibody against S6 were from Cell Signaling Technology . Monoclonal mouse antibodies against cyclin D1, IL6, and HSP90 , GAPDH , β-actin , and His  were also used.2·6H2O were obtained from Sigma-Aldrich. Lipofectamine and PLUS reagent were purchased from Invitrogen Life Technologies. The wild-type prokaryotic IDH2 expression vector (EX-C0462-B01) was from FulenGen Co., Ltd. . The empty eukaryotic vector pCMV6-AC-Myc-His was purchased from Origene Technologies . The restriction endonucleases HindIII and MluI were obtained from New England Biolabs (MA). Premix PrimeSTAR HS, QuickCut enzyme, T4 DNA ligase, DNA Marker, and DNA Loading Buffer were purchased from Takara . Primer synthesis , and 17-AAG and CoClis Table  and sequ2 humidified atmosphere. The HEK293T cell line and BV2 cell line were regularly screened for mycoplasma contamination using a Mycoplasma Stain Assay Kit .HEK293T cell line was a kind gift from Dr. Jonathan A. Fletcher in the Laboratory of Oncology at Brigham and Women’s Hospital and Harvard Medical School. The mouse microglial cell line BV2 was purchased from the cell library of the Chinese Academy of Sciences in Shanghai. The HEK293T cell line was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) supplemented with 1% (v/v) penicillin/streptomycin and 1% (v/v) L-glutamine, and the mouse microglial BV2 cell line was maintained in DMEM/F-12 (Gibco) with 10% FBS supplemented with 1% (v/v) penicillin/streptomycin and 1% (v/v) L-glutamine at 37 °C in a 5% COIDH2 point mutants and PCR primers are listed in Table IDH2 target fragment was obtained by PCR, and IDH2 was cloned into the HindIII and MluI restriction sites of the mammalian vector (pCMV6-AC-Myc-His). IDH2 point mutants, including R140G, R140Q, R140W, R172S, R172K, R172M, R172W, R172G, R172C, and R172P, were constructed by nested PCR based on the WT. All constructs were verified to contain the correct point mutation by restriction analysis and sequencing  for 15 min at room temperature (RT), then the Lipofectamine was diluted with 100 µl of SFM and added to DNA and PLUS complexes, and incubated for another 15 min at RT. Finally, the DNA-PLUS-Lipofectamine complexes were added to 60% confluent cultures of 293T or BV2 cells in 800 µL of SFM in six-well plates. The DNA-PLUS-Lipofectamine complexes in SFM were completely replaced with serum-containing regular media after 3 h of incubation. Cells were lysed for immunoblotting or functional analysis, including measurement of IDH2 enzymatic activity, glucose, and lactic acid levels at 48 or 72 h post-transfection.Transfection of 18. The hybridization signals were detected by chemiluminescence  and captured using a GE ImageQuant LAS4000 chemiluminescence imaging system.Immunoblotting was performed 48 h post-transfection or after treatment with 17-AAG or bortezomib for 72 h. Whole cell lysates were prepared using lysis buffer . Lysates were cleared by centrifugation at 15,000 rpm for 30 min at 4 °C, and the supernatant protein concentrations were determined using a Bio–Rad protein assay . Electrophoresis and western blotting were performed as described previouslyFirst, 1 mg of protein lysate was precleared for 30 min at 4 °C using 20 μL of protein G/A beads. Three micrograms of primary mouse antibodies against His and primary rabbit antibodies against HSP90 were rocked with the lysates for 2–3 h at 4 °C. Next, 20 μL of Sepharose-protein G/A beads was added and incubated overnight at 4 °C and then centrifuged at 10,000 rpm for 2 min, after which the Sepharose beads were washed 3 times with 750 μL of IP buffer for 10 min and once with 750 μL of 10 mM Tris–HCl buffer (pH 7.6). Finally, loading buffer (20 μL) was added to the beads and they were boiled for 5 min at 95 °C. The interaction of IDH2 and HSP90 was subsequently evaluated by immunoblotting with specific antibodies.2, 0.33 mM EDTA, 0.1 mM β-NADP+, and d-(+)-threo-isocitrate in a total volume of 200 μL. All assays were performed in triplicate.293T cells were lysed 48 h post-transfection. The lysates were sonicated and centrifuged at 12,000 rpm at 4 °C. Supernatants were then collected and normalized to the total protein concentration. The oxidative activity of IDH2 was determined by monitoring the increase in the absorbance of NAPDH at 340 nm over a period of 60 min on a Thermo Scientific Microplate Reader. The reaction mixtures consisted of 5.2 μg of lysate protein, 100 mM Tris–Cl buffer (pH 7.5), 1.3 mM MnClIDH2 WT and mutant constructs under normal and/or hypoxic conditions (200 µM CoCl2 treatment). Changes in intracellular lactic acid and glucose levels were determined with the Lactic Acid Assay Kit , the Glucose Assay Kit , and the ATP Detection Kit (Beyotime Biotechnology Co. Shanghai). According to the manufacturer`s instructions, the changes in the absorption value at 505 nm (glucose) or 530 nm (lactic acid) were measured by UV spectrophotometry  after the lysate proteins (5.2 μg/μL) were added to the working solution. ATP levels were measured by Luminometer  after the lysate proteins (2 μg/μL) were added to the working solution. All assays were performed in triplicate. The data were finally analyzed and exported by GraphPad Prism 9.0.0 .HEK293T cells were lysed 48 h post-transfection with 2 treatment). Namely, the oxidative conversion of cell-permeable dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) indicates changes in intracellular ROS levels. Briefly, 293 T cells in 6-well culture dishes were incubated with 10 μM DCFH-DA at 37 °C for 20 min at 48 h post-transfection using IDH2 WT and point mutant constructs, and then the DCF fluorescence distribution of the cells was detected at a 488 nm excitation wavelength and a 535 nm emission wavelength by a fluorospectrophotometer  or at FITC channel by a flow cytometry  after the cells were washed twice with PBS. All assays were performed in triplicate. The data were finally analyzed and exported by NovoExpress .According to the manufacturer`s protocol, ROS levels were measured using the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology Co. Shanghai) under normal or hypoxic conditions . The data were normalized to WT. All the assays were performed in triplicate wells, and at least three independent experiments were performed.293T cells were plated at 3000 cells per well in a 96-well flat-bottomed plate  and cultured in RPMI 1640 for 24 h before transfection with 6 using a modeler. Structural images of IDH2 WT and mutant proteins were generated using PYMOL.Tertiary structure predictions were made with the published crystal structure of porcine heart mitochondrial isocitrate dehydrogenase p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Student’s t-tests were performed on data from cells treated with empty vector or IDH2 WT/point mutants. Statistically significant differences between WT and mutations were defined as *IDH2 mutations occur frequently in a variety of tumors, including AML, brain tumors, and glioma. Point mutations occur in the active-site arginine residues of IDH2, such as R140 and R172. To demonstrate the IDH2 function associated with the conformational changes and stability of the mutant protein, IDH2 WT and ten cancer-associated IDH2 variants (R140G/Q/W and R172S/K/M/W/G/C/P) were overexpressed in 293T and BV2 cell lines by transfection. Immunoblotting showed that the expression of IDH2 WT and R140/R172 mutants was increased compared to that of the pCMV6 empty vector. According to the gray value of the IDH2 protein bands, the overexpression level of each IDH2 mutant was comparable to that of the WT protein  in 293T and BV2 cell lines, and cyclin D1 , but R140Q and R172K point mutations had little affect on p-AKT expression in both cell lines  in 293T cells reduced ∼50–70% in cell viability at 6 days after transfection with mutation constructs, compared with WT control  for 72 h. Treatment with bortezomib markedly inhibited the expression of the IDH2 point mutation, including R140G, R140Q, R140W, R172S and R172K, but it barely affected the expression of IDH2 WT and mutants  , a molecular chaperone, optimizes and maintains folding and localization for many client proteins and prevents their proteasomal degradation2 h Fig. B. ComparIDH2 WT construct followed by His and HSP90 immunostaining 11. Mutated IDH2 promotes cancer progression through metabolic reprogramming and epigenetic deregulation of gene expression. These mutations result in a product conversion from α-KG to 2-HG, which plays a pivotal role in the regulation of methylation, metabolism, the PI3K/AKT/mTOR signaling pathway and protein stability20. To date, the exact role of IDH2 mutations in carcinogenesis is not completely understood. Associations among the structure, function and stability of IDH2 mutants have rarely been reported, and these parameters are closely related to the occurrence and proliferation of cancer. Therefore, ten pathogenic IDH2 point mutants (R140G/Q/W and R172S/K/M/W/G/C/P)  Fig. , express/P) Fig.  were com+ to NADPH15. IDH2 mutations resulted in dysregulation of a set of cellular metabolisms, including an increase in glucose  IDH2 mutations reduced cell viability through inhibition of PI3K/AKT signaling and slowed cell metabolism in 293 T and BV2 cells  to reduced glutathione (GSH), which is a major antioxidant against oxidative stress induced by reactive oxygen species (ROS), and this may lead to ROS accumulation and oxidative DNA damage to promote tumor cell growthons Fig. , which i28. In BV2 cells, overexpression of IDH2 mutants resulted in upregulation of TNF-α and IL-6  have been demonstrated to accumulate 2-HG and promote tumor proliferation by altering a number of downstream cellular activities in the previous studies. The present data showed that there are some differences in protein conformational changes, stability, glycolysis, mitochondrial oxidative phosphorylation, and PI3K/AKT signaling among these IDH2 point mutations, specifically for the most common IDH2 point mutations R140Q and R172K in cancer cells, indicating that each IDH2 point mutation possibly plays a different biological role in tumorigenesis. However, this conception needs further investigation and analysis among these point mutants in detail. These novel findings provide important information for understanding the molecular mechanisms that underlie the pathogenesis of IDH2-mutated tumors.Supplementary Information.


Hepatitis C Virus NS3/NS4A, a serine protease complex, has been found to interact with many host proteins and cause various adverse effects on cellular function and immune response. For example, the cleavage of important immune factors by NS3/NS4A has been suggested as a mechanism for the hepatitis C virus to evade innate immunity. The spectrum of susceptible substrates for NS3/NS4A cleavage certainly includes important immune modulator kinases such as IKKα, IKKβ, IKKε, and TBK1, as demonstrated in this paper. We show that the kinase activities of these four host kinases were transformed in unexpected ways by NS3/NS4A. Treatment with NS3/NS4A caused a significant reduction in the kinase activities of both IKKα and IKKβ, suggesting that HCV might use its NS3/NS4A protease activity to deactivate the NF-κB-associated innate immune responses. In contrast, the kinase activities of both IKKε and TBK1 were enhanced after NS3/NS4A treatment, and more strikingly, the enhancement was more than 10-fold within 20 min of treatment. Our mass spectroscopic results suggested that the cleavage after Cys89 in the kinase domain of IKKε by NS3/NS4A led to their higher kinase activities, and three potential mechanisms were discussed. The observed kinase activity enhancement might facilitate the activation of both IKKε- and TBK1-dependent cellular antiviral pathways, likely contributing to spontaneous clearance of the virus and observed acute HCV infection. After longer than 20 min cleavage, both IKKε- and TBK1 gradually lost their kinase activities and the relevant antiviral pathways were expected to be inactivated, facilitating the establishment of chronic HCV infection. Liver diseases caused by the hepatitis C virus (HCV) are a serious health problem. About 30% of HCV infection is acute infection since infected people can clear the virus within 6 months of infection without any treatment. The remaining 70% of HCV infections are chronic infections that may persist for decades. It is estimated that 10% to 20% of chronically infected people will develop liver cirrhosis, while 1% to 5% will develop hepatocellular carcinoma .The HCV genome is a plus-stranded RNA about 10 kb in length, and its organization is similar to that of members of the family Flaviviridae ,6,7. BasIntracellularly, NS3 has been shown to bind to the catalytic subunit of protein kinase A (PKA) which prevents the nuclear translocation of this subunit and affects many PKA functions . The actInterestingly, the protease activity of NS3/NS4A has been implicated in the inhibition of cellular antiviral pathways, leading to persistent HCV infections. Foy et al. first demonstrated that the NS3/4A protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3) . They suIn addition to these signaling molecules, we hypothesized that NS3/NS4A may cleave other proteins involved in innate immunity and antiviral pathways, e.g., cellular kinases such as Inhibitor of κB kinase α (IKKα), IKKβ, IKKε, and TBK1. These kinases are known to phosphorylate IκB proteins to activate transcription factors nuclear factor-κB (NF-κB), IRF-3, and IRF-7 ,33, whicAll peptides utilized in this study were chemically synthesized and HPLC purified by Peptides International Inc. . All synthetic peptides were confirmed to be the desired ones by analyses of amino acid composition and electron spray mass spectrometry. Their purities exceeded 95%, and they were stored frozen at −80 °C as 10 mM stock solutions in deionized water or 100% dimethyl sulfoxide.Escherichia coli BL21 (DE3) Gold . The truncated NS3 protein also contains a C-terminus His6 tag to facilitate the purification process. The cells were induced with 0.4 mM IPTG at room temperature to increase the solubility of NS3 in the cytoplasmic extract  ATP) at 23 °C for 30 min. The reactions were stopped by adding SDS loading dye. The samples were separated by SDS-PAGE, and the gel was stained with Coomassie blue staining solution. Band intensities were determined to ensure equal sample loading. The gel was dried and then quantitated with a PhosphorImager 445 SI (Molecular Dynamics). Radioactive intensities were determined from the bands  to measure kinase activity. Relative kinase activity was determined by calculating the ratio of the kinase activity of NS3/NS4A-treated kinase over the control kinase (in digestion buffer without NS3/NS4A) from the same digestion duration.Kinase activity assays were performed by incubating 1.5 μL of the digested kinases with a 10 μL cocktail  was used as the NS4A cofactor in all in vitro digestion assays in this paper. To examine the protease activity of the NS3/NS4A complex, an internally quenched fluorogenic substrate (sequence: Ac-Asp-Glu-Asp(Edans)-Glu-Glu-Abu ψ[COO]-Ala-Ser-Lys(Dabcyl)-NH2; Abu, 2-aminobutyric acid; Ac, acetylation; Dabcyl, 4-[[4′-(dimethylamino)phenyl]azo]benzoic acid; Edans, 5-[(2′-aminoethyl)amino]naphthalenesulfonic acid; ψ[COO], the ester bond between Abu and L-(+)-lactic acid) [−1s−1) of the NS3/NS4A complex was close to 345,000 M−1s−1 measured by Taliani et al. [The consensus sequence of (D/E)XXXXC(A/S)  has been determined for all trans cleavage sites  in viral% purity . Since tic acid)  was usedi et al. , indicatSulfolobus solfataricus [The four human kinases were expressed and purified from baculovirus-infected insect cells . ataricus ,51,52 thobsk) at IKKε concentrations of 0.1 and 0.5 μM were determined to be 0.0075 and 0.0145 s−1, respectively . These low micromolar dK values indicate the binding between the protease complex NS3/NS4A and IKKε is moderately strong. The substrate specificity obsk/dK for the degradation of 0.1 and 0.5 μM IKKε was estimated to be 1641 and 8631 M−1s−1, respectively. Previously, steady-state kinetic analysis has been used to estimate the cleavage efficiency (catk/mK) of peptide substrates representing the viral NS4A/NS4B (1600 M−1s−1) and NS4B/NS5A (110 M−1s−1) cleavage sites catalyzed by NS3/NS4A at 30 °C [obsk/dK and catk/mK values suggest that IKKε is as good a substrate, if not better, to NS3/NS4A as those HCV junction-mimicking substrates. Thus, HCV NS3/NS4A protease is capable of cleaving IKKε, and possibly IKKα, IKKβ, and TBK1, in vivo.To examine if NS3/NS4A cleaves these four kinases with reasonable efficiency and to avoid the complication of product inhibition, we measured the cleavage efficiency of IKKε at 23 °C under single-turnover conditions. At least a 10-fold excess of NS3/NS4A over IKKε was used in the kinetic assay. As a kinetic strategy, the single-turnover kinetic assay was employed to estimate the cleavage rate of a human kinase by NS3/NS4A. It does not mean that the single-turnover reaction conditions would be certainly encountered in vivo. The single-turnover assay was performed by using 5.0 μM NS3/NS4A with one of two different IKKε concentrations and the data were fit into a single exponential equation, Equation (1) . The observed cleavage rate constants  and the NS4A peptide (325 μM) were used. Each of those kinases was reacted with NS3/NS4A at 23 °C for 35 min and the reaction was stopped by flash-freezing the reaction in a dry ice/methanol bath. The treated kinases (with or without NS3/NS4A) were then tested for their kinase activities to phosphorylate GST-IκBα (residues 5–55). To test the stability of the kinases in the NS3/NS4A digestion buffer condition, a time-course digestion assay was set up at 23 °C. In the control reactions (without NS3/NS4A), the duration of incubation from 1 min to 20 h significantly reduced the kinase activity of IKKβ B, suggesEnhancement of the kinase activities of IKKε and TBK1 during cleavage could be due to either NS3/NS4A mediated cleavage or protein/protein interactions such as the binding of IKKε to NS3/NS4A, or both. To examine these possibilities, we used Pefabloc SC, a broad-spectrum serine protease inhibitor, to covalently modify NS3 and permanently inhibit its serine protease activity. There are many reports about the interactions of HCV proteins with host cell factors involved in cell signaling, apoptosis, transcriptional regulation, transformation, membrane rearrangements, and immuno-modulation, which lead to dysregulation of cellular functions . Among tcatk and MK from these assays are difficult to kinetically interpret because they are complex functions of all reactions occurring at the enzyme active site over multiple turnovers [In this paper, we used pre-steady-state kinetic methods to examine the cleavage of NS3/NS4A under single turnover reactions, in which the molar excess of the protease complex was reacted with each of the four human kinases . The single turnover conditions ensured that most of the examined kinase were bound and cleaved by NS3/NS4A during the first turnover. In contrast, many reports used steady-state kinetic assays in which a small amount of NS3/NS4A was used to digest the molar excess of a protein substrate (kinase) for long times ,41,47,56urnovers ,59,60,61urnovers  makes thurnovers . In theiurnovers .dK in the low micromolar range  by NS3/NS4A enhanced or abolished their kinase activities . Human IHuman IKKα and IKKβ are highly homologous canonical IKK kinases . NotablyIn this paper, we discovered that the HCV protease complex of NS3/NS4A was able to cleave human IKKα, IKKβ, IKKε, and TBK1. The cleavage of IKKα and IKKβ decreased their kinase activities in a time-dependent manner. Surprisingly, the short cleavage time of IKKε and TBK1 enhanced their phosphorylation of the protein substrate IκBα and the scenarios for such an increase in the kinase activities were discussed. After a long cleavage time by NS3/NS4A, IKKε and TBK1 were cleaved into small peptides and their kinase activities were gradually decreased and eventually lost.


Such efficient UC is dominated by excited state absorption, accompanied by the advantage of long lifetimes  of excited state levels of Er3+, instead of the well-recognized energy transfer UC between sensitizer and activator. NaYS2:Er3+ phosphors are further developed for high-performance underwater communication and narrowband NIR photodetectors. Our findings suggest a novel approach for developing NIR II response UC materials, and simulate new applications, eg., simultaneous NIR and visible optical communication.The second near-infrared (NIR II) response photon up-conversion (UC) materials show great application prospects in the fields of biology and optical communication. However, it is still an enormous challenge to obtain efficient NIR II response materials. Herein, we develop a series of Er 3+ doped ternary sulfides phosphors with visible UC efficiency up to 2.6% and long emission lifetimes under 1532 nm irradiation, via an excited state absorption mechanism.Development of efficient upconversion (UC) phosphors that emit under irradiation in the NIR II region is challenging - most UC materials rely on the presence of sensitizers absorbing at shorter wavelengths. Here, authors synthesize Er The first discovery of an effective UC host material (NaYF4) was made by Pierce in 19726. Until 2004, the hexagonal phase NaYF4 (β-NaYF4) with high UC efficiency was demonstrated7. With the development of nanotechnology, lanthanide doped UC materials experienced the explosive growth, especially in biological applications10, photon-conversion devices12, super-resolution nanoscopy15, and information storage and security18. Much efforts have been devoted to manipulate UC, and Yb3+, Nd3+_sensitized energy transfer (ET) and interfacial energy transfer (IET) are recognized as the promising ways to achieve efficient UC luminescence24.Lanthanide doped photon up-conversion (UC) materials can absorb two or more low-energy photons, and emit high-energy photons, which have attracted extensive attention due to the superior optical performance, such as large anti-Stokes shift, excellent stability against photo-bleaching, and no auto-fluorescence28. Particularly, 1530 nm is also the wavelength of optical communication, exhibits a low loss in transmission, and is widely used in metro, long-distance, ultra-long-distance, and submarine optical cable systems32. However, the development of the highly efficient NIR II 1530 nm response photon UC materials still faces great challenges. Up to date, the recognized efficient UC materials  are based on the sensitized ET, owing to the efficient ET process from sensitizers  to activators . Despite the great progress made in UC, the existing UC materials strongly depend on the excitation of short near-infrared wavelengths (such as 808 and 980 nm). Er3+ has a well spectral response around 1530 nm with a relatively large absorption cross-section, but it is not suitable for sensitizer owing to the lack of matchable activators. A number of reports has announced Er3+ doped UC materials pumping around 1530 nm realized via the excited state absorption process of Er3+ themselves39. Disappointedly, their up-conversion quantum yields (UCQYs) are low, less than 0.5%, limited by the deleterious quenching interactions, and strong electron-phonon coupling etc. To date, because of the lack of appropriate host materials, it still remains a challenge to achieve efficient NIR II 1530 nm response UC.The near-infrared second spectral region (NIR II), especially for 1530 nm, shows great potential with respect to the information storage and security, 3D display, and high-resolution imaging, etc2; where M is an alkali metal ion, and Ln is a rare earth ion) materials process the layer structure, low unit cell symmetry, strong covalence and low phonon energy40, which are promising materials for efficient NIR II response UC. It was discovered in 1970s41, and Güdel et al. reported the UC emissions with UCQY of 21% in NaYS2:Er3+ crystals under the excitation of 980 nm, but this was obtained at a high incident power of 0.79 kW cm−2 at low temperature of 12 K42. Since then, little studies have paid attention to these materials.Ternary rare rarth sulfide . They exhibit cubic (α-) and trigonal (β-) phases depending on the radius ratios between the trivalent lanthanide ions and the monovalent alkali metal ions (RLn3+/RM+). It should be highlighted that β-NaYS2 presents a particularly high efficiency UC emissions with the UCQY as high as 2.6% under 1532 nm excitation (~4.5 W cm−2), originating from the exceptionally long lifetimes  of excited state levels of Er3+. Compared to the commercial NaYF4:Yb3+, Er3+ phosphor, the NaYS2:Er3+ owns much higher UCQY, brightness, and spectral stability of illumination and temperature under the same conditions. Then, the NaYS2:Er3+ phosphors were employed to achieve multiband responsive NIR photodetectors , and green light underwater communication.In this work, we fabricated a series of efficient NIR II 1532 nm response UC materials: MLnS2:Er3+ UC phosphors were synthesized by the gas-solid reaction method, in which the IA alkali metal elements  and trivalent rare earth elements  were selected to occupy the M and Ln sites, respectively (See Methods). Figures 2 and NaLnS2 prepared at 1173 K, which coincide well with the corresponding standard cards. The XRD patterns and UC luminescence spectra further reveal that the optimized temperature for synthesis is 1173 K, otherwise the impurity phase of Y2O2S is formed at 1273 and 1373 K , while KYS2, NaGdS2, NaLuS2, and NaYS2 are indexed as trigonal phase (β-) under the same conditions 43. When the RLn3+/RM+ ratio is larger than 1.0, MLnS2 tends to form cubic phase (NaCl or Th3P4 type). Conversely, the β-phase structure is dominant. The α-NaLaS2 and β-NaYS2 were taken as examples to illustrate the representative structure of MLnS2 family 6 octahedrons44. In β-NaYS2, both Na+ and Y3+ are six-fold coordinated by S2− in form of a regular octahedron and contain empty voids on the special 3a and 3b sites, respectively. The S2− are surrounded by three Na+ and three Y3+. Both MS6 and LnS6 distorted octahedra are more precisely described as trigonal anti-prisms with centro-symmetric D3d symmetry, where all six bonds M–S in the octahedron possess identical length (as in Oh symmetry)46. Therefore, Na+ and Y3+ are orderly situated in alternating NaS6 and YS6 octahedral layers.A series of MLnSons Fig. . As reprily Fig. . In α-Na2 with various doping concentration of Er3+ are recorded in Fig. 2 is obtained according to the standard cards (JCPDS No. 46-1051), and two characteristic peaks located at 26.34° and 31.64° correspond to the (101) and (104) crystal planes, respectively. The diffraction peaks of NaYS2:Er3+ shift to large angle on increasing the doping concentration of Er3+ owing to the replacement of Y3+ (89.3 pm) by Er3+ (88.1 pm) with a smaller radius. The similar morphologies of MLnS2:Er3+ are obtained as displayed in Supplementary Fig. 2:Er3+ and NaYS2:Er3+ phosphors are presented in Fig. 2  and NaYS2 , respectively. The selected area electron diffraction (SAED) patterns  in NaYS2 is determined to be 3.9808 Å. Since the probability of energy transfer between luminous centers (concentration quenching) is proportional to 1/d6, the large adjacent distance d often causes a high doping level and intense luminescence. Crystal structural data for NaYS2 and NaYF4 are summarized in Supplementary Table 2 compared to NaYF4 are mainly ascribed to the layer structure, lower unit cell symmetry , longer minimum Y3+ separation distance, stronger covalence, and lower phonon energy. These advantages of NaYS2 all contribute to the efficient UC of NaYS2:Er3+.The UC intensity ratios of green-to-red emissions in β-MLnS2:Er3+ illuminated at 1532 nm was further investigated to clarify the populating process. Five UC emission bands spanning from 400–1100 nm are given in Fig. 4I9/2, 4I11/2 → 4I15/2 transitions of Er3+, respectively. A schematic illustration of populating mechanism for NaYS2:Er3+ pumping under 1532 nm is provided in Fig. 4I15/2) of Er3+ are pumped to 4I13/2, and further excited to 4I9/2 via resonant illumination, generating 4I9/2 → 4I15/2 transition. The electrons on 4I9/2 jump to the higher excited levels (4S3/2/2H11/2) through continuous three-photon absorption, realizing the green emissions. A fraction of electrons on 4I9/2 decays non-radiatively to 4I11/2, then populates the 4F9/2 state and thereby generating 4I11/2 → 4I15/2 and red (4F9/2 → 4I15/2) emissions, respectively. ET-sensitized UC is always considered as the most efficient strategy to achieve UC emissions, while the excited state absorption is basically limited by the weak absorption cross-section. However, the efficient UC emissions in MLnS2:Er3+ are realized through an ESA process. It is different from the UC from typical energy transfer systems containing Yb3+, Er3+, where Yb3+ serves as a sensitizer and Er3+ as an activator. Thus, the visible and NIR emissions are ascribed to three- and two-photon UCs, as evidenced by the power dependence of the integral UC emission intensity of NaYS2:Er3+  change from single exponential to double exponential, suggesting that cooperative UC among Er3+ is involved. While they decrease for 4I9/2 → 4I15/2 (800 nm), 4I11/2 → 4I15/2 (1000 nm), and 4I13/2 → 4I15/2 (1500 nm) on increasing the Er3+ doping concentration from 2 to 15 mol%. According to the previous reports51, the cross-relaxation should dominate the above process among Er3+. Three possible cross-relaxation processes  are added in Fig. As displayed in Fig. 4I13/2 and 4I9/2 play a crucial role in UC emissions for MLnS2:Er3+. To deeply understand the essence of highly efficient UC emissions for NaYS2:Er3+, the decay time curves of MLnS2:Er3+ were measured  are significantly shorter than that of β-MLnS2:Er3+ . M+ and Ln3+ are randomly disordered on an identical lattice site in α-MLnS2 with cubic structure, inevitably leading to the intensified negative energy exchange. While the Er3+ in β-MLnS2 are ordered separated by NaS6 octahedral layers along the c-axis direction, which blocks the cross-relaxation between each other to a certain extent. Therefore, in virtue of different cation distribution, the long decay time of the 4I13/2 level in the β compared to the α polymorph is observed. Excitingly, the NaYS2:Er3+ phosphors have the longest lifetimes in all the samples, reaching 30.27 ms and 9.24 ms for 4I13/2 → 4I15/2 and 4I9/2 → 4I15/2 transitions, respectively. As mentioned in Fig. 2:Er3+ mean that the electrons on 4I13/2 and 4I9/2 levels stay longer, which is in favor of being re-simulated to higher green energy levels (4S3/2/2H11/2), bringing about efficient green UC emissions. This is in line with the results in Fig. 4I13/2 → 4I15/2 and 4I9/2 → 4I15/2 transitions in MLnS2:Er3+ phosphors. From the UC emission spectra and luminescent decay curves as a function of temperature ranging from 10 - 300 K in Supplementary Figs. 3+ in NaYS2 are significantly shorter than that of the other UC materials52. It can be concluded that the long lifetimes of NaYS2:Er3+ phosphors originate from the long radiative and non-radiative lifetimes of Er3+  of 4I13/2, 4I9/2 levels. From the UC spectra and dynamics at low temperature  has been recognized as the most efficient UC material. Therefore, the commercial NaYF4:Yb3+, Er3+ phosphor of similar size . It is amazing to observe that the green emission intensity of NaYS2:Er3+ is 3.4 times stronger than that of NaYF4:Yb3+, Er3+  and NaYF4:Yb3+, Er3+ excited with 980 nm (0.45-1.45 W cm−2) are compared, as shown in Supplementary Fig. 2:Er3+ is also higher than that of NaYF4:Yb3+, Er3+. The UCQYs of MLnS2:Er3+  are measured using the typical method with two detectors in an integrating sphere  under the illumination of 980 nm with the same power density  at 980 nm of Yb3+ in NaYF4:Yb3+, Er3+ than that of Er3+ , NaYS2:Er3+ is indeed efficient and a promising UC material for 980 nm pumping. It should be noted that the NaYS2:Er3+-based ESA process not only obtains the more efficient UC emissions, but also expands its pumping wavelength to NIR II region. It should be highlighted that green and red UC emissions in NaYS2:Er3+ are from a three-photon process under 1532 nm excitation, while they are a two-photon process in the NaYF4:Yb3+, Er3+ phosphor under 980 nm excitation. It is difficult for the former one to obtain highly efficient UC. In fact, the multi-photons UC is also possible to be more effective than two-photon UC, owing to the enriched energy levels and diversity of transitions in UC59. Such efficient UC emissions-induced ESA may be ascribed to the significantly longer lifetimes of 4I9/2 and 4I13/2 levels of Er3+ in NaYS2:Er3+, which are 9.4 fold and 3.05 fold longer than that of NaYF4:Yb3+, Er3+  to red  region, while NaYS2:Er3+ shows higher brightness and excellent color stability with concentrating in the green emission region  by varying the pumping power. In addition, the UC luminescence ratio of red to green emissions in NaYF4:Yb3+, Er3+ significantly increases, whereas it changes little for NaYS2:Er3+  using a simple NaYS2:Er3+/MAPbI3 hybrid. As illustrated in Fig. 3 film acting as the photon-to-current material was spin-coated on the top of a NaYS2:Er3+ film , thereby producing photocurrents. Figure I-t) response curves of the NaYS2:Er3+/MAPbI3 device separately under 808, 980, and 1532 nm illumination with an incident light intensity of 5 mW cm−2 at the bias voltage of 1 V. The photocurrents are 1.26, 2.19, and 3.78 µA in NaYS2:Er3+/MAPbI3 PDs for 808, 980, and 1532 nm. This is in well agreement with the UC luminescence intensities for the NaYS2:Er3+ phosphor under corresponding excitations, respectively, and shown in Supplementary Fig. R), detectivity (D*) and external quantum efficiency (EQE). As exhibited in the insets of Fig. EQE of the NaYS2:Er3+/MAPbI3 PDs are determined to be 0.26 A/W, 0.44 A/W, and 0.73 A/W; 0.4610 Jones, 0.5610 Jones, and 0.8410 Jones; 39%, 55%, and 59% for the 808, 980, and 1532 nm light, respectively. For the photodetector, EQE is equal to the number of electron-hole pairs per second collected to produce the photocurrentIph, divided by the number of incident photons per second. In a detector with 100% EQE, R = 1 A/W for photon energy Eph = 1 eV63. The photoconductive gain leads to the EQE > 50% when applied a bias in PDs, similar to previous reports66. In general, UC visible emissions of NaYS2:Er3+ under 1532 nm excitation excite the semiconductor (MAPbI3) to produce electron-hole (e-h) pairs. Since holes generally move more slowly than electrons in semiconductors, N electrons have already been collected by the electrode when the slower-moving hole is searched by the electrode under an applied electric field. The device at 1532 nm shows the best performance owing to the most effective UC at this excitation wavelength. The Fig. 2:Er3+/MAPbI3 PD exhibits response times in the range of 310–570 ms. As shown in Supplementary Fig. 2:Er3+/MAPbI3 PDs reached below 2 mW cm−2 for 808 and 980 nm light, particularly below 1 mW cm−2 for 1532 nm light. Compared to other representative NIR PDs, our PDs based on NaYS2:Er3+ phosphors demonstrate the excellent capability for multi-wavelength detection and good performance  of seawater is suitable for under seawater communication, which usually requires visible light source with high power for long-distance seawater transmission68. As known, optical fiber communication based on 1532 nm is mature technology, which can be easily coupled into optical fiber with low coupling and transmission losses. Based on the above results, NaYS2:Er3+ phosphors can efficiently convert 1532 nm to green emissions, which simultaneously combines the advantages of NIR optical fiber communication and visible optical communication. As a proof of concept demonstration, the NIR-II responsive NaYS2:Er3+ phosphors are used to construct the UC underwater information transmission system, as illustrated in Fig. 2:Er3+ phosphors mixed with epoxy resin were packaged on the optical fiber, and 1532 nm laser diode (LD) was coupled into the optical fiber which further simulated the NaYS2:Er3+ phosphors, then generating UC green emission lights. The input signal of 1532 nm with different frequencies was adjusted by the signal generator, Then, the emitted UC lights carrying information passed through the seawater, and collected by receivers. As shown in Fig. 2:Er3+ excited by the 1532 nm. The green UC emission lights remain the frequency after passing seawater over a distance of 0.6 m  phosphors were successfully prepared through the gas-solid reaction method. All of them demonstrate the efficient UC emissions under the illumination of NIR II 1532 nm based on the ESA process. By comparison, β-NaYS2 phosphor is recognized as the most UC material in MLnS2:Er3+ under 1532 nm excitation. It can be explained by ordered layer structure of β phase instead of disordered structure of α phase, as well as exceptionally long lifetimes of excited state levels of NaYS2:Er3+. β-NaYS2:Er3+ displays remarkably higher UCQY and brightness, and much better spectral stability of lights illumination and temperature than those of commercial NaYF4:Yb3+, Er3+ phosphor. β-NaYS2:Er3+ realizes a breakthrough UC efficiency as high as 2.6% under 1532 nm excitation, and the brightness of NaYS2:Er3+ is 8224 cd/m2 under 1532 nm irradiation. Such high optical performances can be assigned to low non-radiative transition and electron-phonon coupling in NaYS2:Er3+ phosphor. Furthermore, we designed and fabricated the sensitive narrowband responsive NIR PDs at wavelength of 808, 980, and 1532 nm, and the UC green light underwater communication application. Our work provides a strategy for constructing NIR II responsive UC materials and expanding the scope of applications.In summary, the Er2O3, Lu2O3, La2O3, Gd2O3, or Er2O3; 99.99%), and alkaline metal carbonates  were used as the primary materials. Carbon disulfide  was employed as the sulfur source.Lanthanide oxides  phosphors were fabricated by the gas-solid reaction method. In the typical synthesis process, the Ln2O3 (15 mmol) and M2CO3 (16.5 mmol) were mixed and thoroughly ground in an agate mortar for 30 min according to the 1:1.1 ratio, and the doping concentrations of Er3+ were 2, 5, 10, and 15 mol%, respectively. The mixture was transferred to the quartz boat and placed into the tube furnace. The samples were heated to 773 K under argon (Ar) atmosphere, and then saturated vapor of CS2 (40 kPa) was flowed in Ar at a flow rate of 50 mL/min. The as-prepared samples were obtained after calcining the mixture at 1073, 1173, 1273, or 1373 K for two hours and washing by water and ethanol for several times.MLnSThe X-ray diffraction (XRD) patterns of samples were characterized by the Shimadzu XRD-6000 X-ray diffractometer with a Cu Kα (λ = 0.1541 nm). Scanning electron microscopic (SEM) images of samples were performed by the Hitachi S-4800 scanning electron microscope. Transmission electron microscopic (TEM) and high-resolution transmission electron microscopic (HRTEM) images of samples were measured by using a JEM-2100F transmission electron microscope.−1 for λ = 532 nm. The UC luminescence spectra and decay curves were measured by a Jobin Yvon iHR550 monochromator equipped with R928 and H10220B-75 photomultiplier tubes from Hamamatsu Photonics using the light sources of laser diodes , and a pulsed work Horizon OPO laser, for signal collection from 400 nm to 1700 nm with a step length of 0.2 nm. The decay profiles were collected using a Tektronix DPO 5104 digital oscilloscope. A closed-cycle helium gas cryostat  equipped with a Lakeshore Model 331 temperature controller was used to control the sample temperature in the 10–300 K range. The underwater optical communication test was performed by a self-designed system, the signal transmitter was composed of a signal generator and 1532 nm LD, Jobin Yvon iHR550 monochromator and oscilloscope constitute the signal receiver. The UCQYs of MLnS2:Er3+ were directly measured using a commercial setup  equipped with an integrated sphere .All samples were pressed into the plate shape with the thickness of 2 mm for optical characterization. The absorption spectra of the samples were recorded on a Lambda 750 UV–Vis-NIR spectrophotometer . The Raman spectra were measured by the Renishaw InVia Raman Microscope (maximum power: 150 W) equipped 532 nm laser as a source of excitation, and the absolute accuracy is 0.55 cmSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Movie1Movie2


Gemtuzumab ozogamicin (GO) is indicated for treatment of relapsed/refractory (R/R) acute myeloid leukemia (AML). The QT interval, pharmacokinetics (PK), and immunogenicity following the fractionated GO dosing regimen have not been previously assessed. This phase IV study was designed to obtain this information in patients with R/R AML.2 on Days 1, 4, and 7 of each cycle, up to 2 cycles. The primary endpoint was mean change from baseline in QT interval corrected for heart rate (QTc).Patients aged ≥ 18 years with R/R AML received the fractionated dosing regimen of GO 3 mg/mFifty patients received ≥ 1 dose of GO during Cycle 1. The upper limit of the 2-sided 90% confidence interval for least squares mean differences in QTc using Fridericia’s formula (QTcF) was < 10 ms for all time points during Cycle 1. No patients had a post-baseline QTcF > 480 ms or a change from baseline > 60 ms. Treatment-emergent adverse events (TEAEs) occurred in 98% of patients; 54% were grade 3–4. The most common grade 3–4 TEAEs were febrile neutropenia (36%) and thrombocytopenia (18%). The PK profiles of both conjugated and unconjugated calicheamicin mirror that of total hP67.6 antibody. The incidence of antidrug antibodies (ADAs) and neutralizing antibodies was 12% and 2%, respectively.2/dose) is not predicted to pose a clinically significant safety risk for QT interval prolongation in patients with R/R AML. TEAEs are consistent with GO’s known safety profile, and ADA presence appears unassociated with potential safety issues.Fractionated GO dosing regimen .The online version contains supplementary material available at 10.1007/s00280-023-04516-9. Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid clonal expansion of undifferentiated myeloblasts, leading to anemia, neutropenia, and thrombocytopenia , 2. It iGemtuzumab ozogamicin (GO) is a CD33-directed antibody–drug conjugate consisting of a monoclonal antibody targeting CD33 linked to a cytotoxic derivative of calicheamicin. GO binds to CD33, an antigen expressed on most AML blast cells. Once bound, the GO–CD33 complex is internalized, and the cytotoxic component is released intracellularly, leading to DNA damage and cell death .GO is indicated in the United States for the treatment of newly diagnosed CD33-positive AML in adult and pediatric (≥ 1 month) patients, and for relapsed/refractory (R/R) CD33-positive AML in adult and pediatric (≥ 2 years) patients . In the 2 doses administered as 2-h intravenous infusions every 14 days  age of 67 [22–82] years) were enrolled in the study. Among enrolled patients, 27 (52.9%) had received 1 prior induction regimen; 11 (21.6%), 8 (15.7%), and 4 (7.8%) had received 2, 3, or > 3 prior induction regimens, respectively (see Supplementary Table 1 in the Online Resource). Five (9.8%) patients had received ≥ 1 allogeneic HSCT prior to GO treatment. There were 19 (37.3%) patients classified as adverse-risk according to the ELN 2017 recommendations . Of 51 pThe upper limit of the 2-sided 90% confidence interval (CI) for least squares (LS) mean differences in QTc using Fridericia’s formula (QTcF) was < 10 ms for all time points during Cycle 1, including those on D4 and D7 assessed in the primary analysis  were reported in 49 (98.0%) patients, the most common being febrile neutropenia (40.0%) and thrombocytopenia  patients had infections; 10 (20.0%) patients experienced grade 3–4 infections while 6 (12.0%) patients experienced grade 5 infections. Sixteen (32.0%) patients experienced hemorrhage; 2 (4.0%) patients experienced grade 3–4 hemorrhage, and no patients experienced grade 5 hemorrhage. The most-reported (≥ 10.0%) infectious TEAE was sepsis, occurring in 7 (14.0%) patients. The most-reported (≥ 5.0%) grade 3–4 infections were sepsis and pneumonia, in 3 (6.0%) patients each. The most-reported (≥ 5.0%) grade 5 infection was sepsis, in 4 (8.0%) patients. One (2.0%) patient experienced an infection (grade 3) that resulted in temporary discontinuation of GO and grade 4 sepsis that resulted in permanent discontinuation. Both infection and sepsis resulting in septicemia occurred simultaneously in the same patient; sepsis progressed, and the patient died. Both events were assessed as unrelated to GO by the investigator. The most-reported (≥ 10.0%) hemorrhage TEAE was epistaxis in 5 (10.0%) patients. One (2.0%) patient had grade 3 gastric hemorrhage and 1 (2.0%) patient had grade 4 traumatic intracranial hemorrhage. No hemorrhage resulted in permanent or temporary discontinuation of GO.Three (6.0%) patients temporarily discontinued GO due to TEAEs, including increased alanine aminotransferase, increased transaminases, and infection, each in 1 (2.0%) patient. Two (4.0%) patients permanently discontinued GO due to TEAEs, including pyrexia and sepsis, each in 1 (2.0%) patient.Grade 5 TEAEs occurred in 16 (32.0%) patients, of whom 5 (10.0%) died of disease progression and 4 (8.0%) died of sepsis. Multiple organ dysfunction, pyrexia, atypical pneumonia, COVID-19 pneumonia, AML (consistent with disease progression), respiratory failure, and capillary leak syndrome  accounted for the deaths of the remaining patients.One patient experienced grade 3 atrial fibrillation and supraventricular tachycardia, which were considered unrelated to GO. No patients experienced grade ≥ 4 cardiac conduction TEAEs. VOD/SOS was not reported; however, 1 patient experienced treatment-related grade 5 CLS associated with pleural effusion, ascites, hyperbilirubinemia, and endothelial syndrome.A total of 43 (86.0%) patients experienced a shift from grade ≤ 2 at baseline to grade 3 or 4 post-baseline in hematology and coagulation laboratory parameters. The most common were decreased white blood cell count in 25/49 (51.0%) patients, decreased lymphocyte count in 24/49 (49.0%) patients, and anemia in 21/50 (42.0%) patients.2 , exposures as measured by geometric mean area under the plasma concentration–time profile (AUC) from time zero to 336 h post-dose and maximum plasma concentration (Cmax) were 461,500 pg h/mL and 11,740 pg/mL; 1639 pg h/mL and 58.8 pg/mL; and 26,820 ng h/mL and 585.6 ng/mL for conjugated calicheamicin, unconjugated calicheamicin, and hP67.6 antibody, respectively (see Supplementary Table 2 in the Online Resource). In general, the concentration–time profiles of conjugated calicheamicin and unconjugated calicheamicin are similar to that of total hP67.6 antibody for Cycle 1, Day 1 and Cycle 1, Day 7.Following administration of multiple fractionated infusions of GO at 3 mg/mOf 50 patients treated with GO, 12 (24.0%) had positive ADAs against GO at baseline. This was likely due to pre-existing host antibodies that were cross-reactive with GO. There was no treatment-boosted ADA response.Treatment-induced ADA was detected in 6 (12.0%) patients. No patients experienced anaphylaxis, hypersensitivity, or other clinical sequelae related to ADA. Among the 6 patients positive for treatment-induced ADA, 2 (33.3%) experienced an infusion-related reaction, both pyrexia (grade 1 and grade 3). Among the 44 patients negative for treatment-induced ADA, 7 (15.9%) patients experienced infusion-related reactions. All were grade 1 or 2, except for 1 instance of grade 3 urticaria.Of the 18 patients who had positive ADAs against GO, none were positive for NAbs at baseline; therefore, no patients had a treatment-boosted response. Treatment-induced NAbs were detected in 1 (2.0%) patient.The best overall response was CR in 2 (3.9% [95% CI 0.5–13.5]) patients and CRi in 3 (5.9% [95% CI 1.2–16.2]) patients (see Supplementary Table 3 in the Online Resource), with an overall rate of 9.8% (95% CI 3.3–21.4). CR + CRi was achieved by 3 (6.0%) of the 50 patients in Cycle 1. Nine of the 50 patients receiving Cycle 1 proceeded to Cycle 2, of whom 3 (33.3%) patients achieved CR + CRi in Cycle 2. Among the 5 patients who achieved CR or CRi (see Supplementary Table 3 in the Online Resource), there were 2 and 3 patients stratified with ELN favorable or intermediate risk, respectively. One patient who achieved CR/CRi had previously received 2 HSCTs, 1 had previously been treated with 3 induction regimens, and the remaining patients had received 1 or 2 prior induction regimens.The median OS was 2.8 (95% CI 1.7–4.2) months, with 45 deaths reported . Of the 45 deaths, disease progression was the most common cause in 35 (77.8%) patients.2 on Days 1, 4, and 7) is not predicted to pose a clinically significant safety risk for QT interval prolongation in patients with R/R AML. The primary endpoint of post–baseline dose QTc was non-inferior to baseline, with the upper limit of the 2-sided 90% CI for the LS mean change from baseline falling below the 20 ms threshold of clinical concern for oncology drugs [n = 2743 observations). Patients with R/R ALL received 1.8 mg/m2 per cycle in divided doses (considered therapeutic) and patients with R/R NHL received 1.8 mg/m2 per cycle as a single dose (considered supratherapeutic). Although the study revealed that QTc had a small but positive correlation with inotuzumab ozogamicin concentration, it was not predicted to pose a clinically significant safety risk for QT interval prolongation in patients with R/R ALL and NHL [This study showed that fractionated dosing of GO (3 mg/mgy drugs . There wgy drugs . This cogy drugs  that use and NHL .2), total AUC of hP67.6 antibody over the course of treatment was 25%, and Cmax was 24% of the original dosing regimen. Since Cmax has been associated with risk of certain AEs , the fractionated regimen was expected to have an improved safety profile versus the previously used regimen.As part of a review of the PK for the prior marketing application of GO, a population PK model was used to simulate exposure for the fractionated GO regimen, based on 8 previous trials in patients with R/R AML or de novo AML . The PK Cmax of total antibody following the third dose from the current clinical study were 26,820 ng·h/mL and 586 ng/mL  versus 27,997 ng·h/mL and 654 ng/mL (simulated PK), suggesting that the PK parameters were comparable between simulated and estimated approaches.This current study enables comparison of the data from the simulated PK model with clinical PK data. The geometric mean AUC and Safety data were consistent with the known safety profile of the fractionated regimen of GO , 9. TEAEIn this study, the incidence of ADAs and NAbs was 12% and 2%, respectively. None of the patients experienced anaphylaxis, hypersensitivity, or other clinical sequelae related to ADAs, suggesting that the presence of ADAs to GO did not appear to be associated with any potential safety issues following GO treatment.A best overall response based on CR + CRi was achieved in 9.8% of patients, with a median OS of 2.8 months; both inferior than previously reported in the MyloFrance-1 study [2/dose) on QT interval prolongation is minimal in patients with R/R AML. TEAEs were consistent with the known safety profile for GO, and the presence of ADAs did not appear to be associated with any potential safety issues.In conclusion, this study showed that the effect of fractionated dosing of GO (3 mg/mSupplementary file1 (DOCX 69 KB)Below is the link to the electronic supplementary material.


Pregnancy comprises a period in a woman’s life in which the circulatory system is subjected to hemodynamical and biochemical changes. During this period, while restructuring blood vessels and exchanging maternal-fetal products there is an increased risk of developing chronic venous disease (CVD), which may have an echo in life after childbirth for both mother and child. Previously, we investigated that pregnancy-associated CVD involves changes in placental architecture at angiogenesis, lymphangiogenesis and villi morphology compared with healthy controls (HC) with no history of CVD. We aimed to more deeply investigate the oxidative stress response in placenta from women with CVD versus HC through several markers . An observational, analytical, and prospective cohort study was conducted on 114 women in their third trimester of pregnancy (32 weeks). A total of 62 participants were clinically diagnosed with CVD. In parallel, 52 controls with no history of CVD (HC) were studied. Gene and protein expressions of NRF2, KEAP1, CUL3, GSK-3β were analyzed by real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry. Nrf2 gene and protein expression was significantly greater in placental villi of women with CVD, while Keap1, CUL-3 and GSK-3β gene and protein expressions were significantly lower. Our results defined an aberrant gene and protein expression of Nrf2 and some of their main regulators Keap1, CUL-3 and GSK-3 β in the placenta of women with CVD, which could be an indicator of an oxidative environment observed in this tissue. Pregnancy comprises a period in a woman’s life in which the circulatory system is subjected to hemodynamical, mechanical and biochemical changes ,2,3,4. DPrevious studies have noticed that CVD supposes a systemic stress during pregnancy that impairs placenta structure and function. For instance, it has been observed that pregnancy-associated CVD is associated with changes at placental villous architecture and enhanced apoptotic death . LikewisNFE2L2) is a critical transcription factor that is involved in cellular stress response, modulating the expression of several gene products [In this sense, the nuclear factor erythroid 2-related factor 2  in placenta from women with CVD and compare them with placenta from HC for a deeper understanding of the pathophysiological network in CVD and its possible implications, especially in pregnancy.In the present work, an observational, analytical, prospective study was carried out with 114 women in the last trimester of pregnancy. Of them, 62 with a clinical diagnosis of CVD according to the CEAP classification  and 52 wPrior to enrolment, each patient was properly informed, providing signed written consent. This work passed the Clinical Research Ethics Committee of the Central University Hospital of Defense University of Alcalá (37/17) and was performed according to the ethical principles of autonomy, beneficence, non-maleficence and distributive justice, as well as following the regulations of Good Clinical Practice, the principles of the Declaration of Helsinki (2013) and the Oviedo Convention (1997).Women included in our study were >18 years old, with medical presentation of CVD (CEAP ≥ 1) in their lower limbs during the third trimester. Exclusion criteria included women with previous evidence of (1) arterial hypertension; (2) venous malformations; (3) heart, kidney or lung failure; (4) BMI ≥ 25; (5) clinical diagnosis of type 1/type 2/gestational diabetes mellitus or other endocrine diseases; (6) autoimmune diseases; (7) active infectious processes; (8) habitual alcohol (≥1 unit/day), tobacco (≥1 cigarette/day), or drug consumption; (9) preeclampsia and/or HELLP syndrome; (10) fetal growth restriction of known causes; (11) Presence of placental infarction, avascular villi, delayed villus maturation, or chronic villitis; (12) Evidence of CVD before pregnancy and (13) the appearance of any exclusion criteria until delivery.In third trimester consultation, clinical history for each woman was revised and physical examinations were accomplished. Lower limbs ultrasounds were evaluated with Eco-Doppler  at 7.5 MHz.With respect to the sociodemographic characteristics of the sample studied, there were no significant differences between CVD and their HC regarding the number of pregnancies: 33 (53.2%) in the event of women with CVD and 19 (36.5%) for the HC group . Further® solution . Thereafter, the samples were processed in a class II laminar flow hood  in a sterile environment. Afterwards, samples were preserved in 1 mL of RNAlater® at −80 °C for later processing for gene expression study.Postpartum placental biopsies were collected from all 114 patients. For each patient sample, five placental pieces were obtained using a scalpel to include diverse mixed cotyledons and then they were separated in two different sterile tubes: one containing minimal essential medium  with 1% antibiotic/antimycotic  and another with RNAlaterThen, conserved MEM samples were washed and rehydrated five times in MEM free of antibiotics to eliminate erythrocytes. Then, by using a scalpel they were divided into 2 cm fragments and fixed in F13 , according to collected protocols . SubsequFirst, we extracted RNA through the guanidinium thiocyanate-phenol-chloroform method as described in previous studies . This me2) ; 1 µL RT enzyme ; 2 µL of 10 mM deoxyribonucleotide triphosphate; 2 µL of 0.1 M dithiothreitol; 1.7 µL of DNase and RNase free water; 0.5 µL of RNase inhibitor (RNase Out).Complementary DNA (cDNA) was synthesized by reverse transcription (RT) from 50 ng/µL of RNA samples. A total of 4 µL of each sample was mixed with 4 µL of 0.25 µg/µL oligo-dT solution  and afterwards placed at 65 °C for 10 min in a dry bath , leading to the denaturation of RNA. The samples were then placed on ice and 10 µL of reverse transcription mix containing the following products: 2.8 µL of First Strand Buffer 5X . The samples were then incubated at 37 °C for one hour and fifteen min in order to facilitate cDNA synthesis. At this point, the temperature was increased to 70 °C and held for 15 min, causing reverse transcriptase denaturation. Subsequently, the temperature was gradually lowered to 4 °C. Negative reverse transcription was equally carried out to ensure the absence of genomic DNA contamination in RNA samples in which the M-MLV RT enzyme was substituted by DNase- and RNase-free water. cDNA produced at room temperature was diluted 1:20 in DNase- and RNase-free water and stored at −20 °C until further use.® Green Supermix —was mixed with 1 µL each forward and reverse primers, and 3 µL of DNase and RNase-free water, which were then added to a MicroAmp® 96-well plate . Thermocycling conditions were used: Initial denaturation for 10 min at 95 °C, denaturation for 15 s at 95 °C, annealing at variable temperatures depending on the melting temperature of each primer pair for 30 s, and elongation at 72 °C for 1 min, for 40–45 cycles. Then, a dissociation curve for 15 s at 95 °C, 1 min 60 °C, 15 s 95 °C, and 15 s 60 °C was developed. Fluorescence detection was performed at the end of every repeat cycle (amplification) and at the different steps of the dissociation curve. The data collected from the aforementioned genes were included in a standard curve made by serial dilutions of a mixture of the samples, that were included in each plate according to the constitutive expression of TBP (in agreement with the manufacturer’s protocols). This RT-qPCR was performed twice in all samples of placenta tissue.Specific primers for the selected genes  were desAvidin-biotin complex with avidin peroxidase was used for antigen/antibody reactions detection . Immunoh®-Peroxidase  was added for one hour at room temperature (1:200 dilution in PBS). Finally, chromogenic diaminobenzidine (DAB) substrate kit , was used to determine protein expression level. This kit was prepared immediately before exposure .Placental tissues were incubated with the primary antibody for one hour and a half . SubsequThe use of the peroxidase chromogenic substrate for 15 min at room temperature allowed for the detection of the signal in form of brown stains. For each section, negative controls were assigned for the different proteins, replacing incubation with primary antibody for a PBS solution. Carazzi hematoxylin was used for 15 min to achieve contrast in all tissues.A total of 5 different sections and 10 areas of view were randomly examined for every patient of CVD and HC groups. A patient was reported as positive when the stained mean area in the studied sample was ≥5% of the total, following the immunoreactive score (IRS) as established in previous studies . Immunos® v6.0  program was utilized. Mann–Whitney U test allow the comparison between both groups, being expressed as the median ± SEM. Significant values were established as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).For the treatment of statistical data, GraphPad Prismp < 0.0001 [CVD = 8.863 ± 2.893, HC = 5.734 ± 1.168, p < 0.0001 [CVD = 1.976 ± 0.617, HC = 1.115 ± 0.450, Our results show a statistically significative augmentation in NRF2 gene expression in the placental tissue of women undergoing CVD during pregnancy, *** p = 0.0015 [CVD = 4.816 ± 1.238, HC = 5.728 ± 1.158, p = 0.0021 [CVD = 1.173 ± 0.583, HC = 1.538 ± 0.576, We observed how the placental villi of women with CVD showed a significant decrease in KEAP1 gene expression compared to HC, ** p < 0.0001 [CVD = 4.904 ± 1.181, HC = 5.952 ± 1.376, p < 0.0001 [CVD = 1.060 ± 0.533, HC = 1.740 ± 0.686, Our results show a significant decrease in CUL3 gene expression in the placental villi of women with CVD compared to HC, *** p = 0.0015 [CVD = 4.816 ± 1.238, HC = 5.728 ± 1.158, p = 0.0021 [CVD = 1.173 ± 0.583, HC = 1.538 ± 0.576, In this sense, we have observed how the placental villi of women with CVD showed a significant decrease in the gene expression of GSK-3β compared to HC, ** Our results indicate an aberrant expression of the transcription factor Nrf2 and its main regulators CUL-3, Keap1 and GSK-3β in the placenta tissue of women with CVD. This study gains further insights into the pathophysiological changes occurred in this organ during pregnancy, supporting the relevance of suffering from CVD as a possible obstetric complication for maternofetal wellbeing, as suggested by previous studies ,33.Firstly, we detected an enhanced Nrf2 expression in the placenta of women with CVD in comparison with their healthy controls. Nrf2 is a major transcription factor which regulates the expression of several genes involved in the cellular stress response. Specifically, Nrf2 participates in the “phase 2 response”, also known as “the electrophile counterattack response”, modulating the expression of some “vitagenes”, such as heme oxygenase 1, thioredoxin or thioredoxin reductase, and counteracting the damaging effects of oxidants and electrophiles . Thus, NThere are several products that can regulate Nrf2 activation and function. For instance, the PI3K/Akt/mTOR pathway appears to be a major activator of basal and induced Nrf2 activities . An enhaConversely, there are some prominent inhibitors of Nrf2 activation, including CUL-3, Keap1 and GSK-3β , which aFinally, GSK-3β is a pivotal molecule during pregnancy involved in several biological processes in the placenta . DiffereOverall, our study has defined an aberrant gene and protein expression of Nrf2 and some of their main regulators Keap1, CUL-3 and GSK-3β in the placenta of women with CVD. As showed in


P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expression, whereas it induced a decrease in the dual specificity phosphatase 6 transcripts, suggesting that BCI-dependent inhibition of dual specificity phosphatase 1 may be responsible for the increase in p38 and JNK phosphorylation. The weaker ERK phosphorylation induced by BCI was reversed by p38 inhibition, indicating that this MAPK is involved in the regulatory loop that dampens ERK activity. The PP2A phosphatase appears to be implicated in the p38-dependent dephosphorylation of ERK1/2. In addition, the PTEN phosphatase inhibition also prevented ERK1/2 dephosphorylation, probably through p38 downregulation. By contrast, inhibition of the p53 nuclear factor decreased ERK phosphorylation, probably enhancing the activity of p38. Finally, the inhibition of either p38 or Sp1-dependent transcription halved the increase in P2X7 receptor expression induced by BCI. Moreover, the combined inhibition of both p38 and Sp1 completely prevented the effect exerted by BCI. Together, our results indicate that dual specificity phosphatase 1 acts as a novel negative regulator of P2X7 receptor expression in neuroblastoma cells due to the downregulation of the p38 pathway. The activity of the classic ERK1/2 group is mainly triggered by growth factors and mitogens that regulate cell growth, differentiation and development (Among the intracellular pathways activated by P2RX7 and associated to cell proliferation are the cascades of mitogen-activated protein kinases (MAPKs). MAPKs are Ser/Thr kinases that can phosphorylate a large number of downstream effectors located in the cell nucleus and cytoplasm, but also associated with membranes or the cytoskeleton . In mammelopment . By contelopment . The magelopment . Althougelopment . MKPs beelopment .MKPs, and particularly DUSP1 and DUSP6, have been proposed as cancer biomarkers, since aberrant expression of these phosphatases has been found in a wide variety of human cancers, including neuroblastoma. . Importa® conjugate antibodies (A31572 and A11001) were from Molecular Probes.The commercial antibodies used in the study were raised against: P2RX7 ; Sp1 ; SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (#4668), p38 (#9212), phospho-p38 (Thr180/Tyr182) (#4511) and phospho-Erk1/2 (Thr202/Tyr204) (#4370), all from Cell Signaling; Erk (sc-1647) and DUSP1 (sc-370) from Santa Cruz Biotechnology; DUSP6 (ab76310) from Abcam; GAPDH (G9545) and α-tubulin (T5168) from Sigma-Aldrich. The horseradish peroxidase-conjugated secondary antibodies were from Dako (P0448 and P0260) and the secondary Alexa Fluorα (p53) and VO-OH Pic (PTEN), all from Tocris Bioscience; BCI (DUSP1/DUSP6), okadaic acid (PP2A) and BBG (P2RX7) were supplied by Merck-Millipore; mithramycin A (Sp1) and actinomycin D were purchased from Sigma-Aldrich. Other chemicals were supplied by Merck Millipore or Sigma-Aldrich.The specific inhibitors of MAPKs and other proteins used were: SP600125 (JNK), SB202190 (p38), U0126 (MEK), 666-15 (CREB), SR11302 (AP-1), KJ Pyr 9 (Myc), cyclic Pifithrin ® and grown in DMEM (Gibco), supplemented with 10% FBS (Invitrogen), Glutamax®, 100 U/ml penicillin and 100 μg/ml streptomycin, all purchased from Gibco. Cell cultures were grown at 37°C in a humidified atmosphere containing 5% CO2. To carry out the experiments, the cells were seeded in complete DMEM medium for 12 h and then transferred to serum-free medium (SFM) for the time periods indicated.Mouse Neuro-2a  and human SH-SY5Y (ATCC HTB-11) neuroblastoma cell lines were obtained from ATCC® qPCR readymix (Sigma), 5 µl of the cDNA generated, gene-specific primers and TaqMan MGB probes for mouse P2RX7, Sp1, DUSP1, DUSP6, and GAPDH . Fast thermal cycling was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) as follows: pre-denaturation at 95°C for 20 s, followed by 40 cycles each of 95°C for 1 s and 60°C for 20 s. The results were normalized by parallel amplification of the endogenous control GAPDH.Total RNA was extracted from cells using Speedtools total RNA extraction kit (Biotools), according to the manufacturer’s instructions. After digestion with TURBO DNase (Ambion), the total RNA was quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific), and 1 µg of RNA was reversed transcribed using M-MLV reverse transcriptase in the presence of 6 µg of random primers and 350 µM dNTPs (Invitrogen). Quantitative real-time PCR reactions (qPCR) were carried out using LuminoCtL-Lysine (Biochrom) and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). The cells were then washed in PBS and incubated for 1 h at RT in blocking solution . Subsequently, the cells were incubated for 2 h at RT with the primary antibodies against P2RX7 (1:100), DUSP1 (1:100), DUSP6 (1:100), or α-tubulin . After washing twice in PBS containing 2% BSA, the cells were incubated for 1 h at RT with appropriate Alexa Fluor™ conjugated secondary antibodies: donkey anti-rabbit IgG and goat anti-mouse IgG at a 1:500 dilution. Finally, the cells were washed in PBS, the nuclei were counterstained with DAPI, the coverslips were mounted with Aqua Poly/Mount reagent (Polysciences) and confocal images were acquired on a TCS SPE microscope with a 63× Apochromat NA = 1.3 oil objective lens (Leica Microsystems) or with an Eclipse TE2000-E microscope with a 20x Nikon Fluor NA = .45 air objective lens (Nikon). Images were quantified using the ImageJ free software.N2a were cultured on coverslips precoated with 0.01 mg/ml poly-2 at 37°C. Subsequently, an equal volume of solubilization buffer was added (10% Triton X-100 plus 0.1 N HCl in anhydrous isopropanol) and after mixing smoothly for 30 min at RT using an orbital shaker, the cell extracts were finally collected, and the absorbance was quantified at 570 nm. The values were normalized to those obtained from untreated cells, set as 100% metabolic activity.N2a were cultured overnight in complete DMEM and on the following day, the cells were transferred to SFM containing either 5 µM or 10 µM BCI. After 24 h, the metabolic activity was quantified using a MTT assay as follows. The culture medium was replaced with PBS containing the tetrazolium salt 3--2,5-diphenyltetrazolium bromide  and the cells were maintained for 2 h in a humidified atmosphere containing 5% CO® Viability/Cytotoxicity Assay (Invitrogen), replacing the culture medium with PBS containing live-cell staining dye  and dead-cell staining dye (2 μM ethidium homodimer-1). Then, cells were incubated for 30 min in a humidified atmosphere containing 5% CO2 at 37°C, and fluorescence images were acquired on an Eclipse TE2000-E microscope with a 20x Nikon Fluor NA = .45 air objective lens (Nikon).N2a cells were cultured overnight in complete DMEM and then transferred on the following day to SFM containing either 5 µM or 10 µM BCI. After 24 h, cell viability was analyzed with the LIVE/DEADTo estimate the relative protein phosphorylation, N2a cells cultured overnight in complete DMEM were transferred to SFM for 30 min and then treated for 1 h with BCI (10 μM) or with the vehicle alone. Cell extracts were prepared using the Human Phospho-Kinase Array Kit  following the manufacturer’s instructions. Although this array was initially developed to identify human phosphoproteins, excellent cross-reactivity with rodent proteins has been demonstrated . After wN2a cells were lysed for 1 h at 4°C in lysis buffer containing 50 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P40 (pH 7.4), Complete™ Protease Inhibitor Cocktail Tablets (Roche Diagnostics), 1 mM Sodium Orthovanadate (Sigma) and 1.5 µM Okadaic Acid . The supernatant was harvested by centrifugation, quantified, and equal amounts of protein extract (20 μg) were resolved by 10% Tris-Glycine SDS-PAGE and the proteins transferred to nitrocellulose membranes (Amersham GE). The membranes were then blocked for 1 h at RT with PBS + 0.1% Tween-20 (PBST) buffer containing either 5% skimmed milk or 3% BSA depending on the primary antibody used. The membranes were probed overnight at 4°C with primary antibodies against P2RX7 , Sp1 , JNK , phospho-JNK , p38 , phospho-p38 , Erk , phospho-Erk1/2 , and GAPDH . The next day the membrane was washed twice with PBST and antibody binding was detected with HRP-conjugated secondary antibodies . Protein bands were visualized with an ECL HRP Chemiluminescent Substrate (Perkin Elmer) on an ImageQuant LAS 500 Model and densitometric analysis was performed with ImageQuant software (Amersham GE) using GAPDH as an internal reference.t-test for two-group comparisons or one way-ANOVA with the Dunnett’s post hoc test. For multiple comparisons, one-way ANOVA analyses were corrected with Sidak’s post hoc test . A value of p ≤ .05 was considered statistically significant.All the data are shown as the mean ± standard error of the mean (SEM) of a minimum of three independent experiments performed in duplicate or triplicate. The data were analyzed using unpaired P2rx7 gene expression, reaching transcript levels up to 8-fold those in the control cells exposed to the vehicle alone  for 24 h or 48 h, or with the vehicle alone (control), and the levels of P2RX7 were quantified by immunofluorescence  and immuTo shed light on the intracellular signaling coupled to the effect of BCI, we examined cell-signaling in an unbiased manner using an array platform that detects multiple protein phosphorylation events . N2a celAs the array analysis was carried out only once, the phosphorylation state of MAPKs was validated in conventional Western blots . As suchAs mentioned previously, BCI is an allosteric inhibitor of DUSP1/6 phosphatases that mainly control the activity of MAPKs through the dephosphorylation of their activation residues. When the DUSP1 and DUSP6 transcripts in N2a cells exposed to BCI (10 µM) for 1, 2, 4, 6, 8 or 24 h in SFM were evaluated, BCI provoked a significant increase in the DUSP1 transcripts relative to the cells exposed to the vehicle alone. Moreover, BCI fully impaired the upregulation of DUSP6 expression induced by serum deprivation in control cells . As expeThe dephosphorylation of ERK induced by BCI in N2a cells must involve the participation of protein phosphatases other than DUSP1/6 and several studies reported that p38 inhibits ERK activity by enhancing MEK1/2 dephosphorylation through protein phosphatase PP2A . MoreoveThe data obtained that far demonstrated that BCI produces a strong increase in P2RX7 transcripts and protein in N2a cells, and the inhibition of DUSP1 by BCI appears to be responsible for the increase in p38 and JNK phosphorylation. To further explore the signaling pathway involved in the upregulation of P2RX7 expression induced by BCI, N2a cells in SFM were exposed for 10 min to inhibitors of either p38  or JNK  before treating them with BCI (5 µM) for 24 h. Remarkably, inhibition of p38 partially impaired the upregulation of P2RX7 induced by BCI , whereasP2rx7 gene expression in neuroblastoma (de novo synthesis of the transcript.Specificity protein 1 (Sp1) is a transcription factor that plays a crucial role in the control of blastoma . Mithramblastoma . Hence, P2rx7 gene, showing that this 5′-regulatory region not only contains several Sp1 motifs but also, it contains other putative regulatory elements including c-AMP-responsive element binding proteins (CREB), activator protein 1 (AP-1), and Myc/Max heterodimer binding sites , AP-1 , Myc  or p53  before treating them for 24 h with BCI (5 µM). However, none of the drugs tested diminished the upregulation of P2RX7 expression provoked by BCI  binding sites (The signaling pathway involved in the upregulation of P2RX7 expression induced by BCI was also explored here. The increase of P2RX7 expression required ng sites . Furtherng sites . Howeverng sites . Other png sites . Alternang sites . We alsong sites . Furtherng sites . Here, tng sites . Alternang sites .P2rx7 gene expression. Based on preclinical studies reporting a strong oncogenic role of this receptor, an increase of P2RX7 facilitates cell proliferation even in scenarios of trophic deprivation, promoting tumor cell proliferation, energy production, migration and invasiveness of cancer cells (In summary, our data highlight DUSP1 as a novel negative regulator of P2RX7 expression in neuroblastoma cells due to the downregulation of the p38 pathway. This negative feedback mechanism could be particularly relevant in tumor microenvironment, where the activation of the p38 may upregulate er cells . The p38er cells . Moreoveer cells . Enhanceer cells . Unfortuer cells . The coner cells . To conc


The oxidation behavior of TaWSiN films was investigated after annealing at 600 °C in a 1%O2–Ar atmosphere, and cone-like Ta0.3W0.7O2.85 oxides formed and extruded from the TaWSiN films.This study explored the structural characteristics, mechanical properties, and oxidation behavior of W-enriched TaWSiN films prepared through co-sputtering. The atomic ratios [W/(W + Ta)] of the as-deposited films maintained a range of 0.77–0.81. The TaWSiN films with a Si content of 0–13 at.% were crystalline, whereas the film with 20 at.% Si was amorphous. The hardness and Young’s modulus of crystalline TaWSiN films maintained high levels of 26.5–29.9 GPa and 286–381 GPa, respectively, whereas the hardness and Young’s modulus of the amorphous Ta The Ta17W55N28 and Ta8W68N24 films with [W/(Ta + W)] ratios of 0.76 and 0.89, respectively, formed a W2N-dominant structure and exhibited a high hardness level of 29.9–31.9 GPa and a high Young’s modulus of 381–391 GPa. In contrast, transition metal nitride films with Si addition formed nanocomposite structures, such as TiSiN  phase.The crystalline T2 texture, high hardness values of 26.5–29.9 GPa, and Young’s modulus values of 286–381 GPa. Moreover, the mechanical properties of these W2N-dominant TaWSiN films followed the Hall–Petch relationship according to the variation in average grain size. In contrast, the amorphous Ta7W33Si20N40 films with a high Si content of 20 at.% exhibited low hardness and Young’s modulus values of 18.2 and 229 GPa, respectively.The crystalline TaWSiN films with a W/(W + Ta) ratio of 0.77–0.81 and a Si content of 0–13 at.% exhibited a W0.3W0.7O2.85 oxides formed on both the surfaces of the crystalline Ta10W38Si13N39 films and amorphous Ta7W33Si20N40 films after they were annealed at 600 °C in a 1% O2–Ar atmosphere. The cone oxides consisted of laminated sublayers and each sublayer comprised granular grains with diameters of 10–20 nm. Beneath these oxides, the films maintained structures similar to those of the as-deposited state.Cone-like Ta2N-dominant TaWSiN films possess superior mechanical properties to TaSiN and WSiN films, and can be utilized as hard coatings.W


Portulaca oleracea L. (known as purslane), is a nutritious facultative C4 halophyte. Recently, it has been successfully grown indoors under LED lightings by our team. However, basic understanding about the impacts of light on purslanes are lacking. This study aimed to investigate the effects of light intensity and duration on productivity, photosynthetic light use efficiency, nitrogen metabolism and nutritional quality of indoor grown purslanes. All plants were grown in 10% artificial seawater hydroponically under different photosynthetic photon flux densities (PPFDs) and durations and thus different daily light integrals (DLI). They are, L1 ; L2 ; L3 ; L4 , respectively. Compared to L1, higher DLI promoted root and shoot growth and thus increased shoot productivity by 2.63-,1.96-, 3.83-folds, respectively for purslane grown under L2, L3, L4. However, under the same DLI, L3 plants  had significantly lower shoot and root productivities compared those with higher PPFDs but shorter durations (L2 and L4). While all plants had similar total chlorophyll and carotenoid concentrations, CL (L3) plants had significantly lower light use efficiency (Fv/Fm ratio), electron transport rate, effective quantum yield of PSII, photochemical- and non-photochemical quenching. Compared to L1, higher DLI with higher PPFDs (L2 and L4) increased leaf maximum nitrate reductase activity while longer durations increased leaf  Portulaca oleracea L. (known as purslane) is one of the most nutritious halophytes owing to its high content of antioxidants , supplied by red-LED to blue-LED ratio of 2.2 (defined as LED R/B 2.2). Our findings suggest that a 100 mM of NaCl is the most suitable saline condition to grow purslane in order to achieve higher productivity and better quality using our indoor farming systems . Using three different vegetables namely Kai Lan , Nai Bai (B. chinensis L.) and mizuna (B. juncea var. japonica), we , we  carried uctivity .-2 day-1), which describes the number of photosynthetically active photons that are delivered to a specific area over a 24-hour period. Many plant growth traits are better related to DLI than to instantaneous PPFD levels at any given time (Ocimum basilicum) grown under higher DLIs of 12.9, 16.5, or 17.8 mol m-2 day-1 had higher shoot FW, photosynthesis, stomatal conductance and transpiration than under lower DLI of 9.3 mol m-2 day-1. It was also found increases in soluble sugar and phenolic contents in basil plants grown under higher DLIs than under lower DLIs (Lactuca sativa L. cv. Ziwei), there was a positive linear correlation between DLI and leaf biomass with increasing tDLI from 6.48 to 17.28 mol m-2 day-1 (Both light intensity and duration (photoperiod) determine daily light integral, DLI , CL enhanced yield and nutritional quality measured by pigments, antioxidant compounds. However, nitrate (NO3-) was significantly reduced in rocket leaves grown under CL. This result implies that CL may increase nitrate reductase activity (NRA) and the reduction of Tropaeolum majus L.) under the same DLI but different photoperiods with different PPFDs, Continuous lighting (CL) maximizes the light duration while minimizes the light intensity at the same DLI. The application of CL in a vertical indoor farm is considered an effective method to save both lighting and cooling costs . Howeverf NO3− . In the 4 plant, it is able to adapt to high light conditions via the C4 photosynthetic pathway  were used in this study. Seedlings were inserted into polyurethane cubes after germination for 4 days. All seedlings were placed under a PPFD of 100 μmol m−2 s−1 supplied by high-pressure sodium lamps for 4 weeks before transplanting onto indoor hydroponic systems. Plants were then grown in 10% artificial seawater (ASW) with full strength nutrient solution. To make up a 10% ASW, a salinity of 3.3 ppt was prepared by dissolving 28.8g of Red Sea Salt®  in 8 L full strength nutrient solution. The conductivity of and the pH of the nutrient solutions were 2.2 ± 0.2 mS cm−1 and 6 ± 0.2, respectively. The plants were grown under four different light intensities and durations: L1 ; L2 ; L3 ; L4 . The same LED spectral quality with red/blue LED ratios of 2.2  was used for all the different light conditions. The light spectral distribution was reported by °C/23°C and 60%/80% (day/night) respectively.Seeds of purslane /leaf dry weight (g)  and root were separated for FW measurements. The total leaf number was recorded before determining the total leaf area (TLA) of each plant using leaf area meter . To obtain DW, shoot and roots were wrapped separately in aluminium foils and dried in the oven at 80°C for 4 days. Specific leaf area (SLA) was calculated using leaf area (cmight (g) .v/Fm ratios  of dark-adapted attached leaves were measured using the Plant Efficiency Analyser  during mid-photoperiod. The ETR, ΔF/Fm’, qP and NPQ were determined from detached leaf using the IMAGING PAM MAXI  at 25°C in the laboratory. The details of Fv/Fm ratio, ETR, ΔF/Fm′, qP and NPQ measurements were described by FChl a, b, and Car were calculated according to Leaf samples (0.05 g) were harvested on day 12 after transplanting and soaked in 5 ml of N, N-dimethylformamide in the dark for 48 h at 4°C . The abs3- to nitrite ( Plant samples were dried at 80°C for 4 days. Dried samples (0.01 g) were grounded with 10 ml deionised water and were then incubated at 37°C for two hours. Sample turbidity was then removed by vacuum filtering the mixture through a 0.45 µm-pore-diameter membrane. The flow injection analyser  was used to measures The frozen sample stored at −80°C was powdered in liquid nitrogen and ground with 4 ml of ice-cold extractiPon buffer with the presence of 0.2 g/g FW PVPP. The extraction buffer included 0.25 M Tris-HCl (pH 8.5), 3 mM dithiothreitol (DTT), 10 µM flavin adenine dinucleotide (FAD), 1 µM sodium molybdate, 1 mM Ethylenediamine-tetra-aceticacid (EDTA). The extracts were centrifuged at 18,000 g for 20 min at 4°C. The supernatant was used to determine the NRA immediately.In vitro NADH:NRA assay was derived from act was determined by assaying NR either with Mg2+ (10 mM), or with EDTA (15 mM). In all cases, the total reaction medium was 700 µl which contained 50 mM Hepes-KOH (pH 7.5), 1 mM DTT, 10 µM FAD, 10 mM KNO3, 0.2 mM NADH, NR extraction, and 10 mM MgCl2 or 15 mM EDTA. The reaction was started by adding of 100 – 200 µl NR extraction. Incubation was performed at 25°C for 20 min, and the reaction was then terminated by the addition of an equal volume (700µl) of Sulfanilamide (1%(w/v) in 3 N HCl) and the naphthylethylens-diamine dihydrochloride (0.02% w/v). After 30 min at room temperature, the absorbance was read at wavelength of 540nm. The blank was identical to the samples, but the NR extracts were boiled for 5 min before the addition. NRAmax was expressed as µmol nitrite (-1 g-1 FW. The NRact is defined as the activity measured in the presence of 10 mM MgCl2 divided by the activity measured in the presence of 15 mM EDTA (expressed as a percentage).The details of leaf TSP extraction were described in Dried plant tissues of 0.01 g were added to 4 ml of 80% ethanol and heated in a 65°C water bath for 30 min. The homogenate was centrifuged at 4000 rpm for 5 min before collecting the supernatant. The pellet was resuspended with another 2 ml of 80% ethanol and the process was repeated twice. TSS concentration was determined at 490 nm using a spectrophotometer  according to The same amount of froze plant tissues (0.5 g) was used to extract proline, ASC and TPC, separately. The details of extraction processes for these three phytochemicals were described in Dried samples (0.2 g) were digested in 4 ml of 65% nitric acid using UltraWAVE single reaction chamber microwave digestion system . The digested solution was diluted with Milli-Q water to a final volume of 25 ml. The Optima 8300 ICP-OES  and Syngistix software  were used to measure and calculate the concentrations of dietary minerals.One-way (ANOVA) was used to test for significant differences among the different treatments. Tukey’s multiple comparison tests were carried out to discriminate among the means of the different groups (MINITAB 19).-2 s-1, 18 h, DLI of 20.736 mol m-2 day-1) and L4  plants were similar but significantly greater than those of L1  and L3 plants .  or higher PPFD with longer duration (L2 and L3 but with the same DLI of 20.736 mol m-2 day-1). Grown under the same DLI, L2, and L4 plants with non-CL had significantly higher shoot and root productivity than L3 plants with CL. On day 16 after transplanting, however, L4 plants had the highest values for shoot and root productivity, followed by L2 and L3 plants. The L1 plants had the lowest values but no significant differences were observed between L2 and L3 plants  as those of L2 and L4, they had shorter total root length, smaller total root surface area and fewer number of root tips than those of L2 and L4 plants , plants increase the thickness of leaves by synthesising more palisade cell layers to prevent light damage caused by excessive light energy  with lower PPFD had lower productivity compared to L2 and L4 conditions which had higher PPFDs but shorter photoperiods. Controversial results such as CL promoted growth were reported in lettuce (Lactuca sativa L. cv. ‘Zishan’) plants accumulated more biomass under one CL (red and blue). However, with the alternation of temporally overlapped red and blue light biomass accumulation decreased with the increase of alternating light frequency.Summing up, the above discussion suggests that light intensity, duration and DLI all play important roles in enhancing plant growth and productivity in both early and later stages of growth . In the  lettuce , medicin lettuce  and rock lettuce . Recentl-2 s-1, an increasing trend was observed in total Chl and Car concentrations. However, grown under a PPFD of 350 μmol m-2 s-1, sweet potato seedlings had lower total Chl concentration compared to other conditions   . Both ETynthesis . The greficiency . Theoret  and L4 plants (480 µmol photon m-2 s-1) than L1 and L3 plants (240 µmol photon m-2 s-1)   and upreased NRA . A decrexidation  upon CL n levels . While sproteins . Althougm’ and qP in L3 plants (−2 s−1 to 350 μmol m−2 s−1 and then decreased at 450 μmol ·m−2 s−1. Plants grown under CL conditions could have higher concentrations of insoluble sugars which is mainly starch (Ocimum basilicum) grown under higher DLIs had higher TPC contents than under lower DLIs. In this study, under the same DLI, purslane grown under L3 with CL also had the higher TPC than L2 and L4 plants in both leaves and stems , red light at 635 nm enhanced the accumulations of Ca and Mg while the highest N content was found in the plants grown under green light and blue light, respectively at 522 nm and 455 nm , an overall two times increase in DLI, 20.736 mol m-2 day-1  through either increasing light intensity and/or extending photoperiod, displayed positive effects on the growth characteristics of purslane. Under the same higher DLI (20.736 mol m-2 day-1), L2 condition is the most suitable lighting strategy for enhancing productivity and quality of purslane. At an early stage of development, light treatments L2  and L4  exerted the most positive effects on purslane productivity. At later stages, light treatment L4 was observed to be the most favourable. However, given that high light intensity is often costly and difficult to maintain, the L2 treatment would be more preferable. L3 treatment with continuous light , however, resulted in lower shoot and root growth compared to L2 and L4 conditions under the same DLI. A lowered photosynthetic efficiency, as indicated by the lowered ETR, ΔF/Fm’ and qP values, was also observed in L3 plants. Thus, L3 treatment may not be favourable for the productivity of purslane. Purslane grown under L2 treatment had the highest concentration of proline, ASC and TSS in the leaves and stems, suggesting that purslane grown under L2 treatment is the most nutritious. Furthermore, leaf dietary minerals such as K, Ca, Mg and Fe concentrations were the highest in purslane grown under L2 compared to the other conditions. No clear trends were observed in stem dietary mineral concentrations among purslane plants grown under the different light conditions. Nevertheless, the results of this study provide growers of purslane a better understanding of the optimal light conditions to enhance its productivity and quality.Compared to 10.368 mol mhttps://researchdata.nie.edu.sg/dataverse/he-jie.The original contributions presented in the study are publicly available. This data can be found here: st draft of the manuscript. JH and LQ revised the manuscript. All authors contributed to the article and approved the submitted version.JH initiated and funded the expenses for the project. JH and LQ planned the experiments and carried out some parts of the experiments. SG carried out most measurements, analysed the data and plotted the graphs under supervision of JH and LQ. JH wrote the 1