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IntroductionAdoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD.MethodsThe subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence.ResultsOur protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the “foreign” antigen without causing GVHD after they were transferred into the recipients.DiscussionOur strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.

The poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) Olaparib is a widely used targeted therapy for a variety of solid tumors with homologous recombination deficiency (HRD) caused by mutation of BRCA1/2 or other DNA repair genes. The anti-tumor activity of Olaparib has been largely attributed to its ability to inhibit PARP enzymes and block DNA single-strand break (SSB) repair, which eventually leads to the most detrimental DNA damage, double-strand breaks (DSB), in HRD cells. Although PARPi was found to induce p53-dependent cell death, the underlying molecular mechanism remains incompletely understood. Here, we report that Olaparib treatment leads to p53 stabilization and activation of its downstream target genes in a dose- and time-dependent manner. Mechanistically, Olaparib triggers nucleolar stress by inhibiting biosynthesis of the precursor of ribosomal RNAs (pre-rRNA), resulting in enhanced interaction between ribosomal proteins (RPs), RPL5 and RPL11, and MDM2. Consistently, knockdown of RPL5 and RPL11 prevents Olaparib-induced p53 activation. More importantly, Olaparib efficiently suppresses breast and colorectal cancer cell survival and proliferation through activation of p53. Altogether, our study demonstrates that Olaparib activates the nucleolar stress-RPs-p53 pathway, suggesting rRNA biogenesis as a novel target for PARPi.

The high cost, potential toxicity, and possible enzyme inhibition ability of artificial mediators have limited the large-scale application of laccase (Lac)/mediator systems. Here, sinapic acid (SA), a natural mediator, was covalently attached to amino-functionalized magnetic nanoparticles (MNPs) via amide bond formation. The as-prepared SA@MNPs were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, cyclic voltammetry, and thermogravimetric analysis. The SA@MNPs were then applied to evaluate the activity of the immobilized mediator for Lac-catalyzed dye decolorization using indigo carmine (IC) as a model dye. When SA and SA@MNPs were used as Lac mediators, IC decolorization yields of ∼93% and 96%, respectively, were obtained after 60 min. Moreover, SA@MNPs exhibited an IC decolorization yield of ∼90% after being reused for 8 cycles. The Lac/SA@MNP system was shown to degrade IC by breaking down the chromophoric group. The easy recyclability, good reusability, nontoxicity, and relatively low cost of SA@MNPs make this immobilized natural mediator a promising tool for dye treatment.

Efficient reuse and regeneration of spent mediators are highly desired for many of the laccases’ biotechnology applications. This investigation demonstrates that a redox mediator 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) covalently attached to silica nanoparticles (SNPs) effectively mediated dye decolorization catalyzed by laccase. Characteristics of ABTS-modified silica nanoparticles (ABTS-SNPs) were researched by scanning electron microscopy and Fourier-transformed infrared spectroscopy. When ABTS and ABTS-SNPs were used as laccase mediators, the decolorization yields of 96 and 95% were, respectively, obtained for indigo carmine dye. The results suggest that ABTS immobilized on SNPs can be used as laccase mediators as they retain almost the same efficiency as the free ABTS. The oxidized ABTS-SNPs were regenerated by their reduction reaction with ascorbic acid. Decolorization efficiency of regenerated ABTS-SNPs and their initial forms were found to be almost equivalent. Six reuse cycles for spent ABTS-SNPs were run for the treatment of indigo carmine, providing decolorization yields of 96–77%. Compared with free mediator, the immobilized mediators have the advantage of being easily recovered, regenerated, and reused making the whole process environmentally friendly.

The use of chelators for cancer treatment has been an alternative option. Dithiocarbamates have recently attracted considerable attention owning to their diverse biological activities; thus, the preparation of new dithiocarbamate derivatives with improved antitumor activity and selectivity as well as probing the underlying molecular mechanism are required. In this study, di-2-pyridylhydrazone dithiocarbamate S-propionic acid (DpdtpA) and its copper complex were prepared and characterized, and its antiproliferative activity was evaluated. The proliferation inhibition assay showed that DpdtpA exhibited excellent antiproliferative effect in hepatocellular carcinoma (IC50 = 1.3 ± 0.3 μM for HepG2, and 2.5 ± 0.6 μM for Bel-7402). However, in the presence of copper ion, the antiproliferative activity of DpdtpA was dramatically attenuated (20–30 fold) owing to the formation of copper chelate. A preliminarily mechanistic study revealed that reactive oxygen species (ROS) generation mediated the antiproliferative activity of DpdtpA, and accordingly induced apoptosis, DNA cleavage, and autophagy. Surprisingly, the cytotoxicity of DpdtpA copper complex (DpdtpA–Cu) was also involved in ROS generation; however, a paradoxical relation between cellular ROS level and cytotoxicity was observed. Further investigation indicated that DpdtpA could induce cell cycle arrest at the S phase; however, DpdtpA–Cu lacked this effect, which explained the difference in their antiproliferative activity.

Magnetic poly(p-phenylenediamine) (PpPD) nanocomposite was synthesized via mixing p-phenylenediamine solution and Fe3O4 nanoparticles and used as a carrier for immobilized enzymes. Successful synthesis of PpPD/Fe3O4 nanofiber was confirmed by transmission electron microscopy and Fourier transform infrared spectroscopy. Laccase (Lac) was immobilized on the surface of PpPD/Fe3O4 nanofiber through covalent bonding for reactive blue 19 dye removal. The immobilized Lac-nanofiber conjugates could be recovered from the reaction solution using a magnet. The optimum reaction pH and temperature for the immobilized Lac were 3.5 and 65 °C, respectively. The storage, operational stability, and thermal stability of the immobilized Lac were higher than those of its free counterpart. The dye removal efficiency of immobilized Lac was about 80% in the first 1 h of incubation, while that of free Lac was about 20%. It was found that the unique electronic properties of PpPD might underlie the high dye removal efficiency of immobilized Lac. Over a period of repeated operation, the dye removal efficiency was above 90% during the first two cycles and remained at about 43% after eight cycles. Immobilized Lac on PpPD/Fe3O4 nanofiber showed high stability, easy recovery, reuse capabilities, and a high removal efficiency for reactive blue 19 dye; therefore, it provides an optional tool for dye removal from wastewater.

The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity.

The successful encapsulation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a well-known laccase mediator, within a mesoporous metal-organic framework sample (i.e., MIL-100(Fe)) was achieved using a one-pot hydrothermal synthetic method. The as-prepared ABTS@MIL-100(Fe) was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, nitrogen sorption, and cyclic voltammetry (CV). Our ABTS@MIL-100(Fe)-based electrode exhibited an excellent electrochemical response, indicating that MIL-100(Fe) provides an appropriate microenvironment for the immobilization and electroactivity of ABTS molecules. ABTS@MIL-100(Fe) was then evaluated as an immobilized laccase mediator for dye removal using indigo carmine (IC) as a model dye. Through the application of laccase in combination with a free (ABTS) or immobilized (ABTS@MIL-100(Fe)) mediator, decolorization yields of 95% and 94%, respectively, were obtained for IC after 50 min. In addition, following seven reuse cycles of ABTS@MIL-100(Fe) for dye treatment, a decolorization yield of 74% was obtained. Dye decolorization occurred through the breakdown of the chromophoric group by the Laccase/ABTS@MIL-100(Fe) system, and a catalytic mechanism was proposed. We therefore expect that the stability, reusability, and validity of ABTS@MIL-100(Fe) as a laccase mediator potentially render it a promising tool for dye removal, in addition to reducing the high running costs and potential toxicity associated with synthetic mediators. [ABSTRACT FROM AUTHOR]

This response refers to: Xu, Z.; Liu, Y.; Zhou, S.; Fu, Y.; Li, C. Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques. Int. J. Mol. Sci. 2016, 17, 1042. Merlot, A.M.; Sahni, S.; Lane, D.J.R.; Richardson, V.; Huang, M.L.H.; Kalinowski, D.S.; Richardson, D.R. Letter to the Editor: Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques and. Int. J. Mol. Sci. 2016, 17, 1916. [ABSTRACT FROM AUTHOR]

Magnetic poly(p-phenylenediamine) (PpPD) nanocomposite was synthesized via mixing p-phenylenediamine solution and Fe3O4 nanoparticles and used as a carrier for immobilized enzymes. Successful synthesis of PpPD/Fe3O4 nanofiber was confirmed by transmission electron microscopy and Fourier transform infrared spectroscopy. Laccase (Lac) was immobilized on the surface of PpPD/Fe3O4 nanofiber through covalent bonding for reactive blue 19 dye removal. The immobilized Lac-nanofiber conjugates could be recovered from the reaction solution using a magnet. The optimum reaction pH and temperature for the immobilized Lac were 3.5 and 65 °C, respectively. The storage, operational stability, and thermal stability of the immobilized Lac were higher than those of its free counterpart. The dye removal efficiency of immobilized Lac was about 80% in the first 1 h of incubation, while that of free Lac was about 20%. It was found that the unique electronic properties of PpPD might underlie the high dye removal efficiency of immobilized Lac. Over a period of repeated operation, the dye removal efficiency was above 90% during the first two cycles and remained at about 43% after eight cycles. Immobilized Lac on PpPD/Fe3O4 nanofiber showed high stability, easy recovery, reuse capabilities, and a high removal efficiency for reactive blue 19 dye; therefore, it provides an optional tool for dye removal from wastewater. [ABSTRACT FROM AUTHOR]

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (Ka = approximately 104 M-1). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (KEB = approximately 104 M-1). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches. [ABSTRACT FROM AUTHOR]

The antitumor property of iron chelators and aromatic nitrogen mustard derivatives has been well documented. Combination of the two pharmacophores in one molecule in drug designation is worth to be explored. We reported previously the syntheses and preliminary cytotoxicity evaluation of benzaldehyde nitrogen mustard pyridine carboxyl acid hydrazones (BNMPH) as extended study, more tumor cell lines (IC50 for HepG2: 26.1 ± 3.5 μM,HCT-116: 57.5 ± 5.3 μM, K562: 48.2 ± 4.0 μM, and PC-12: 19.4 ± 2.2 μM) were used to investigate its cytotoxicity and potential mechanism. In vitro experimental data showed that the BNMPH chelating Fe2+ caused a large number of ROS formations which led to DNA cleavage, and this was further supported by comet assay, implying that ROS might be involved in the cytotoxicity of BNMPH. The ROS induced changes of apoptosis related genes, but the TFR1 and NDRG1 metastatic genes were not obviously regulated, prompting that BNMPH might not be able to deprive Fe2+ of ribonucleotide reductase. The BNMPH induced S phase arrest was different from that of iron chelators (G1) and alkylating agents (G2). BNMPH also exhibited its inhibition of human topoisomerase IIα.Those revealed that the cytotoxic mechanism of the BNMPH could stem from both the topoisomerase II inhibition, ROS generation and DNA alkylation. [ABSTRACT FROM AUTHOR]

Bilirubin oxidase (BOX) from Myrothecium sp. IMER1 was immobilized on chitosan using glutaraldehyde as a cross-linking agent. FTIR spectra of the chitosan before and after BOX immobilization proved that the enzyme had been successfully immobilized to the cross-linked chi-tosan. The stabilities of the immobilized BOX were improved compared to free counterpart. The apparent Km for the immobilized enzyme was 1,097.1 μM, which increased by about 1.74-fold compared to the free enzyme. The immobilized BOX was operated in a batch mode for the de-colorization of indigo carmine from aqueous solution. De-colorization rates of the dye by the immobilized BOX (92%) were higher than that by the free enzyme (83%) in 150 min. Immobilized BOX could be used repeatedly, and its removal efficiency for indigo carmine remained about 79% for up to ten usages. The results demonstrated that immobilized BOX has potential application in dyestuff treatment. [ABSTRACT FROM AUTHOR]

In the present research, the potential of pellets of live Streptoinyces sp. LH1 was investigated as a biosorbents for removal of an anionic disazo direct dye Congo red, from arm aqueous solution. Live biomass of strain LH1 was found to be more effective with a removal rate of 74% compared with 65% of dead biomass for Congo red. The visual observation and ultraviolet-visible (UV-VIS) spectral analysis showed that dye removal using live biomass mainly involved adsorption by the cell surface including the intracellular bioaccumulation. Batch pH, initial dye concentration and isotherm studies were conducted to evaluate the biosorption capacity of live biomass. A maximum removal of 97.5% for 50 mg/L of Congo red by Streptomyces sp. LHI pellets was observed at pH 2.0. The pellets seemed to ,exhijit high biosorption activity in the low pH range. Equilibrium was reached, in 1 h. Increasing the initial dye concentration resulted in decreasing the color removal efficiency while higher adsorption yields were observed at lower concentrations of dye. Isotherm studies indicated that bicsorption of Congo red on live biomass fitted both the Langmuir and Freundlich models well. Biomass of strain LH1 was found to be effective in removing the other five dyes from an aqueous solution. This study showed that it is possible to develop systems for dye removal using Streptomyces biomass which occurs as a byproduct in waste streams of fermentation industries. [ABSTRACT FROM AUTHOR]

In this study, dye decolorizing potential of crude enzyme from Trametes sp. LH-3 was demonstrated towards crystal violet (CV) by polymerization reactions. Trametes sp. LH-3 produced laccase as the dominant lignolytic enzyme during solid state fermentation (SSF) of wheat straw, an agroindustry waste. Native polyacrylamide gel electrophoresis (PAGE) confirms that crude enzyme contained two laccase isoforms which were the major enzymes involved in decolorization of CV. The dye (5 mg L-1) was decolorized by 50% within 24 h by the crude laccase (10 U. ml-1) alone, but more efficiently in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a redox mediator, showing 93.5% decolorization within 12 h. Increasing the ABTS concentration can improve the decolorization rates, but 10 μM ABTS was sufficient for maximum decolorization of CV (5 mg L-1) by the crude laccase and excessive redox mediators resulting in activity inhibition of laccase. During the decolorization reaction, the characteristic absorption peak of the dye around 589 nm decreased rapidly with slight blue shifts (559 nm), and some dark-colored precipitates were formed in the samples. Characterization of CV transformation products by UV-VIS and FT-IR analyses revealed that N-demethylation and poly-merization were the key mechanisms of decolorization of CV by laccase. The results of the present study suggested that the application of crude laccase and effective mediators that are easily available and inexpensive could overcome its drawbacks in dye effluent treatment. [ABSTRACT FROM AUTHOR]

A non-ligninolytic fungus Myrothecium sp. IMER1 and its bilirubin oxidase (BOX) were evaluated for their ability to decolorize an alkaline dye effluent. The strain was capable of decolorizing dye effluent at various concentrations of 20, 33.3, 50 and 66.7% (v/v) in the pH basic range of 9.5-10.0, but high dye effluent concentration in the medium resulted in low colour removal rate. The biomass production and BOX activity of Myrothecium sp. IMER1 were inhibited during decolorization. Spectra analyses of culture supernatants and colour changes in fungal cells suggested that dyes in the effluent were removed by Myrothecium sp. IMER1 mainly due to enzymatic reaction. Thus, biodegradation was the major dye decolorization mechanism, rather than inactive surface adsorption. BOX was able to decolorize dye effluent effectively over a pH range of 4-10, and maximum decolorization of dye effluent was observed at pH 7.0. In addition, colour removal efficiency was higher at neutral and alkaline pH than at acid pH. In the presence of 2,2-Azinobis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) as a redox mediator, a marked increase was obtained in decolorization at the various pH values tested. The germination of Phaseolus mungo seeds was inhibited with dye effluent but not with the dye effluent treated by Myrothecium sp. IMER1 or BOX, indicating the less toxic nature of the treated dye effluent to the plants. Therefore, both this non-ligninolytic strain and its extracellular BOX will be potential candidates for wastewater treatment of dye effluents, especially alkaline dye effluents. [ABSTRACT FROM AUTHOR]

In this study, the crude filtrate from Myrothecium sp., at its maximum bilirubin oxidase (BOD) activity, was found to have an efficient decolorizing ability towards indigo carmine, a highly toxic indigoid class of dye. Maximum BOD synthesis was detected in 5-days old cultures. After 140 min of incubation, the decolorization by the crude enzyme was 81% at pH 7.0. SDS polyacrylamide gel electrophoresis (PAGE) showed that the crude enzymes contained two main proteins. On the other hand, when native-PAGE was stained with BOD substrates (bilirubin and ABTS) or the dye indigo carmine, it showed a single BOD activity zone or a single decolorized zone. These results demonstrated that BOD was the major enzyme of the crude ones involved in dye decolorization. The optimum pH and temperature for enzymatic decolorization were determined to be 4.5 and 40 °C, respectively. Interestingly, the crude BOD decolorized indigo carmine in a broad pH range between 4-9. The UV-visible spectrophotometric analysis demonstrated the decolorization and degradation of indigo carmine by BOD. The results suggest that BOD has a potential application to remove dyes from effluents. [ABSTRACT FROM AUTHOR]