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To overcome the problem that Bi3+-activated phosphors suffer from, it is an urgent need to realize narrow-band light emission of Bi3+-activated phosphors, which not only improves their luminescence...

Purpose:Prunella vulgaris (PV), a traditional Chinese medicine, has been used to treat patients with thyroid disease for centuries in China. The purpose of the present study was to investigate its bioactive ingredients and mechanisms against Hashimoto’s thyroiditis (HT) using network pharmacology and molecular docking technology to provide some basis for experimental research.Methods: Ingredients of the PV formula were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Additionally, HT-related genes were retrieved from the UniProt and GeneCards databases. Cytoscape constructed networks for visualization. A protein–protein interaction (PPI) network analysis was constructed, and a PPI network was built using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These key targets of PV were enriched and analyzed by molecular docking verification, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment.Results: The compound–target network included 11 compounds and 66 target genes. Key targets contained Jun proto-oncogene (JUN), hsp90aa1.1 (AKI), mitogen-activated protein kinase 1 (MAPK1), and tumor protein p53 (TP53). The main pathways included the AGE-RAGE signaling pathway, the TNF signaling pathway, the PI3K–Akt signaling pathway, and the mitogen-activated protein kinase signaling pathway. The molecular docking results revealed that the main compound identified in the Prunella vulgaris was luteolin, followed by kaempferol, which had a strong affinity for HT.Conclusion: Molecular docking studies indicated that luteolin and kaempferol were bioactive compounds of PV and might play an essential role in treating HT by regulating multiple signaling pathways.

Ag3PO4/Cu2O composites were prepared by a facile wet chemical method. The specimens were examined by X-ray diffraction, scanning electron microscopy, transmission electron microscopy and UV–Vis diffuse reflectance spectroscopy. The specimens exhibited an excellent photocatalytic activity on the degradation of RhB than pure Ag3PO4 and Cu2O. Furthermore, the photocatalyst specimen was found to exhibit higher efficiency in hydrogen conversion. It was found that a 30 wt% Ag3PO4-loaded specimen showed an excellent hydrogen generation performance. The excellent photocatalytic activity was ascribed to the separation of e–h pairs via the Z-scheme method composed of Ag, Ag3PO4 and Cu2O. This study demonstrated the potential approach to the photocatalytic splitting water to release hydrogen as well as the environmental purification of organic pollutants under visible-light irradiation.

Zinc oxide nanorods with germanium doping were prepared by ion implantation and annealing treatment method, the microstructural and optical properties of which were studied by means of the X-ray diffraction, photoluminescence, and transmission electron microscopy measurements. The pristine sample exhibited a remarkable ultraviolet emission owing to the band edge emission from the zinc oxide matrix, indicated a good crystalline quality. Three emission peaks appeared after germanium ions were doped into zinc oxide nanorods. The 513 nm photoluminescence peak was ascribed to the transition between the vacancy defect in zinc oxide matrix. Photoluminescence peaks located at 548 and 778 nm were ascribed to germanium ions luminescence center in zinc germanate grain and germanium monoxide luminescence center. The light emission tuning was obtained by germanium doping and annealing treatment, which may help the development of the practical optoelectronic devices based on zinc oxide nanomaterials.

Roof rocks in coal mines are subjected to the combination of in situ stresses and dynamic stresses induced by mining activities. Understanding the mechanical properties of roof rocks under static and dynamic loads at medium strain rates is of great significance to revealing the mechanism of rock bursts. In this study, we employ the digital image correlation (DIC) technique to investigate the energy concentration and dissipation behaviors, failure mode, and deformation characteristics of roof rocks under combined static and dynamic loads. Our results show that both the static pre-stress and dynamic loading rate have significant effects on the uniaxial compressive strength of rock specimens. From the energy principle, when the static pre-stress is the same, both elastic strain energy density and dissipated energy density increase with increasing dynamic loading rate. The hazard of rock bursts increases with decreasing static pre-stress and increasing dynamic loading rate. At higher dynamic loading rates, more cracks are generated, and the failure becomes more violent. The crack initiation, propagation and coalescence processes are identified, and the failure mode is closely related to the evolution of the global principal strain field of the rock specimens.

Haloxylon ammodendron, a C4 perennial, succulent and xero-halophytic shrub, is highly resistant to harsh environments, therefore, exploring the stress resistance mechanism will be beneficial for the use of xerophytes to prevent desertification. To determine osmotic adjustment (OA) and antioxidase functions under simulated drought stress, 8-week-old seedlings were treated with sorbitol solutions to maintain osmotic potentials (Ψs) at a control and −0.5 and −1.0 MPa. Under −0.5 MPa osmotic stress, H. ammodendron stably maintained the water content of assimilating branches, a result that was not significantly different from the result of the control group. Moreover, the Ψs decreased significantly, which helped plants absorb water efficiently from the environment, as H. ammodendron accumulated massive osmotic regulators in its assimilating branches to adjust shoot Ψs. Specifically, the contribution of Na+ to shoot Ψs was up to 45%, and Na+ became the main osmotic regulator of OA. During the treatments, the content and contribution of K+ remained stable. However, the total contribution of three organic osmotic regulators (free proline, betaine and soluble sugar) was only 20%, and betaine was the main organic osmotic regulator, accounting for approximately 15% of the 20% contribution. Moreover, H. ammodendron seedlings presented strong antioxidases, especially when there was a high activity level of superoxide dismutase, and with an increase in treatment time and degree of osmotic stress, the activity of peroxidase and catalase increased significantly. Substantial accumulation of osmotic adjustment substances was an important strategy for H. ammodendron to cope with simulated drought stress, in particular, H. ammodendron absorbed much Na+ and transported Na+ into the assimilating branch for OA. The scavenging of reactive oxygen species by antioxidases was another adaptation strategy for H. ammodendron to adapt to simulated drought stress.

Spiro[fluorene-9,9′-xanthene] (SFX) bipolar hosts bearing one, two and three quinolyl substituents, namely SFX-bPy, SFX-DbPy and SFX-TbPy, were designed and synthesized for phosphorescent organic light emitting diodes (PhOLEDs). The successive substitution of quinoline at 2′, 2 and 7′ positions of SFX results in reduced LUMO energy levels while leaving the HOMO energy levels nearly intact. The impact of quinoline substitution in these SFX-based hosts on PhOLED performance was investigated in detail through green and red model devices. For the green emitting devices, the device based on SFX-bPy host showed better performance (23.6 cd A−1, 23.4 lm W−1, 6.3%) due to high triplet energy level (T1) and balanced carriers-transporting ability. In contrast, for the red PhOLED devices, the device hosted by SFX-DbPy displayed higher performance (15.8 cd A−1, 16.0 lm W−1, 9.1%), attributable to the well matched T1 and separated frontier molecular orbitals. This work thus sheds light on the rational design of SFX-based bipolar hosts for more efficient PhOLEDs.

A vertical flow constructed wetland was combined with a biological aerated filter to develop an ecological filter, and to obtain the optimal operating parameters: The hydraulic loading was 1.55 m 3 /(m 2 ·d), carbon–nitrogen ratio was 10, and gas–water ratio was 6. The experimental results demonstrated considerable removal efficiency of chemical oxygen demand (COD), ammonia nitrogen (NH 4 + -N), total nitrogen (TN), and total phosphorus (TP) in wastewater by the ecological filter, with average removal rates of 83.79%, 93.10%, 52.90%, and 79.07%, respectively. Concentration of NH 4 + -N after treatment met the level-A discharge standard of GB18918-2002. Compared with non-plant filter, the ecological filter improved average removal efficiency of COD, NH 4 + -N, TN, and TP by 13.03%, 25.30%, 14.80%, and 2.32%, respectively: thus, plants significantly contribute to the removal of organic pollutants and nitrogen. Through microporous aeration and O 2 secretion of plants, the ecological filter formed an aerobic–anaerobic–aerobic alternating environment; thus aerobic and anaerobic microbes were active and effectively removed organic pollutants. Meanwhile, nitrogen and phosphorus were directly assimilated by plants and as nutrients of microorganisms. Meanwhile, pollutants were removed through nitrification, denitrification, filtration, adsorption, and interception by the filler. High removal rates of pollutants on the ecological filter proved that it is an effective wastewater-treatment technology for decentralized wastewater of mountainous towns.

In this paper, an optimal semi-continuous process for vinegar production from edible alcohol through biotransformation by acetic acid bacteria (AAB) WUST-01 was developed. The optimized medium composition for the starting-up stage was glucose 5.1 g/L, yeast extract 26.2 g/L, and ethanol 11.9 mL/L, and the optimal ethanol for the following semi-continuous stage was 50 mL/L. In the semi-continuous biotransformation process, the optimal withdraw ratio was 50% of working volume with 12 h cycle time. With these conditions, the total acidity could reach to 77.3 g/L and the acidity productivity could reach to 3.0 g/(L h) in a 5 L reactor. Furthermore, it was investigated to strengthen vinegar synthesis through enhancing alcohol dehydrogenase and aldehyde dehydrogenase activity in AAB by ferrous ion and pueraria flower extract as the enzyme regulators. With these regulators, the vinegar synthesis efficiency can be improved 16.3 and 13.2% respectively.

Background This study aims to investigate the imprecision of internal quality control of routine chemistry analytes so that we can have an overall knowledge of imprecision level of measurement in laboratories in China. Methods The internal quality control information including two kinds of coefficient of variations (CVs) of 22 analytes were collected via an on-line questionnaire. Then, the percentages of laboratories meeting the quality requirement for each analyte were calculated according to five imprecision criteria. Results Among these 22 analytes, creatine kinase got the most satisfied result, the percentages of laboratories whose current CV met 1/3 total allowable error (1/3TEa), 1/4TEa, and the specifications based on biological variation were 99.3%, 95.9%, 99.6%, 99.9%, and 90.6%, respectively, while Na got the lowest satisfaction rate according to these specifications, which were 4.15%, 22.4%, 4.2%, 9.0%, and 1.3%, respectively. From an overall perspective, most of the 22 analytes got a satisfied result when applying the criteria defined by CLIA'88, while only a small amount of laboratories can meet the requirement when applying the specifications derived from biological variation. Conclusions There is huge space for us to enhance and substantial effort is needed in improving and implementing quality management in China.

The high concentration vinegar plays an important role in condiment industry and daily life. Its demand significantly increase. In this work, an optimized process for high concentration vinegar production by AAB.WUST-01 using edible alcohol as feedstock was developed. The influences of carbon source, nitrogen source and ethanol concentrations on the biomass growth were investigated in starting-up stage in vinegar fermentation. The optimized medium composition was obtained. The optimum value for glucose, yeast extract and ethanol were about 5.1 g/L, yeast extract 26.2 g/L, and ethanol 10 mL/L respectively. In such a condition, the biomass is increased 4.34 fold from 0.780 to 4.16 g/L. Also, we discussed the influences of ethanol concentrations to the vinegar acidity in the acid producing stage. The result showed that ethanol in the fermentation would be 50 mL/L, the corresponding total acid could reach 45.71 g/L, and the yield reached up to 88.76%.

To suppress concentration quenching and to improve charge-carrier injection/transport in the emission layer (EML) of phosphorescent organic light-emitting diodes (PhOLEDs), a facial homoleptic iridium(III) complex emitter with amorphous characteristics was designed and prepared in one step from a multifunctional spiro ligand containing spiro[fluorene-9,9′-xanthene] (SFX) unit. Single-crystal X-ray analysis of the resulting fac-Ir(SFXpy)3 complex revealed an enlarged Ir···Ir distance and negligible intermolecular π–π interactions between the spiro ligands. The emitter exhibits yellow emission and almost equal energy levels compared to the commercial phosphor iridium(III) bis(4-phenylthieno[3,2-c]pyridinato-N,C2′)acetylacetonate (PO-01). Dry-processed devices using a common host, 4,4′-bis(N-carbazolyl)-1,1′-biphenyl, and the fac-Ir(SFXpy)3 emitter at a doping concentration of 15 wt % exhibited a peak performance of 46.2 cd A–1, 36.3 lm W–1, and 12.1% for the current efficiency (CE), power efficiency (PE), a...

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H2O2) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H2O2, increased SA and callose accumulation and higher defence gene expression.

Erbium–nitrogen codoped zinc oxide nanowires of ytterbium-doped are prepared by thermal evaporation and ion implantation methods. Ytterbium ions are doped into nanowires at a fluence of (0, 1, 3, 5, and 9) × 1015 cm−2. Microstructural and optical properties of specimen are investigated by X-ray diffractometer, absorption spectra, Raman, and upconversion photoluminescence examinations. Upconversion photoluminescence emissions at 550 nm and 660 nm are obtained under 980-nm light excitation. Both intensities of green and red peaks are enhanced by the introduction of ytterbium ions. When ytterbium ion fluence is 5 × 1015 cm−2, light emission intensity reaches maximum value. The energy transfer and cross-relaxation processes are responsible for the change of emission intensity.

ZnO quantum dots (QDs) embedded in SiO2 matrix are fabricated by ion implantation and annealing treatment methods. When the Zn-doping dose is (2, 3, 5, and 7) × 1016 cm−2, the size of quantum dots is in the range of ∼4–10 nm in diameter according to the XRD and HR-TEM results. Ultraviolet and green light emissions from the specimen are obtained at room temperature. With the increase of the Zn-doping dose, the PL peak in the ultraviolet region red shifts from 3.32 to 3.10 eV. This PL peak is related to the size of ZnO QDs, which is ascribed to the free exciton recombination in QDs. The green light emissions centered at 2.43 and 2.25 eV are independent of the Zn-doping dose and annealing temperature, which are attributed to the deep-level defect and the small peroxy radical (SPR) defect, respectively.

Background Clinical laboratory tests are important for clinicians to make diagnostic decisions, but discrepancies may directly lead to incorrect diagnosis. We would like to introduce some statistical methods to evaluate the comparability of chemistry analytes while comparing the performances of different measurement systems. Methods We used a panel of 10 fresh-frozen single donation serum samples to assess assays for the measurement of glucose and other 13 analytes. Statistical methods used in this article include traditional statistical analysis, robust statistics, regression analysis and differences on medical decision levels (MDL). All the statistical analysis results would be evaluated. 20 Chinese tertiary hospitals accredited to ISO 15189 took part in this work. The commercial random access platforms included: Olympus (8 labs), Hitachi (6 labs) and Roche (6 labs). To compare the acceptable rates, Chi square test was used. Results The statistical analysis results are as follows: (1) Coefficient of variations are between 2.8 and 3.9 %, with the slopes and intercepts of regression functions between 0.928 to 1.064 and −0.174 to 0.630, respectively. (2) The percentage of robust z-scores between −2 and 2 is bigger than 90 %. (3) The total percentages of differences on all the MDLs are: less than optimal was 31.7 % (19/60); less than desirable was 60.0 % (36/60); less than minimum was 65.0 % (39/60); more than minimum was 35.0 % (21/60). In this study, 2 laboratories (Nos. 8 and 16) were considered as poor performance by z-scores. 10 laboratories (Nos. 4, 5, 7, 8, 9, 10, 11, 14, 16 and 19) have unacceptable measurement errors on MDLs. 10 laboratories (Nos. 1, 2, 3, 6, 12, 13, 15, 17, 18, 20) can achieve mutual recognition of serum glucose testing results, including: 5 (5/8) Olympus, 2 (2/6) Hitachi and 3 (3/6) Roche. There was no significant difference among acceptable rates of the three measurements systems for the serum glucose assay. Conclusions Traditional statistical analysis, robust statistics and robust z-score, fitting linear regression equations and calculating differences on different MDLs can be used on studying the comparability and mutual recognition of clinical chemistry analytes among hospitals or laboratories in China. The mutual recognition and interchangeability of results remains jeopardized even among tertiary hospitals in China. More works and efforts should be done for improvement of the current situation of interchangeability of results in clinical laboratories in China.

Plackett-burman (P-B) and response surface methodology (RSM) were used to optimize the conditions of Poly (β-L-malic acid) production by Aureobasidium pullulans. Results of the statistical analysis showed that the concentration of Poly (β-L-malic acid ) was influenced by the concentrations of initial glucose, (NH4)2SO4, CaCO3 and agitation speed, liquid loading, and incubation time. The optimized fermentation conditions performed in shaking flasks were glucose 100.428g/L, (NH4)2SO4 3.06g/L, succinic acid 2g/L, K2CO3 0.4g/L, KH2PO4 0.1g/L, CaCO3 27.7g/L, ZnSO4·7H2O 5×10-5g/L, MgSO4·7H2O 0.1g/L, and corn steep liquor 0.1% (v/v), liquid loading 55mL/500mL, temperature 25°C, agitation speed 207 r/min, and incubation time 152h. Under which the maximum Poly (β-L-malic acid) concentration of 3.897±0.472g/L was obtained at the optimum conditions, which significantly increased by 218.8% compared with the control (1.781±0.342 g/L).

Nitrogen and erbium co-doped of ZnO nanowires (NWs) are fabricated by ion implantation and subsequent annealing in air. The incorporation of Er 3+ and N + ions is verified by energy dispersive X-ray spectroscopy (EDS) and Raman spectra. The samples exhibit upconversion photoluminescence around ∼550 nm and ∼660 nm under an excitation at 980 nm. It is discovered that the N-doped can drastically increase the upconversion photoluminescence intensity by modifying the local structure around Er 3+ in ZnO matrix. The enhancement of the PL intensity by the N-doped is caused by the formation of ErO 6− x N x octahedron complexes. With the increase of the annealing temperature ( T a ), the Er 3+ ions diffuse towards the surface of the NWs, which benefits the red emission and evokes the variation of intensity ratio owing to the existence of some organic groups.

Silicon–germanium nanoparticles are prepared by ion implantation and annealing method. Further annealing treatment is carried out in forming gas to passivate nonradiative defects around nanoparticles. Time-resolved photoluminescence measurements were examined to reveal the local environment surrounding the nanoparticles. With the increase in oxygen gas content, photoluminescence intensity from specimen is enhanced by ∼5 times and its decay time τ decreases from 23.2 to 1.6 µs. Oxygen gas is considered to be an effective passivating gas for this kind of specimen.

Silicon ions were implanted into SiO2 thin films with various doses and energies. For the films implanted with various ion doses the photoluminescence (PL) intensity of 470 nm firstly increased with the increase of Si ion dose, which is similar to the variation trend of displacement per atom (DPA) number during ion radiation. Further increasing Si ion dose the PL intensity of 470 nm decreased gradually since the neutral oxygen vacancy centers were destroyed. For the samples implanted with different energy the variation trend of PL intensity for 470 nm peak is similar to the result of DPA under different radiation energy according to SRIM2006 simulation. With the increase of radiation energy a new PL peak at 550 nm appeared because of the variation of defect type. Combining with the simulation results and PL spectra the radiation effect on Si/SiO2 thin films were proposed.

Er and Pr ions were implanted into silicon-rich silicon oxide (SRSO) thin films with Si crystals embedded in SiO2 matrix. The 525 and 546 nm luminescence peaks were clearly observed in Er-only doped film, but disappeared in the photoluminescence (PL) spectra of Er-Pr codoped films. Instead, a broad PL spectrum extending from 450 to 700 nm was obtained for Er-Pr codoped films with Er/Pr concentration ratio of 1. Concentration profiles of Si, Er and Pr ions in films were simulated by SRIM2006 and related radiation effect on PL response was also discussed. Our results indicate that this material is a potential candidate for the development of white light-emitting diode (LED) and field emission displays for its visible luminescence.

Catechins are antioxidants known to exhibit beneficial biological activities. Five main catechins are found in green tea and each catechin has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new pharmaceuticals based on catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, or by chemical synthesis, or by biotransformation reactions with high yield and specificity. This study shows that epigallocatechin gallate (EGCG) can be hydrolysed to epigallocatechin (EGC) by a hydrolase produced by Aspergillus niger after induction by addition of EGCG to the fungal cultures. Cultures without EGCG induction and cultures, to which olive oil had been added as an inducer, did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 56%. Methods based on thin-layer chromatography and high performance liquid chromatography, were used for separation and quantification of EGCG and EGC. ...

The effect of a single break in the phosphodiester backbone of Bacillus subtilis RNAse P RNA (P RNA) was examined using circular permutation analysis (CPA). This method reveals that many of the phosphodiester bonds in this catalytic RNA can be broken with little or no effect on substrate binding. Phosphate positions that show strong effects are located mostly in regions conserved among all RNAse P RNAs, or they are in regions known to interact directly with the pre-tRNA substrate. Two circularly permuted isomers of P RNA were constructed and analyzed in detail. The KM for both circularly permuted isomers is nearly identical to that of the wild-type P RNA. Since the KM of the P RNA is essentially the same as the binding constant to the substrate, this finding confirms the CPA results. The implications of backbone breakage are discussed with respect to folding and catalysis of the RNAse P RNA.

Catechins are a group of polyphenolic compounds that are antioxidants having beneficial biological activities. There are four main catechins in green tea, and each has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new medicine from catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, by chemical synthesis, or by biotransformation reactions with high yield and specificity. In this study epigallocatechin gallate (EGCG) can be hydrolyzed to epigallocatechin (EGC) by a hydrolase from Aspergillus oryzae after induction by addition of EGCG to the cultures. However, cultures without EGCG induction did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 70%. Thin layer chromatography and high performance liquid chromatography were applied to separate and quantify EGCG and EGC.

Several diaryl ethers were synthesized and tested in vitro against seven phytopathogenic fungi, namely Fusarium graminearum, Alternaria alternate, Helminthosporium sorokinianum, Pyricularia oryzae, Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. cucumarinum and Alternaria brassicae. Compared to a commercial agricultural fungicide, hymexazol, especially compounds a, b, e, g and k were found to be more effective at 50 mug/ml against F. graminearum, F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumarinum. Meantime, some structure-activity relationships were also observed.

Objective To develop a novel method of generating multiple autologous acute myeloid leukemia (AML) reactive T-cell lines as a step toward adoptive immunotherapy for AML. Materials and Methods AML peripheral blood mononuclear cells (MNC), including >90% AML blasts and 1% to 3% T cells, were seeded in limiting dilution culture in which AML blasts were induced to undergo dendritic cell (DC) differentiation. T cells were primed and activated with the addition of a cytokine combination. Results Highly reactive anti-AML T-cell lines (both CD4 + and CD8 + ) were generated, selected, and expanded. The estimated average frequency of AML-reactive T cells or precursors was 6 ± 3/1,000,000 AML peripheral blood mononuclear cells (n = 11). Robust intracellular interferon-γ (IFN-γ) release from T-cell lines was demonstrated by flow cytometry after stimulation by autologous AML cells, but not an autologous B-lymphoblastoid cell line (LCL). These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. Most CD4 + T-cell lines exerted strong proapoptotic effects on AML cells. AML cell apoptosis by CD4 + T-cell lines correlated with IFN-γ secretion. Conclusion This study demonstrates a methodology for generating large numbers of AML-reactive cytotoxic T cell lines (either class I or II restricted) that may be useful clinically in adoptive immunotherapy. This study also provides estimates of AML-reactive T-cell frequency in patients with AML.

Purpose: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. Experimental Design: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Results: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNγ production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule–armed human T cells significantly reduced SCLC tumor burdens in a mouse model. Conclusion: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor–positive tumors, such as SCLC, are warranted.

A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.

Traditionally, the focus of anti-tumor immunity has been on CD8+ CTL responses. It is now realized that CD4+ T cells play a relevant role in protective anti-tumor responses. HLA Class II molecules are expressed on AML blasts, predicting that AML cells may stimulate CD4+ T cells. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we developed a novel method of generating multiple autologous AML reactive T cell lines by competitive limiting dilution (LD) AML MNC culture. Most autologous AML reactive T cell lines generated from this culture were CD4+. These CD4+ T cell lines with high IFN-gamma secretion in response to autologous AML cells showed low to moderate specific lysis of autologous AML cells in 4-hour 51Cr release assays. However, co-culture assays demonstrated that these CD4+ AML reactive T cell lines exerted intense cytotoxicity toward autologous AML cells, depleting more than 95% of autologous AML line cells in 2 days, and more than 99% in 7 days, with no or weak toxicity to allogeneic AML cells and cell lines, HL60, NB4, U937 KG1a and ak LCL cell line. Anti-HLA class II, Dr, Dp, Dq mAb (67±16% inhibition) and anti-HLA-Dr mAb (69±11% inhibition) but not anti-HLA class I mAb significantly inhibited IFN-gamma secretion of six CD4+ T cell lines stimulated by autologous AML cells confirmed the HLA class II restriction of reactivity of these CD4+ T cell lines. Flow cytometry analysis showed that these CD4+ T cell lines induced significant Annexin-V expression on autologous AML cells. The induction of Annexin-V+ on AML cells by CD4+ T cell lines correlated significantly with IFN-gamma secretion in response to autologous AML cells (n=11; r=0.84). This result suggested that the most important mechanism of autologous AML cell elimination by CD4+ AML reactive T cells generated from LD-AML-MNC cultures was apoptosis induction related to IFN-gamma secretion.