α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Ethyl pyruvate attenuates ventilation‐induced diaphragm dysfunction through high‐mobility group box‐1 in a murine endotoxaemia model

Mechanical ventilation (MV) can save the lives of patients with sepsis. However, MV in both animal and human studies has resulted in ventilator‐induced diaphragm dysfunction (VIDD). Sepsis may promote skeletal muscle atrophy in critically ill patients. Elevated high‐mobility group box‐1 (HMGB1) levels are associated with patients requiring long‐term MV. Ethyl pyruvate (EP) has been demonstrated to lengthen survival in patients with severe sepsis. We hypothesized that the administration of HMGB1 inhibitor EP or anti‐HMGB1 antibody could attenuate sepsis‐exacerbated VIDD by repressing HMGB1 signalling. Male C57BL/6 mice with or without endotoxaemia were exposed to MV (10 mL/kg) for 8 hours after administrating either 100 mg/kg of EP or 100 mg/kg of anti‐HMGB1 antibody. Mice exposed to MV with endotoxaemia experienced augmented VIDD, as indicated by elevated proteolytic, apoptotic and autophagic parameters. Additionally, disarrayed myofibrils and disrupted mitochondrial ultrastructures, as well as increased HMGB1 mRNA and protein expression, and plasminogen activator inhibitor‐1 protein, oxidative stress, autophagosomes and myonuclear apoptosis were also observed. However, MV suppressed mitochondrial cytochrome C and diaphragm contractility in mice with endotoxaemia (P < 0.05). These deleterious effects were alleviated by pharmacologic inhibition with EP or anti‐HMGB1 antibody (P < 0.05). Our data suggest that EP attenuates endotoxin‐enhanced VIDD by inhibiting HMGB1 signalling pathway.     

Divergent long‐term trends and interannual variation in ecosystem resource use efficiencies of a southern boreal old black spruce forest 1999–2017

Long‐term trends in ecosystem resource use efficiencies (RUEs) and their controlling factors are key pieces of information for understanding how an ecosystem responds to climate change. We used continuous eddy covariance and microclimate data over the period 1999–2017 from a 120‐year‐old black spruce stand in central Saskatchewan, Canada, to assess interannual variability, long‐term trends, and key controlling factors of gross ecosystem production (GEP) and the RUEs of carbon (CUE = net primary production [NPP]/GEP), light (LUE = GEP/absorbed photosynthetic radiation [APAR]), and water (WUE = GEP/evapotranspiration [E]). At this site, annual GEP has shown an increasing trend over the 19 years (p < 0.01), which may be attributed to rising atmospheric CO₂ concentration. Interannual variability in GEP, aside from its increasing trend, was most strongly related to spring temperatures. Associated with the significant increase in annual GEP were relatively small changes in NPP, APAR, and E, so that annual CUE showed a decreasing trend and annual LUE and WUE showed increasing trends over the 19 years. The long‐term trends in the RUEs were related to the increasing CO₂ concentration. Further analysis of detrended RUEs showed that their interannual variation was impacted most strongly by air temperature. Two‐factor linear models combining CO₂ concentration and air temperature performed well (R²~0.60) in simulating annual RUEs. LUE and WUE were positively correlated both annually and seasonally, while LUE and CUE were mostly negatively correlated. Our results showed divergent long‐term trends among CUE, LUE, and WUE and highlighted the need to account for the combined effects of climatic controls and the ‘CO₂ fertilization effect’ on long‐term variations in RUEs. Since most RUE‐based models rely primarily on one resource limitation, the observed patterns of relative change among the three RUEs may have important implications for RUE‐based modeling of C fluxes.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Interaction of β‐cyclodextrin‐capped CdSe quantum dots with inorganic anions and cations

A facile method was developed for the preparation of water soluble β‐Cyclodextrin (β‐CD)‐modified CdSe quantum dots (QDs) (β‐CD‐QDs) by directly replacing the oleic acid ligands on the QDs surface with β‐CD in an alkaline aqueous solution. The as‐prepared QDs show good stability in aqueous solution for several months. Oxoanions, including phosphoric acid ion, sulphite acid ion and carbonic acid ion, affect the fluorescence of β‐CD‐QDs. Among them, H₂PO₄^– exhibited the largest quenching effect. For the polyprotic acids (HO)₃AO, the effect of acidic anions on the fluorescence of β‐CD‐QDs was in the order: monoanion (HO)₂AO₂^– > dianion (HO)AO₃^2– >> trianion AO₄^3–. After photoactivation for several days in the presence of anions at alkaline pH, the β‐CD‐QDs exhibited strong fluorescence emission. The effect of various heavy and transition metal ions on the fluorescence properties of the β‐CD‐QDs was investigated further. It was found that Ag⁺, Hg²⁺ and Co²⁺ have significant quenching effect on the fluorescence of the β‐CD‐QDs. The Stern–Volmer quenching constants increased in the order: Hg²⁺ < Co²⁺ <Ag⁺. The adsorption model of metal ions on β‐CD‐QDs was explored. Copyright © 2011 John Wiley & Sons, Ltd.     

Pro‐apoptotic effect of haem oxygenase‐1 in human colorectal carcinoma cells via endoplasmic reticular stress

Several biological effects of haem oxygenase (HO)‐1, including anti‐inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO‐1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO‐1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)‐3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT‐15, LOVO and HT‐29 cells in serum‐free (SF) conditions with increased HO‐1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP‐induced apoptosis and HO‐1 protein expression in human colon cancer cells. CoPP‐induced apoptosis of colon cancer cells was prevented by the addition of the pan‐caspase inhibitor, Z‐VAD‐FMK (VAD), and the Casp‐3 inhibitor, Z‐DEVD‐FMK (DEVD). N‐Acetyl cysteine inhibited reactive oxygen species‐generated H₂O₂‐induced cell death with reduced intracellular peroxide production, but did not affect CoPP‐induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO‐1 expression by those cells, and knockdown of HO‐1 protein expression by HO‐1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO₄ or biliverdin, induced DNA ladders, and cleavage of Casp‐3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R‐like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM‐ and CoPP‐induced apoptosis. Increased GRP78 level and formation of the HO‐1/GRP78 complex were detected in CORM‐ and CoPP‐treated human CRC cells. A pro‐apoptotic role of HO‐1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.     

Dy3+‐, Sm3+‐, Ce3+‐ and Tb3+‐activated optical properties of microcrystalline BaMgP2O7 phosphors

Photoluminescence (PL) and thermoluminescence (TL) properties of rare earth (RE) ion (RE = Dy³⁺, Sm³⁺, Ce³⁺, Tb³⁺) activated microcrystalline BaMgP₂O₇ phosphors are presented in this work. Non‐doped and doped samples of BaMgP₂O₇ were prepared using a solid state diffusion method and characterized by X‐ray diffraction (XRD), scanning electron microscopy (SEM), PL and TL. The XRD measurement confirmed the phase purity of the BaMgP₂O₇ host matrix. The average particle size was found through SEM measurement to be around 2 μm. All activators using the PL technique displayed characteristic excitation and emission spectra that corresponded to their typical f → f and f → d transitions respectively. Thermoluminescence measurements showed that BaMgP₂O₇:RE (RE = Dy³⁺, Sm³⁺, Tb³⁺, Ce³⁺) and co‐doped BaMgP₂O₇:Ce³⁺,Tb³⁺ phosphors have also TL behaviour.     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).     

Concomitant Induction of Heme Oxygenase‐1 Attenuates the Cytotoxicity of Arsenic Species from Lumbricus Extract in Human Liver HepG2 Cells

Heme oxygenase‐1 (HO‐1) is an inducible antioxidant enzyme that degrades heme to three products, biliverdin, carbon monoxide (CO), and iron ion. The present study was originally designed to characterize the HO‐1 induction by Lumbricus extract as a potential cytoprotective mechanism. Through bioactivity‐guided fractionation, with human HepG2 cells as the cellular detector, surprisingly, we found that arsenic was enriched in the active fractions isolated from Lumbricus extract. Arsenic speciation was further carried out by liquid chromatography with inductively coupled plasma mass spectrometry (LC/ICP‐MS). Our results showed that Lumbricus extract contained two major arsenic species, arsenite (As^III; 53.7%) and arsenate (As^V; 34.2%), and six minor arsenic species. Commercial sodium arsenite (NaAsO₂) was used to verify the effects of Lumbricus extract on HO‐1 expression and related intracellular signaling pathways. Both p38 MAP kinase and NF‐E2‐related factor 2 (Nrf2) pathways were found to modulate HO‐1 induction by Lumbricus extract and NaAsO₂. The cytotoxicity of arsenite was augmented by p38 MAP kinase inhibitor SB202190 and HO‐1 inhibitor tin protoporphyrin IX (SnPP), whereas p38 MAP kinase inhibitor SB202190 also inhibited HO‐1 induction by NaAsO₂. These results suggest that arsenic‐containing compounds are responsible for HO‐1 induction by Lumbricus extract. Although the exact role of toxic arsenic compounds in the treatment of oxidative injury remains unclear, concomitant HO‐1 induction may be a key mechanism to antagonize the cytotoxicity of arsenic compounds in human cells.     

Convenient enzymatic resolution of (R,S)‐2‐methylbutyric acid catalyzed by immobilized lipases

The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)‐2‐methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)‐pentyl 2‐methylbutyrate was prepared in 2 h with c 40%, ee_p 90%, and E = 35, while Thermomyces lanuginosus leads to c 18%, ee_p 91%, and E = 26. The (S)‐enantiomer was obtained by the use of Candida rugosa or Rhizopus oryzae (2‐h reaction, c 34% and 35%, ee_p 75 and 49%, and E = 10 and 4, respectively). Under optimal conditions, the effect of the solvent, the molar ratio, and the nucleophile were evaluated.     

Lasing transition at 1.06 μm emission in Nd3+‐doped borate‐based tellurium calcium zinc niobium oxide glasses for high‐power solid‐state lasers

The spectroscopic properties of Tellurium Calcium Zinc Niobium oxide Borate (TCZNB) glasses of composition (in mol%) 10TeO₂ + 15CaO + 5ZnO + 10 Nb₂O₅ + (60 – x)B₂O₃ + Nd₂O₃ (x = 0.1, 0.5, 1.0 or 1.5 mol%) have been investigated experimentally. The three phenomenological intensity parameters Ω₂, Ω_4, Ω₆ have been calculated using the Judd–Ofelt theory and in turn radiative properties such as radiative transition probabilities, emission cross‐sections, branching ratios and radiative lifetimes have been estimated. The trend found in the JO intensity parameter is Ω₂ > Ω₆ > Ω₄ If Ω₆ > Ω₄, the glass system is favourable for the laser emission ⁴F_3/2 → ⁴I_11/2 in the infrared (IR) wavelength. The experimental values of branching ratio of ⁴F_3/2 → ⁴I_11/2 transition indicate favourable lasing action with low threshold power. The evaluated total radiative transition probabilities (A_T), stimulated emission cross‐section (σ_e) and gain bandwidth parameters (σ_e × Δλ_p) were compared with earlier reports. An energy level analysis has been carried out considering the experimental energy positions of the absorption and emission bands.     

Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll‐like receptors (TLRs) that respond to pathogen‐ and damage‐associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4‐mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (B_max) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4‐blocking antibodies. We conclude that the expression, ligand‐binding capacity and pro‐inflammatory function of cardiomyocyte TLR4 are up‐regulated after long‐term MI, which promote inflammation and exacerbate heart failure.     

2‐(4‐Ethoxy phenyl)‐4‐phenyl quinoline organic phosphor for solution processed blue organic light‐emitting diodes

This paper reports the synthesis and characterization of 2‐(4‐ethoxyphenyl)‐4‐phenyl quinoline (OEt‐DPQ) organic phosphor using an acid‐catalyzed Friedlander reaction and the preparation of blended thin films by molecularly doping OEt‐DPQ in poly(methyl methacrylate) (PMMA) at different wt%. The molecular structure of the synthesized phosphor was confirmed by Fourier transform infra‐red (FTIR) spectroscopy and nuclear magnetic resonance spectra (NMR). Surface morphology and percent composition of the elements were assessed by scanning electron microscopy (SEM) and energy dispersive analysis of X‐rays (EDAX). The thermal stability and melting point of OEt‐DPQ and thin films were probed by thermo‐gravimetric analysis (TGA)/differential thermal analysis (DTA) and were found to be 80°C and 113.6°C, respectively. UV–visible optical absorption spectra of OEt‐DPQ in the solid state and blended films produced absorption bands in the range 260–340 nm, while photoluminescence (PL) spectra of OEt‐DPQ in the solid state and blended thin films demonstrated blue emission that was registered at 432 nm when excited at 363–369 nm. However, solvated OEt‐DPQ in chloroform, tetrahydrofuran or dichloromethane showed a blue shift of 31–43 nm. Optical absorption and emission parameters such as molar extinction coefficient (ε), energy gap (E_g), transmittance (T), reflectance (R), refractive index (n), oscillator energy (E₀) and oscillator strength (f), quantum yield (φ_f), oscillator energy (E₀), dispersion energy (E_d), Commission Internationale de l'Éclairage (CIE) co‐ordinates and energy yield fluorescence (E_F) were calculated to assess the phosphor's suitability as a blue emissive material for opto‐electronic applications such as organic light‐emitting diodes (OLEDs), flexible displays and solid‐state lighting technology.     

Low concentration of lipopolysaccharide acts on MC3T3‐E1 osteoblasts and induces proliferation via the COX‐2‐independent NFκB pathway

The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely stuided, Cyclooxygenase‐2 (COX‐2) is a key enzyme for prostaglandin E2 (PGE₂) production, which induces the nuclear factor kappa B (NFκB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3‐E1 osteoblasts were treated with NFκB inhibitor BAY 11‐7082 and sc791 (a selective COX‐2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFκB activation and translocation. This induction was not related to COX‐2 expression, suggesting that LPS‐induced NFκB activiation is independent of COX‐2. It is possible that low concentration of LPS can act as a stimulating factor of the NFκB pathway in nonstimulated cells such as osteoblasts. COX‐2 is not necessary for the NFκB pathway during LPS‐induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation. Copyright © 2009 John Wiley & Sons, Ltd.     

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558_HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558_HS expressing cytoplasmic HSP70, and purified EXO_HSP and EXO_HS from J558_HSP and J558_HS tumour cell culture supernatants by ultracentrifugation. We found that EXO_HSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Ia^b, CD40, CD80 and inflammatory cytokines than EXO_HS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXO_HSP are able to stimulate type 1 CD4⁺ helper T (Th1) cell responses, and more efficient P1A‐specific CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO_HS. In addition, we further elucidate that EXO_HSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8⁺ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.     

Concentration‐dependent studies of Nd3+‐doped zinc phosphate glasses for NIR photoluminescence at 1.05 μm

Nd³⁺‐doped lead‐free zinc phosphate glasses with the chemical compositions (60‐x) NH₄H₂PO₄ + 20ZnO + 10BaF₂ + 10NaF + xNd₂O₃ (where x = 0.5, 1.0, 1.5, 2.0 and 2.5 mol%) were prepared using a melt quenching technique. Vibrational bands were assigned and clearly elucidated by Raman spectral profiles for all the glass samples. Judd–Ofelt (J–O) intensity parameters (Ω_λ: λ = 2, 4, 6) were obtained from the spectral intensities of different absorption bands of Nd³⁺ ions. Radiative properties such as radiative transition probabilities (A_R), radiative lifetimes (τ_R) and branching ratios (β_R) for different excited states were calculated using J–O parameters. The near infrared (NIR) photoluminescence spectra exhibited three emission bands (⁴F_3/2 level to ⁴I_13/2, ⁴I_11/2 and ⁴I_9/2 states) for all the concentrations of Nd³⁺ ions. Various luminescence properties were studied by varying the Nd³⁺ concentration for the three spectral profiles. Fluorescence decay curves of the ⁴F_3/2 level were recorded. The energy transfer mechanism that leads to quenching of the ⁴F_3/2 state lifetimes was discussed at higher concentration of Nd³⁺ ions. These glasses are suggested as suitable hosts to produce efficient lasing action in NIR region at 1.05 μm.     

Eu3+‐tetrakis β‐diketonate complexes for solid‐state lighting application

Eu³⁺–β‐diketonate complexes are used, for example, in solid‐state lighting (SSL) or light‐converting molecular devices. However, their low emission quantum efficiency due to water molecules coordinated to Eu³⁺ and low photostability are still problems to be addressed. To overcome such challenges, we synthesized Eu³⁺ tetrakis complexes based on [Q][Eu(tfaa)₄] and [Q][Eu(dbm)₄] (Q1 = C₂₆H₅₆N⁺, Q2 = C₁₉H₄₂N⁺, and Q3 = C₁₇H₃₈N⁺), replacing the water molecules in the tris stoichiometry. The tetrakis β‐diketonates showed desirable thermal stability for SSL and, under excitation at 390 nm, they displayed the characteristic Eu³⁺ emission in the red spectral region. The quantum efficiencies of the dbm complexes achieved values as high as 51%, while the tfaa complexes exhibited lower quantum efficiencies (28–33%), but which were superior to those reported for the tris complexes. The structures were evaluated using the Sparkle/PM7 model and comparing the theoretical and the experimental Judd–Ofelt parameters. [Q1][Eu(dbm)₄] was used to coat a near‐UV light‐emitting diode (LED), producing a red‐emitting LED prototype that featured the characteristic emission spectrum of [Q1][Eu(dbm)₄]. The emission intensity of this prototype decreased only 7% after 30 h, confirming its high photostability, which is a notable result considering Eu³⁺ complexes, making it a potential candidate for SSL.     

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4⁺ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4⁺CD25⁺FoxP3⁺ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.     

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Secreted phosphoprotein‐24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor‐β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W‐20‐17 mesenchymal stem cells with rhBMP‐2, short‐term Smad1/5 phosphorylation was inhibited, intermediate‐term alkaline phosphatase (ALP) induction was blunted, and long‐term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS–PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non‐reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween‐20 at pH 2.7–8.5. LC–MS/MS analysis of affinity‐purified, non‐reducing SDS–PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α₂‐macroglobulins (α₂‐macroglobulin [α₂M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti‐thrombin III (Serpin C1), which may protect Spp24 from proteolysis. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.