Aims/hypothesis There is convincing evidence that endoplasmic reticulum (ER) stress is implicated in the pathogenesis of diabetes and its complications; however, the mechanisms are not fully understood. This study aimed to dissect the role and signalling pathways of activating transcription factor 4 (ATF4) in ER-stress-associated endothelial inflammation and diabetic retinopathy. Methods ER stress and ATF4 activity were manipulated by complementary pharmacological and genetic approaches in cultured retinal endothelial (TR-iBRB) cells. Diabetes was induced by streptozotocin in heterozygous Atf4 knockout and wild-type mice. ER stress markers, inflammatory cytokines and adhesion molecules, activation of the signal transducer and activator of transcription 3 (STAT3) pathway, and retinal vascular permeability were measured. Results High-glucose treatment resulted in rapid induction of ER stress, activation of ATF4, and increased production of inflammatory factors in TR-iBRB cells. Suppressing ER stress or inhibiting ATF4 activity markedly attenuated high-glucose-induced production of intercellular adhesion molecule 1, TNF-α and vascular endothelial growth factor. Conversely, enhancing ER stress or overexpressing Atf4 was sufficient to induce endothelial inflammation, which was, at least in part, through activation of the STAT3 pathway. Furthermore, knockdown of the Stat3 gene or inhibiting STAT3 activity restored ER homeostasis in cells exposed to high glucose and prevented ATF4 activation, suggesting that STAT3 is required for high-glucose-induced ER stress. Finally, we showed that downregulation of Atf4 significantly ameliorated retinal inflammation, STAT3 activation and vascular leakage in a mouse model of type 1 diabetes. Conclusions/interpretation Taken together, our data reveal a pivotal role of ER stress and the ATF4/STAT3 pathway in retinal endothelial inflammation in diabetic retinopathy.

Aims/hypothesis Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygeninduced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1α (also known as Hif1a) knockout (KO) mice. Methods Conditional Hif-1α KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1α mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1α and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. Results While the Hif-1α KO mice showed significantly decreased HIF-1α levels in the retina, they exhibited no apparent histological or visual functional abnormalities (2011( ) 54:1554( -1566( DOI 10.1007( /s00125-011-2081 under normal conditions. Compared with wild-type counterparts, Hif-1α KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1α in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. Conclusions/interpretation Müller cell-derived HIF-1α is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1α is therefore a promising therapeutic target for diabetic retinopathy.

A simple model for proton relative biological effectiveness (RBE) is proposed. It describes the RBE as a function of proton depth, the dose and the linear energy transfer (LET) when proton passes through tissue-like materials. Radiobiological parameters were first obtained by fitting the published experimental cell survival data. The dose-averaged LET values were calculated for 250-MeV proton beam in a water phantom by using GEANT4 Monte Carlo simulation code and were then used as input values to calculate the values of RBE as function of depths. The model was also applied to proton spread-out Bragg peak, where the increasing RBE with depth causes an extended RBE-weighted dose in the distal fall-off region. This model was found to be able to reproduce the measured RBE values as a function of LET, depth and dose for a specific cell line.

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.

SUMMARYBoth innate and adaptive immune systems are thought to participate in the pathogenesis of rheumatoid arthritis in adults and children. The experiments reported here were undertaken to examine how immune complexes, potent stimulators of inflammation, may regulate cells of the adaptive immune system. Human T cells were prepared from peripheral blood by negative selection and incubated with bovine serum albumin (BSA)-anti-BSA immune complexes that were formed in the presence or absence of human C1q. C1q-bearing immune complexes, but not unopsonized complexes, elicited both TNF-a and IFN-g secretion from human T cells. Secretion of both cytokines was time-and dosedependent. Cross-linking C1q on the cell surface of T cells produced the same results. Cytokine secretion was not inhibited by blocking the C3b receptor (CR1, CD35) on T cells prior to incubation with immune complexes. Reverse transcriptase polymerase chain reaction (RT-PCR) of immune complexstimulated cells revealed accumulation of both TNF-a and IFN-g mRNA within 2 h post-stimulation. IL-2 was not detected in cell culture supernatants, but IL-2 receptor a chain (CD25) was detected in low density on a small proportion of T cells activated by C1q-bearing immune complexes. Secretion of both cytokines was inhibited partially, but not completely, by IL-10. These experiments show that immune complexes, potent inflammatory mediators, may activate T cells through a novel mechanism. These findings have implications for chronic inflammatory diseases in humans.

When high-energy protons interact in beam delivery systems and are stopped in patients, a fraction of beam will undergo nuclear interactions that release secondary particles, in particular, neutrons of different energies. The GEANT4 Monte Carlo Code was used to simulate the interaction of 250 MeV proton beam in tissue and iron to calculate the energy and angular distributions of generated protons, neutrons and photons, and thus provide H* (10), the ambient dose equivalent. A modular physics list by utilising electromagnetic interactions and hadronic interactions was constructed. Three different GEANT4 models that include the low-energy parameterisation, binary cascade and pre-compound model with Bertini cascade for proton inelastic interactions were compared. The findings suggest that the models play critical roles in terms of secondary particle generation. Further benchmarks are necessary to select the best model predicting a realistic scenario.

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Background: There are considerable socioeconomic costs associated with bone defects, making regenerative medicine an increasingly attractive option for treating them. Chitosan is a natural biopolymer; it is used in approaches for sustained slow release and osteogenesis, and metformin has osteoinductivity. Our study aimed to synthesize chitosan and human serum albumin (HSA) with a metformin nanoformulation to evaluate the therapeutic effects of this nanoformulation on bone defects in vitro.Methods: A pluripotent differentiation assay was performed in vitro on mouse bone marrow mesenchymal stem cells (BMSCs). Cell Counting Kit-8 was used to detect whether metformin was toxic to BMSCs. The osteogenesis-related gene expression of osteocalcin (OCN) and osteoprotegerin (OPG) from BMSCs was tested by real-time polymerase chain reaction (PCR). HSA, metformin hydrochloride, and chitosan mixtures were magnetically stirred to finish the assembly of metformin/HSA/chitosan nanoparticles (MHC NPs). The MHC NPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FT-IR). To test the expression of OCN and OPG, western blot were used. MHC NPs were evaluated in vitro for their osteoinductivity using alkaline phosphatase (ALP).Results: BMSCs successfully differentiated into osteogenic and adipogenic lineages in vitro. According to real-time PCR, a 50 µM concentration of metformin promoted osteogenesis in BMSCs most effectively by upregulating the osteogenic markers OCN and OPG. The microstructure of MHC NPs was spherical with an average nanosize of 20 ± 4.7 nm and zeta potential of −8.3 mV. A blueshift and redshift were observed in MHC NPs following exposure to wavelengths of 1,600–1,900 and 2,000–3,700 nm, respectively. The encapsulation (%) of metformin was more than 90%. The simulation study showed that MHC NPs have good stability and it could release metformin slowly in vitro at room temperature. Upon treatment with the studied MHC NPs for 3 days, ALP was significantly elevated in BMSCs. In addition, the MHC NPs-treated BMSCs upregulated the expression of OPG and OCN, as shown by real-time PCR and western blot.Conclusion: MHC NPs have a stable metformin release effect and osteogenic ability. Therefore, as a derived synthetic biopolymer, it is expected to play a role in bone tissue regeneration.