Optogenetic restoration of high sensitivity vision with bReaChES, a red-shifted channelrhodopsin.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Inhibiting the activation of MAPK (ERK1/2) in stressed Müller cells prevents photoreceptor degeneration.

Rationale: Müller cells play an essential role in maintaining the health of retinal photoreceptors. Dysfunction of stressed Müller cells often results in photoreceptor degeneration. However, how these cells communicate under stress and the signalling pathways involved remain unclear. In this study, we inhibited the MAPK (ERK1/2) signalling, mainly activated in Müller cells, evaluated the protective effects on the photoreceptors and further explored the signalling communication between stressed Müller cells and degenerating photoreceptors. Methods: We evaluated the changes of MAPK (ERK1/2) signalling and its downstream targets in human retinal explants treated with PD98059, a specific phosphorylated ERK1/2 inhibitor, by western blot and immunostaining. We further assessed photoreceptor degeneration by TUNEL staining and outer nuclear layer thickness. We also injected PD98059 into the eyes of mice exposed to photo-oxidative stress. We evaluated the protective effects on photoreceptor degeneration by optical coherence tomography (OCT) and electroretinography (ERG). The crosstalk between Müller cells and photoreceptors was further dissected based on the changes of transcription factors by RNA sequencing and protein profiles of multiple signalling pathways. Results: We found that MAPK (ERK1/2) signalling was mainly activated in Müller cells under stress, both ex vivo and in vivo. PD98059 inhibited the phosphorylation of ERK1/2, reduced expression of the gliotic marker glial fibrillary acidic protein (GFAP) in Müller cells and increased levels of the neuroprotective factor, interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors. Inhibition of pERK1/2 also reduced retinal photo-oxidative damage in mice retinas assessed by OCT and ERG. We also identified that the JAK/STAT3 signalling pathway might mediate signalling transduction from Müller cells to photoreceptors. Conclusion: MAPK (ERK1/2) deactivation through chemical inhibition, mainly in stressed Müller cells, can alleviate gliosis in Müller cells and restore the expression of IRBP in photoreceptors, which appears to prevent retinal degeneration. Our findings suggested a new way to prevent photoreceptor degeneration by manipulating the stress response in Müller cells.

Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.

Transketolase in human Müller cells is critical to resist light stress through the pentose phosphate and NRF2 pathways.

The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of "traffic flow" through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.

Hard Tissue Volume Stability Effect beyond the Bony Envelope of a Three-Dimensional Preformed Titanium Mesh with Two Different Collagen Barrier Membranes on Peri-Implant Dehiscence Defects in the Anterior Maxilla: A Randomized Clinical Trial.

This single-blinded, randomized, controlled study aimed to clinically and radiographically evaluate hard tissue volume stability beyond the bony envelope using three-dimensional preformed titanium mesh (3D-PFTM) for peri-implant dehiscence defects in the anterior maxilla. A total of 28 patients who wished to undergo implant surgery combined with guided bone regeneration (GBR) after extraction of a single maxillary anterior tooth were randomly assigned to two groups depending on the type of collagen membrane used, additionally with the 3D-PFTM-test (n = 14, cross-linked collagen membrane; CCM) and control (n = 14, non-cross-linked collagen membrane; NCCM) groups. Each implant was evaluated radiographically using CBCT at baseline, immediately after surgery, and at 6 months postoperatively. The relative position and distances from the bony envelope to the outlines of the augmented ridge were further determined immediately after GBR and 6 months after healing. At the platform level, the mean horizontal hard tissue gain (HG) at all the sites was 2.35 ± 0.68 mm at 6 months postoperatively. The mean HG rate was 84.25% ± 14.19% in the CCM group and 82.56% ± 13.04% in the NCCM group, but the difference was not significant between the groups. In all cases, HG was maintained beyond the bony envelope even after 6 months of GBR. This study suggests that 3D-PFTM should be considered a valuable option for GBR for peri-implant dehiscence defects in the anterior maxilla. In addition, 3D-PFTM may confer predictable hard tissue volume stability even after the healing period of hard tissue augmented outside the bony envelope by GBR.

8-Geranylumbelliferone isolated from Paramignya trimera triggers RIPK1/RIPK3-dependent programmed cell death upon TNFR1 ligation.

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.

Targeting the Notch and TGF-ß signaling pathways to prevent retinal fibrosis in vitro and in vivo.

Rationale: The Notch and transforming growth factor-ß (TGFß) signaling pathways are two intracellular mechanisms that control fibrosis in general but whether they play a major role in retinal fibrosis is less clear. Here we study how these two signaling pathways regulate Müller cell-dominated retinal fibrosis in vitro and in vivo. Methods: Human MIO-M1 Müller cells were treated with Notch ligands and TGFß1, either alone or in combination. Western blots were performed to study changes in γ-secretase proteases, Notch downstream effectors, endogenous TGFß1, phosphorylated Smad3 (p-Smad3) and extracellular matrix (ECM) proteins. We also studied the effects of RO4929097, a selective γ-secretase inhibitor, on expression of ECM proteins after ligand stimulation. Müller cell viability was studied by AlamarBlue and cytotoxicity by lactate cytotoxicity assays. Finally, we studied changes in Notch and TGFß signaling and tested the effect of intravitreal injections of the Notch pathway inhibitor RO4929097 on retinal fibrosis resulted from Sodium iodate (NaIO3)-induced retinal injury in mice. We also studied the safety of intravitreal injections of RO4929097 in normal mice. Results: Treatment of Müller cells with Notch ligands upregulated γ-secretase proteases and Notch downstream effectors, with increased expression of endogenous TGFß1, TGFß receptors and p-Smad3. TGFß1 upregulated the expression of proteins associated with both signaling pathways in a similar manner. Notch ligands and TGFß1 had additive effects on overexpression of ECM proteins in Müller cells which were inhibited by RO4929097. Notch and TGFß ligands stimulated Müller cell proliferation which was inhibited by RO4929097 without damaging the cells. NaIO3-induced retinal injury activated both Notch and TGFß signaling pathways in vivo. Intravitreal injection of RO4929097 prevented Müller cell gliosis and inhibited overexpression of ECM proteins in this murine model. We found no safety concerns for up to 17 days after an intravitreal injection of RO4929097. Conclusions: Inhibiting Notch signaling might be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing a treatment for preventing fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.

Preclinical and clinical studies of photobiomodulation therapy for macular oedema.

AIMS/HYPOTHESIS: Diabetic macular oedema (DME) is the leading cause of visual impairment in people with diabetes. Intravitreal injections of vascular endothelial growth factor inhibitors or corticosteroids prevent loss of vision by reducing DME, but the injections must be given frequently and usually for years. Here we report laboratory and clinical studies on the safety and efficacy of 670 nm photobiomodulation (PBM) for treatment of centre-involving DME. METHODS: The therapeutic effect of PBM delivered via a light-emitting diode (LED) device was tested in transgenic mice in which induced Müller cell disruption led to photoreceptor degeneration and retinal vascular leakage. We also developed a purpose-built 670 nm retinal laser for PBM to treat DME in humans. The effect of laser-delivered PBM on improving mitochondrial function and protecting against oxidative stress was studied in cultured rat Müller cells and its safety was studied in pigmented and non-pigmented rat eyes. We then used the retinal laser to perform PBM in an open-label, dose-escalation Phase IIa clinical trial involving 21 patients with centre-involving DME. Patients received 12 sessions of PBM over 5 weeks for 90 s per treatment at a setting of 25, 100 or 200 mW/cm2 for the three sequential cohorts of 6-8 patients each. Patients were recruited from the Sydney Eye Hospital, over the age of 18 and had centre-involving DME with central macular thickness (CMT) of >300 µm with visual acuity of 75-35 Log minimum angle of resolution (logMAR) letters (Snellen visual acuity equivalent of 20/30-20/200). The objective of this trial was to assess the safety and efficacy of laser-delivered PBM at 2 and 6 months. The primary efficacy outcome was change in CMT at 2 and 6 months. RESULTS: LED-delivered PBM enhanced photoreceptor mitochondrial membrane potential, protected Müller cells and photoreceptors from damage and reduced retinal vascular leakage resulting from induced Müller cell disruption in transgenic mice. PBM delivered via the retinal laser enhanced mitochondrial function and protected against oxidative stress in cultured Müller cells. Laser-delivered PBM did not damage the retina in pigmented rat eyes at 100 mW/cm2. The completed clinical trial found a significant reduction in CMT at 2 months by 59 ± 46 µm (p = 0.03 at 200 mW/cm2) and significant reduction at all three settings at 6 months (25 mW/cm2: 53 ± 24 µm, p = 0.04; 100 mW/cm2: 129 ± 51 µm, p < 0.01; 200 mW/cm2: 114 ± 60 µm, p < 0.01). Laser-delivered PBM was well tolerated in humans at settings up to 200 mW/cm2 with no significant side effects. CONCLUSIONS/INTERPRETATION: PBM results in anatomical improvement of DME over 6 months and may represent a safe and non-invasive treatment. Further testing is warranted in randomised clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02181400 Graphical abstract.

Platelet activity with hemoglobin level in patients with hemodialysis: Prospective study.

BACKGROUND: VerifyNow (VN; Accumetrics, San Diego, CA) P2Y12 reaction unit (PRU) has an inverse relation with hemoglobin level (Hb). Chronic kidney disease (CKD) is associated with low response to clopidogrel and low Hb. Our aim is to investigate the relation between PRU and Hb, and to assess whether Hb directly affects PRU or not in patients with CKD undergoing hemodialysis (HD). METHODS: We analyzed the relation between PRU and Hb in 43 HD patients and compared it with a control group of 127 patients with normal renal function. Both groups underwent percutaneous coronary intervention for stable coronary artery disease. We also compared PRU between the 2 groups considering Hb as a confounding factor. RESULTS: In the control group, Hb and PRU showed a significant inverse correlation (correlation coefficient r = -0.340; P < .001), but not in the HD group (correlation coefficient r = -0.099; P = .53). PRU was higher in the HD group than the control group after adjusting for the influence of Hb (299.2 [95% confidence interval: 278.4-316.7] vs 248.7 [95% confidence interval: 227.7-269.0]; P < .001), even after propensity score matching (299.2 [95% confidence interval: 278.4-316.7] vs 241.7 [95% confidence interval: 221.8-262.2]; P < .001). CONCLUSIONS: PRU was higher regardless of lower Hb in CKD on HD patients than normal renal function patients. Therefore, Hb was not crucial factor to decide PRU in CKD on HD patients in this study.

3-O-acetylrubianol C (3AR-C) induces RIPK1-dependent programmed cell death by selective inhibition of IKKß.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKß. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKß phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKß inhibitor to overcome TNF resistance in cancer therpay.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis.

Plant-derived lignans have numerous biological effects including anti-tumor and anti-inflammatory activities. Screening of purified constituents of Rubia philippinensis from human glioblastoma cells resistant to TNF-related apoptosis-inducing ligand (TRAIL) has suggested that the lignan pinoresinol was a highly active TRAIL sensitizer. Here we show that treatment with nontoxic doses of pinoresinol in combination with TRAIL induced rapid apoptosis and caspase activation in many types of glioblastoma cells, but not in normal astrocytes. Analyses of apoptotic signaling events revealed that pinoresinol enhanced the formation of TRAIL-mediated death-inducing signaling complex (DISC) and complete processing of procaspase-8 within the DISC in glioblastoma cells, in which caspase-8 was inactivated. Mechanistically, pinoresinol downregulated the expression of cellular FLICE-inhibitory protein (cFLIPL) and survivin through proteasome-mediated degradation, without affecting death receptors or downstream intracellular apoptosis-related proteins. Furthermore, the sensitization of TRAIL-mediated apoptosis by pinoresinol strictly depended on the expression level of cFLIPL, which was regulated through de novo protein synthesis, rather than by NF-κB or p53 signaling. Taken together, our results indicate that pinoresinol facilitates DISC-mediated caspase-8 activation by targeting cFLIPL in an early event in apoptotic signaling, which provides a potential therapeutic module for TRAIL-based chemotherapy.

Post-translational modification of the death receptor complex as a potential therapeutic target in cancer.

Programmed cell death is critical to the physiological function of multi-cellular organisms, controlling development, immunity, inflammation, and cancer progression. Death receptor (DR)-mediated regulation of a protease functions as a second messenger to initiate a death signal cascade to induce apoptosis or necroptosis. Recently, it has become clear that post-translational modifications (PTMs) of signaling components in the DR complex are highly complex, temporally controlled, and tightly regulated, and play an important role in cell death signaling. This review focuses on the molecular mechanisms and pathophysiological consequences of PTMs on the formation of the DR signaling complex, especially with respect to tumor necrosis factor receptor 1 (TNFR1). Furthermore, characterization of the role of PTMs in spatially different TNFR1 complexes (complexes I and II), especially with respect to the role of ubiquitination and phosphorylation of receptor interacting protein 1 (RIP1) in programmed cell death in cancer cells, will be reviewed. By integrating recently gained insight of the functional importance of PTMs in complex I or II, this review discusses how the concerted action of PTMs results in life or death upon DR ligation. Finally, the emerging concept of a sequential cell death checkpoint by the PTMs of RIP1, which may reveal novel therapeutic opportunities for the treatment of some cancers, will be discussed.

A Combination Therapy Targeting Endoglin and VEGF-A Prevents Subretinal Fibro-Neovascularization Caused by Induced Müller Cell Disruption.

Purpose: Subretinal fibroneovascularization is one of the most common causes of vision loss in neovascular AMD (nAMD). Anti-VEGF therapy effectively inhibits vascular leak and neovascularization but has little effect on fibrosis. This study aimed to identify a combination therapy to concurrently inhibit subretinal neovascularization and prevent fibrosis. Methods: We generated transgenic mice in which induced disruption of Müller cells leads to subretinal neovascularization, which is reliably accompanied by subretinal fibrosis. We conducted Western blots and immunohistochemistry to study changes in transforming growth factor-ß (TGFß) signaling including endoglin, a coreceptor essential for TGFß signaling, and then tested the effects of monthly intravitreal injection of anti-VEGF-A and anti-endoglin, either alone or in combination, on the development of subretinal fibroneovascularization in our transgenic mice. Results: Müller cell disruption increased expression of TGFß1, TGFß type 1 receptor, and phosphorylated-Smad3. Endoglin was strongly expressed in subretinal fibroneovascular tissue. Fluorescein angiography and measurements of retinal vascular permeability indicated that intravitreal anti-VEGF-A in combination with anti-endoglin treatment more efficiently inhibited vascular leak compared with either monotherapy. Immunostaining of retinal wholemounts with antibodies against glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 indicated that the combination therapy also effectively prevented subretinal fibrosis and inhibited microglial activation. Luminex cytokine assays indicated that intravitreal anti-VEGF-A and anti-endoglin treatment, either alone or in combination, reduced the production of IL33 and macrophage inflammatory protein-3α. Conclusions: Our findings offer a potentially novel combination approach to concurrently managing subretinal neovascularization and fibrosis in nAMD.

C-27-carboxylated oleanane triterpenoids up-regulate TRAIL DISC assembly via p38 MAPK and CHOP-mediated DR5 expression in human glioblastoma cells.

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3ß-hydroxyolean-12-en-27-oic acid (C27OA-1), 3ß,6ß,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3ß-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.

Constituents from Scutellaria barbata Inhibiting Nitric Oxide Production in LPS-Stimulated Microglial Cells.

The arial parts of Scutellaria barbata D. Don (Lamiaceae) efficiently inhibited NO production in BV2 microglial cells, and the active constituents were further isolated based on activity-guided isolation using silica-gel column chromatography, RP-C18 MPLC and prep-HPLC. As the results, 2 flavonoids including 6-methoxynaringenin (1) and 6-O-methylscutellarein (5), and 6 neo-clerodane diterpenes such as scutebarbatine W (2), scutebatas B (3), scutebarbatine B (4), scutebarbatine A (6), 6-O-nicotinolylscutebarbatine G (7), and scutebarbatine X (8) were isolated. The structures of these compounds were elucidated based on NMR and MS data, and the comparison of literature values. All the compounds except compound 7 inhibited NO production efficiently with IC50 values of lower than 50 µm. Particularly, compounds 1 and 8 were the most efficient with IC50 values of 25.8 and 27.4 µm, respectively. This is the first report suggesting the potential of S. barbata on the reduction of neuroinflammation.

Cardioprotective effects of PKG activation by soluble GC activator, BAY 60-2770, in ischemia-reperfusion-injured rat hearts.

Soluble guanylate cyclase (sGC) has been suggested as a therapeutic target for cardiac ischemia-reperfusion (IR) injury. Until now, the molecular mechanism of BAY 60-2770, a sGC activator, in cardiac IR injury has not been assessed. To identify the cardioprotective effects of BAY 60-2770 in IR-injured rat hearts, IR injury was established by occlusion of LAD for 40 min and reperfusion for 7 days, and the effects of BAY 60-2770 on myocardial protection were assessed by echocardiography and TTC staining. 5 nM and 5 µM of BAY 60-2770 were perfused into isolated rat hearts in a Langendorff system. After 10- or 30-min reperfusion with BAY 60-2770, cGMP and cAMP concentrations and PKG activation status were examined. Hearts were also perfused with 1 µM KT5823 or 100 µM 5-HD in conjunction with 5 nM Bay 60-2770 to evaluate the protective role of PKG. Mitochondrial oxidative stress was investigated under hypoxia-reoxygenation in H9c2 cells. In IR-injured rat hearts, BAY 60-2770 oral administration reduced infarct size by TTC staining and improved left ventricular function by echocardiography. Tissue samples from BAY 60-2770-perfused hearts had approximately two-fold higher cGMP levels. BAY 60-2770 increased PKG activity in the myocardium, and the reduced infarct area by BAY 60-2770 was abrogated by KT-5823 in isolated myocardium. In H9c2 cardiac myoblasts, hypoxia-reoxygenation-mediated mitochondrial ROS generation was diminished with BAY 60-2770 treatment, but was recovered by pretreatment with KT-5823. BAY 60-2770 demonstrated a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. BAY 60-2770 has a protective effect against cardiac IR injury via mitoKATP opening and decreased mitoROS by PKG activation. These results demonstrated that BAY 60-2770 may be used as a therapeutic agent for cardiac IR injury.