METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-Methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high-mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Fat mass and obesity-associated protein alleviates Aß1-40 induced retinal pigment epithelial cells degeneration via PKA/CREB signaling pathway.

Amyloid-ß (Aß) is thought to be a critical pathologic factor of retinal pigment epithelium (RPE) degeneration in age-related macular degeneration (AMD). Aß induces inflammatory responses in RPE cells and recent studies demonstrate the N6-methyladenosine (m6A) regulatory role in RPE cell inflammation. m6A is a reversible epigenetic posttranslational modification, but its relationship with Aß-induced RPE degeneration is yet to be thoroughly investigated. The present study explored the role and mechanism of m6A in Aß-induced RPE degeneration model. This model was induced via intravitreally injecting oligomeric Aß and the morphology of its retina was analyzed. One of m6A demethylases, the fat mass and obesity-associated (FTO) gene expression, was assessed. An m6A-messenger RNA (mRNA) epitranscriptomic microarray was employed for further bioinformatic analyses. It was confirmed that Aß induced FTO upregulation within the RPE. Hypopigmentation alterations and structural disorganization were observed in Aß-treated eyes, and inhibition of FTO exacerbated retinal degeneration and RPE impairment. Moreover, the m6A-mRNA epitranscriptomic microarray suggested that protein kinase A (PKA) was a target of FTO, and the PKA/cyclic AMP-responsive element binding (CREB) signaling pathway was involved in Aß-induced RPE degeneration. m6A-RNA binding protein immunoprecipitation confirmed that FTO demethylated PKA within the RPE cells of Aß-treated eyes. Altered expression of PKA and its downstream targets (CREB and brain-derived neurotrophic factor) was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot analyses. Hence, this study's findings shed light on FTO-mediated m6A modification in Aß-induced RPE degeneration and indicate potential therapeutic targets for AMD.

Progression of Polypoidal Lesions Associated with Exudative Recurrence in Polypoidal Choroidal Vasculopathy.

PURPOSE: To investigate the characteristics of the branching vascular network (BVN) and polypoidal lesions in polypoidal choroidal vasculopathy (PCV) to determine near-term indicators that may predict exudative recurrence. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with PCV receiving anti-vascular endothelial growth factor (VEGF) monotherapy or anti-VEGF plus photodynamic therapy were followed for at least 1 year using swept-source OCT angiography (SS-OCTA) imaging. METHODS: Patients were divided into 2 groups based on whether exudative recurrence occurred during follow-up. Multiple parameters were collected and compared between the 2 groups, such as age, gender, visual acuity, number of polypoidal lesions, lesion area at the first SS-OCTA visit, and total lesion area change from the first SS-OCTA visit to the last SS-OCTA visit. To evaluate the association between SS-OCTA imaging-based risk factors and the exudative recurrences, imaging features associated with PCV such as BVN growth and polypoidal lesion progression (enlargement, new appearance, and reappearance) at each follow-up visit were analyzed. The time intervals from the nonexudative visit with lesion progression to the corresponding exudative recurrence visit were documented to explore their association with exudative recurrences. Cox regression and logistic regression analyses were used. MAIN OUTCOME MEASURES: Association between BVN growth and polypoidal lesion progression with exudative recurrence. RESULTS: Thirty-one eyes of 31 patients (61% men) were included. Sixteen eyes had no recurrence of exudation, and 15 eyes had recurrence during follow-up. The average follow-up duration was 20.55 ± 6.86 months (range, 12-36 months). Overall, the recurrence group had worse best-corrected visual acuity (P = 0.019) and a greater increase in lesion area (P = 0.010). Logistical regression analysis showed that polypoidal lesion progression, including new appearance, enlargement, and reappearance of polypoidal lesions, was associated with exudative recurrences within 3 months (odds ratio, 26.67, 95% confidence interval, 3.77-188.54, P = 0.001). CONCLUSIONS: Growth of nonexudative BVN and progression of polypoidal lesions were found to be lesion characteristics associated with exudative recurrences, and progression of polypoidal lesions might serve as a stand-alone indicator for the near-term onset of exudation. In PCV, more frequent follow-up visits are recommended when polypoidal lesions show progression.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Small dome-shaped pigment epithelium detachment in polypoidal choroidal vasculopathy: an under-recognized sign of polypoidal lesions on optical coherence tomography?

PURPOSE: To evaluate the diagnostic accuracy of spectral-domain optical coherence tomography (SD-OCT) and swept-source optical coherence tomographic angiography (SS-OCTA) to identify polypoidal lesions in serous or serosanguinous maculopathy. MATERIALS AND METHODS: A retrospective review of patients presenting pigment epithelial detachments (PEDs) with the diagnosis of polypoidal choroidal vasculopathy (PCV), neovascular age-related macular degeneration (nAMD), and central serous chorioretinopathy (CSC), all of which underwent SD-OCT, SS-OCTA, and indocyanine green angiography (ICGA). Typical features of polypoidal lesions on SD-OCT included sharply peaked PED, notched PED, and hyperreflective ring underneath PED. SS-OCTA feature was vascularized PEDs on cross-sectional images corresponding to cluster-like structures on en face images. The parameters of PEDs were measured for analysis. RESULTS: Of 72 eyes, 30 had PCV, 22 had nAMD, and 20 had CSC. A total of 128 localized PEDs were detected on SD-OCT. Typical features on SD-OCT had a high specificity (94.0%) but a limited sensitivity (73.8%). SS-OCTA features provided a higher sensitivity (96.7%). PEDs of the polypoidal lesions unrecognized by SD-OCT were dome-shaped, with smaller ratio of height to base diameter and less area, and almost had heterogeneous internal reflectivity and a connected double-layer sign. Some lesions misidentified by SS-OCTA developed into ICGA-proven polypoidal lesions at follow-up visits. CONCLUSION: A small dome-shaped PED with heterogeneous internal reflectivity and a connected double-layer sign on SD-OCT may suggest a polypoidal lesion of PCV. SS-OCTA may be a helpful tool to investigate preclinical PCV and observe the formation of polypoidal lesions.

Association of Microvasculature and Macular Sensitivity in Idiopathic Macular Epiretinal Membrane: Using OCT Angiography and Microperimetry.

Purpose: To investigate the correlation between retinal capillary structure and macular function in patients with idiopathic epiretinal membrane (iERM) by using optical coherence tomography angiography (OCTA) and microperimetry. Methods: This retrospective and observational study included 30 idiopathic ERM eyes of 30 consecutive patients. OCTA was performed to evaluate macular microvasculature including the superficial capillary plexus, deep capillary plexus, and foveal avascular zone. Best corrected visual acuity (BCVA) and microperimetry were measured at baseline and 3 months after surgery. Associations between macular microvasculature and visual function were assessed. Results: Visual function including BCVA and macular sensitivity improved significantly at 3 months post-operatively (p < 0.001). At baseline, BCVA was positively correlated with foveal or parafoveal sensitivities and negatively correlated with central foveal thickness (p < 0.05). Pre-operative foveal sensitivity was significantly correlated with the vessel density of foveal or parafoveal superficial capillary plexus (p < 0.05). A multiple regression model revealed that pre-operative vessel density of foveal deep capillary plexus was an independent positive prognostic factor for post-operative BCVA (B = -0.020 ± 0.006, p = 0.006) and macular sensitivity (B = 0.200 ± 0.081, p = 0.027). Conclusion: Integrated evaluation of iERM by using OCTA and microperimetry shows an association between microvasculature and macular sensitivity. Pre-operative vessel density of foveal deep capillary plexus assessed by OCTA may be a potentially valuable prognostic factor for iERM surgery.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Untargeted metabolomics for uncovering plasma biological markers of wet age-related macular degeneration.

Wet age-related macular degeneration (wAMD) causes central vision loss and represents a major health problem in elderly people. Here we have used untargeted metabolomics using UHPLC-MS to profile plasma from 127 patients with wAMD (67 choroidal neovascularization (CNV) and 60 polypoidal choroidal vasculopathy (PCV)) and 50 controls. A total of 545 biochemicals were detected. Among them, 17 metabolites presented difference between patients with wAMD and controls. Most of them were oxidized lipids (N=6, 35.29%). Comparing to controls, 28 and 18 differential metabolites were identified in patients with CNV and PCV, respectively. Two metabolites, hyodeoxycholic acid and L-tryptophanamide, were differently distributed between PCV and CNV. We first investigated the genetic association with metabolites in wet AMD (CFH rs800292 and HTRA1 rs10490924). We identified six differential metabolites between the GG and AA genotypes of CFH rs800292, five differential metabolites between the GG and AA genotypes of HTRA1 rs10490924, and four differential metabolites between the GG and GA genotypes of rs10490924. We selected four metabolites (cyclamic acid, hyodeoxycholic acid, L-tryptophanamide and O-phosphorylethanolamine) for in vitro experiments. Among them, cyclamic acid reduced the activity, inhibited the proliferation, increased the apoptosis and necrosis in human retinal pigment epithelial cells (HRPECs). L-tryptophanamide affected the proliferation, apoptosis and necrosis in HRPECs, and promoted the tube formation and migration in primary human retinal endothelial cells (HRECs). Hyodeoxycholic acid and O-phosphorylethanolamine inhibited the tube formation and migration in HRECs. The results suggested that differential metabolites have certain effects on wAMD pathogenesis-related HRPECs and HRECs.

ROS production and mitochondrial dysfunction driven by PU.1-regulated NOX4-p22phox activation in Aß-induced retinal pigment epithelial cell injury.

Rationale: Amyloid ß (Aß) deposition, an essential pathological process in age-related macular degeneration (AMD), causes retinal pigment epithelium (RPE) degeneration driven mostly by oxidative stress. However, despite intense investigations, the extent to which overoxidation contributes to Aß-mediated RPE damage and its potential mechanism has not been fully elucidated. Methods: We performed tandem mass-tagged (TMT) mass spectrometry (MS) and bioinformatic analysis of the RPE-choroid complex in an Aß1-40-induced mouse model of retinal degeneration to obtain a comprehensive proteomic profile. Key regulators in this model were confirmed by reactive oxygen species (ROS) detection, mitochondrial ROS assay, oxygen consumption rate (OCR) measurement, gene knockout experiment, chromatin immunoprecipitation (ChIP), and luciferase assay. Results: A total of 4243 proteins were identified, 1069 of which were significantly affected by Aß1-40 and found to be enriched in oxidation-related pathways by bioinformatic analysis. Moreover, NADPH oxidases were identified as hub proteins in Aß1-40-mediated oxidative stress, as evidenced by mitochondrial dysfunction and reactive oxygen species overproduction. By motif and binding site analyses, we found that the transcription factor PU.1/Spi1 acted as a master regulator of the activation of NADPH oxidases, especially the NOX4-p22phox complex. Also, PU.1 silencing impeded RPE oxidative stress and mitochondrial dysfunction and rescued the retinal structure and function. Conclusion: Our study suggests that PU.1 is a novel therapeutic target for AMD, and the regulation of PU.1 expression represents a potentially novel approach against excessive oxidative stress in Aß-driven RPE injury.

TRIM31 inhibits NLRP3 inflammasome and pyroptosis of retinal pigment epithelial cells through ubiquitination of NLRP3.

NOD-like receptor protein 3 (NLRP3) is associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells serve as the immune defense of macula, and their dysfunction causes clinically relevant changes in AMD. In the present study, oxidized low-density lipoprotein (ox-LDL) activated the NLRP3 inflammasome in human RPE cell line ARPE-19. Our data showed that the expression of NLRP3, interleukin-1ß (IL-1ß), and caspase-1 and the release of IL-1ß in ARPE-19 cells were substantially increased by ox-LDL, whereas the addition of NLRP3 inhibitor INF39 dose-dependently reversed the effect of ox-LDL. Overexpression of tripartite motif-containing protein 31 (TRIM31) also suppressed the effect of ox-LDL in ARPE-19 cells. TRIM31 knockdown had similar effects with ox-LDL but INF39 could block the effect of TRIM31 knockdown. Moreover, TRIM31 could interact with NLRP3 in ARPE-19 cells. Overexpression of TRIM31 increased NLRP3 ubiquitination. In conclusion, the results propose that TRIM31 could enhance NLRP3 ubiquitination, therefore inhibiting NLRP3 inflammasome and pyroptosis in human RPE cells.

Functional evaluation with microperimetry in large idiopathic macular holes treated by a free internal limiting membrane flap tamponade technique.

BACKGROUND: Free internal limiting membrane (ILM) flap tamponade technique is an alternative choice for treating large idiopathic macular holes (IMHs). However, the functional recovery related to this surgical approach is not well-characterized. This study aimed to evaluate morphological and microperimetric outcomes 6 months after free ILM flap tamponade technique for large IMHs. METHODS: Twenty-two patients (22 eyes) with large IMHs (minimal diameter > 400 µm) were retrospectively enrolled in this study. All patients underwent 23-gauge pars plana vitrectomy with ILM peeling and free ILM flap tamponade procedures. Snellen best-corrected visual acuity (BCVA), optical coherence tomography (OCT), and MP-1 microperimetry were measured at baseline and 6 months after surgery. Associations of postoperative BCVA with retinal sensitivity were detected. RESULTS: Macular hole closure was achieved in 21 eyes (95.5%). Dislodgement of free ILM flap was found in non-closed eye. Mean logMAR BCVA improved from 1.10 ± 0.33 at baseline to 0.67 ± 0.32 at 6 months postoperatively (P < 0.001). The mean overall macular sensitivity and foveal fixation stability increased respectively from 8.58 ± 3.05 dB and 65.64 ± 17.28% before surgery to 11.55 ± 2.72 dB and 78.59 ± 13.00% at 6 months after surgery (P < 0.001). The mean change in foveal sensitivity (within 2°) was significantly greater than the change achieved for peri-foveal sensitivity (2° to 10°) by 1.50 ± 2.62 dB (P = 0.014). Linear regression analysis showed that postoperative logMAR BCVA was significantly associated with duration of symptom (B = 0.063, P = 0.001), preoperative logMAR BCVA (B = 0.770, P = 0.000), preoperative peri-foveal (B = - 0.065, P = 0.000) and foveal sensitivity (B = - 0.129, P = 0.000). Moreover, multiple regression model revealed that preoperative foveal sensitivity was independently associated with postoperative logMAR BCVA (B = - 0.430, P = 0.040). CONCLUSIONS: Vitrectomy combined with ILM peeling and free ILM flap tamponade technique results in effective morphological and functional recovery for large IMHs. Preoperative foveal sensitivity might be a prognostic indicator for postoperative BCVA.

Cytolethal distending toxin-induced release of interleukin-1ß by human macrophages is dependent upon activation of glycogen synthase kinase 3ß, spleen tyrosine kinase (Syk) and the noncanonical inflammasome.

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro-inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase-1 (canonical inflammasome), Cdt treatment leads to caspase-4 activation and involvement of the noncanonical inflammasome. Cdt-treated cells exhibit pyroptosis characterised by cleavage of gasdermin-D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase-1) or noncanonical (caspase-4) inflammasome blocks both Cdt-induced release of IL-1ß and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt-induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt-induced activation of GSK3ß. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as A. actinomycetemcomitans.

Heterogeneous Nuclear Ribonucleoproteins A1 and A2 Function in Telomerase-Dependent Maintenance of Telomeres.

The A/B subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs A/B), which includes hnRNP A1, A2/B1, and A3, plays an important role in cell proliferation. The simultaneous suppression of hnRNP A1/A2, but not the suppression of hnRNP A1 or A2 alone, has been shown to inhibit cell proliferation and induce apoptosis in cancer cells, but not in mortal normal cells. However, the molecular basis for such a differential inhibition of cell proliferation remains unknown. Here, we show that the simultaneous suppression of hnRNP A1 and hnRNP A2 resulted in dysfunctional telomeres and induced DNA damage responses in cancer cells. The inhibition of apoptosis did not alleviate the inhibition of cell proliferation nor the formation of dysfunctional telomeres in cancer cells depleted of hnRNP A1/A2. Moreover, while proliferation of mortal normal fibroblasts was not sensitive to the depletion of hnRNP A1/A2, the ectopic expression of hTERT in normal fibroblasts rendered these cells sensitive to proliferation inhibition, which was associated with the production of dysfunctional telomeres. Our study demonstrates that hnRNP A1 and A2 function to maintain telomeres in telomerase-expressing cells only, suggesting that the maintenance of functional telomeres in telomerase-expressing cancer cells employs factors that differ from those used in the telomerase-negative normal cells.

NLRX1 modulates differentially NLRP3 inflammasome activation and NF-κB signaling during Fusobacterium nucleatum infection.

NOD-like receptors (NLRs) play a large role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, enhances mitochondrial ROS (mROS) generation. mROS can activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-induced NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). We found that depletion of NLRX1 by shRNA attenuated ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibited Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Conversely, NLRX1 also acted as a negative regulator of NF-κB signaling and IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, and NLRX1 should decrease their ability to stimulate expression of pro-inflammatory cytokines such as IL-8. Therefore, NLRX1 may act as a potential switch with regards to anti-microbial responses in healthy or diseased states in the oral cavity.

Mineral particles stimulate innate immunity through neutrophil extracellular traps containing HMGB1.

Calcium phosphate-based mineralo-organic particles form spontaneously in the body and may represent precursors of ectopic calcification. We have shown earlier that these particles induce activation of caspase-1 and secretion of IL-1ß by macrophages. However, whether the particles may produce other effects on immune cells is unclear. Here, we show that these particles induce the release of neutrophil extracellular traps (NETs) in a size-dependent manner by human neutrophils. Intracellular production of reactive oxygen species is required for particle-induced NET release by neutrophils. NETs contain the high-mobility group protein B1 (HMGB1), a DNA-binding protein capable of inducing secretion of TNF-α by a monocyte/macrophage cell line and primary macrophages. HMGB1 functions as a ligand of Toll-like receptors 2 and 4 on macrophages, leading to activation of the MyD88 pathway and TNF-α production. Furthermore, HMGB1 is critical to activate the particle-induced pro-inflammatory cascade in the peritoneum of mice. These results indicate that mineral particles promote pro-inflammatory responses by engaging neutrophils and macrophages via signaling of danger signals through NETs.

Cooperation of Rel family members in regulating Aß1-40-mediated pro-inflammatory cytokine secretion by retinal pigment epithelial cells.

Amyloid-beta (Aß) is a hallmark component of age-related macular degeneration (AMD), which induces secretion of pro-inflammatory cytokines from retinal pigment epithelium (RPE). Previous studies have shown that p50/RelA (p65), a member of NF-κB family, is an essential pro-inflammatory transcription factor responding to Aß1-40 stimulation, but few focused on the other two Rel transcription factor members - RelB and c-Rel - and their role in Aß1-40-mediated inflammation. It was reported that RelA, RelB and c-Rel are also implicated in various NF-κB-mediated inflammatory diseases. Therefore, we infer that Aß1-40-mediated inflammation targets not only the classical inflammation regulator, RelA, but also RelB and c-Rel. In this study, we demonstrate that intravitreally injected Aß1-40 mice develop AMD-like pathologic changes, coupled with Rel protein (RelA, RelB and c-Rel) synthesis and nuclear translocation. To focus on the interaction mechanism of Rel proteins, we found that RelB and c-Rel formed a heterodimer with RelA in mice model. We also found that c-Rel silencing decreased the levels of Aß1-40-dependent RelA expression, indicating that RelB and c-Rel may interact with RelA as coactivator and c-Rel is required to activate the expression of RelA. Moreover, Rel protein silencing decreased the expression of distinct pro-inflammatory cytokines. Together, we demonstrate that besides RelA, RelB and c-Rel can also be activated by Aß1-40, all of which mediate pro-inflammatory cytokine transcription and RPE damage. Our findings imply that RPE-mediated inflammation under the stimulation of Aß1-40 is multi-targeted and RelA, RelB and c-Rel proteins may be the new targets of anti-inflammatory agents.

Pleomorphic bacteria-like structures in human blood represent non-living membrane vesicles and protein particles.

Although human blood is believed to be a sterile environment, recent studies suggest that pleomorphic bacteria exist in the blood of healthy humans. These studies have led to the development of "live-blood analysis," a technique used by alternative medicine practitioners to diagnose various human conditions, including allergies, cancer, cardiovascular disease and septicemia. We show here that bacteria-like vesicles and refringent particles form in healthy human blood observed under dark-field microscopy. These structures gradually increase in number during incubation and show morphologies reminiscent of cells undergoing division. Based on lipid analysis and Western blotting, we show that the bacteria-like entities consist of membrane vesicles containing serum and exosome proteins, including albumin, fetuin-A, apolipoprotein-A1, alkaline phosphatase, TNFR1 and CD63. In contrast, the refringent particles represent protein aggregates that contain several blood proteins. 16S rDNA PCR analysis reveals the presence of bacterial DNA in incubated blood samples but also in negative controls, indicating that the amplified sequences represent contaminants. These results suggest that the bacteria-like vesicles and refringent particles observed in human blood represent non-living membrane vesicles and protein aggregates derived from blood. The phenomena observed during live-blood analysis are therefore consistent with time-dependent decay of cells and body fluids during incubation ex vivo.

Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome-dependent secretion of IL-1ß and the danger signals ASC and HMGB1.

Fusobacterium nucleatum is an invasive anaerobic bacterium that is associated with periodontal disease. Previous studies have focused on virulence factors produced by F. nucleatum, but early recognition of the pathogen by the immune system remains poorly understood. Although an inflammasome in gingival epithelial cells (GECs) can be stimulated by danger-associated molecular patterns (DAMPs) (also known as danger signals) such as ATP, inflammasome activation by this periodontal pathogen has yet to be described in these cells. This study therefore examines the effects of F. nucleatum infection on pro-inflammatory cytokine expression and inflammasome activation in GECs. Our results indicate that infection induces translocation of NF-κB into the nucleus, resulting in cytokine gene expression. In addition, infection activates the NLRP3 inflammasome, which in turn activates caspase-1 and stimulates secretion of mature IL-1ß. Unlike other pathogens studied until now, F. nucleatum activates the inflammasome in GECs in the absence of exogenous DAMPs such as ATP. Finally, infection promotes release of other DAMPs that mediate inflammation, such as high-mobility group box 1 protein and apoptosis-associated speck-like protein, with a similar time-course as caspase-1 activation. Thus, F. nucleatum expresses the pathogen-associated molecular patterns necessary to activate NF-κB and also provides an endogenous DAMP to stimulate the inflammasome and further amplify inflammation through secretion of secondary DAMPs.

N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

One-step preparation of hydrophilic carbon nanofiber containing magnetic Ni nanoparticles materials and their application in drug delivery.

A one-step process for the synthesis of hydrophilic carbon nanofibers (CNFs) through CO2 hydrogenation on NiNa/Al2O3 was developed for the loading and targeted delivery of the anticancer drug doxorubicin (DOX). CNFs that were synthesized on NiNa/Al2O3 for 9 h at 500 °C exhibited an adequate magnetic response and a large content of hydrophilic oxygen-containing functional groups on the carbon surface, resulting in excellent colloidal solution. The CNF material exhibited a highly efficient capacity for DOX adsorption, particularly at pH 9.0. The loading and release of DOX was strongly pH dependent, possibly due to electrostatic and π-π stacking interactions between DOX and CNF sample. The Langmuir isotherm and pseudo second-order kinetics of DOX-loaded CNFs were well-modeled for the process of DOX adsorption. DOX-loaded CNF targeted cancer cells more selectively and effectively than free DOX and exhibited a marked tendency to kill HeLa cancer cells and reduced toxicity to normal human primary fibroblast (HPF) cells.