Correlation of Cytokine Levels and Microglial Cell Infiltration during Retinal Degeneration in RCS Rats

Microglial cells, which are immunocompetent cells, are involved in all diseases of the central nervous system. During their activation in various diseases, a variety of soluble factors are released. In the present study, the correlation between cytokine levels and microglial cell migration in the course of retinal degeneration of Royal College of Surgeons (RCS) rats was evaluated. MFG-E8 and CD11b were used to confirm the microglial cells. In the retina of RCS rats, the mRNA expression of seven genes (MFG-E8 and its integrins αυ and ß5, CD11b and the cytokines TNF-α, IL-1ß, and MCP-1) formed almost similar bimodal peak distributions, which were centred at P7 and P45 to P60. In contrast, in rdy rats, which comprised the control group, a unimodal peak distribution centred at P14 was observed. The gene expression accompanied the activation and migration of microglial cells from the inner to the outer layer of the retina during the process of degeneration. Principal component analysis and discriminant function analysis revealed that the expression of these seven genes, especially TNF-α and CD11b, positively correlated with retinal degeneration and microglial activity during retinal degeneration in RCS rats, but not in the control rats. Furthermore, linear regression analysis demonstrated a significant correlation between the expression of these genes and the activation of microglial cells in the dystrophic retina. Our findings suggest that the suppression of microglial cells and the blockade of their cytotoxic effects may constitute a novel therapeutic strategy for treating photoreceptor death in various retinal disorders

Essential Role of MFG-E8 for Phagocytic Properties of Microglial Cells

Milk fat globule factor-E8 (MFG-E8) has been regarded as a key factor involved in the phagocytosis of apoptotic cells. We induced a lentivirus into the microglial cells for the augmentation or abrogation of MFG-E8 expression in mouse microglial cells, and investigated phagocytosis of phosphatidylserine tagged human red blood cells (hRBCs) in co-cultures. Increased MFG-E8 levels were associated with a significant increase in phagocytic activity compared to the controls. Conversely, phagocytosis dramitically decreased due to the abrogation of MFG-E8. In addition, the expression of the inflammatory cytokines, TNF-α and IL-1β, also increased or decreased in the microglial cells with the augmentation or abrogation of MFG-E8, respectively. Our findings indicate that the enhanced expression of MFG-E8 could increase phagocytosis of apoptotic cells; conversely, the rate of phagocytosis and the expression of inflammatory cytokines decreased when MFG-E8 expression was knocked down. Our results confirm that MFG-E8 plays an important role in phagocytosis, and possibly serves as an essential signal molecule for microglial cells

Identification and experimental validation of key m6A modification regulators as potential biomarkers of osteoporosis

Osteoporosis (OP) is a severe systemic bone metabolic disease that occurs worldwide. During the coronavirus pandemic, prioritization of urgent services and delay of elective care attenuated routine screening and monitoring of OP patients. There is an urgent need for novel and effective screening diagnostic biomarkers that require minimal technical and time investments. Several studies have indicated that N6-methyladenosine (m6A) regulators play essential roles in metabolic diseases, including OP. The aim of this study was to identify key m6A regulators as biomarkers of OP through gene expression data analysis and experimental verification. GSE56815 dataset was served as the training dataset for 40 women with high bone mineral density (BMD) and 40 women with low BMD. The expression levels of 14 major m6A regulators were analyzed to screen for differentially expressed m6A regulators in the two groups. The impact of m6A modification on bone metabolism microenvironment characteristics was explored, including osteoblast-related and osteoclast-related gene sets. Most m6A regulators and bone metabolism-related gene sets were dysregulated in the low-BMD samples, and their relationship was also tightly linked. In addition, consensus cluster analysis was performed, and two distinct m6A modification patterns were identified in the low-BMD samples. Subsequently, by univariate and multivariate logistic regression analyses, we identified four key m6A regulators, namely, METTL16, CBLL1, FTO, and YTHDF2. We built a diagnostic model based on the four m6A regulators. CBLL1 and YTHDF2 were protective factors, whereas METTL16 and FTO were risk factors, and the ROC curve and test dataset validated that this model had moderate accuracy in distinguishing high- and low-BMD samples. Furthermore, a regulatory network was constructed of the four hub m6A regulators and 26 m6A target bone metabolism-related genes, which enhanced our understanding of the regulatory mechanisms of m6A modification in OP. Finally, the expression of the four key m6A regulators was validated in vivo and in vitro, which is consistent with the bioinformatic analysis results. Our findings identified four key m6A regulators that are essential for bone metabolism and have specific diagnostic value in OP. These modules could be used as biomarkers of OP in the future

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.Wellcome Trus

Structural design of self-thermal methanol steam reforming microreactor with porous combustion reaction support for hydrogen production

To replace the traditional electric heating mode and increase methanol steam reforming reaction performance in hydrogen production, methanol catalytic combustion was proposed as heat-supply mode for methanol steam reforming microreactor. In this study, the methanol catalytic combustion microreactor and self-thermal methanol steam reforming microreactor for hydrogen production were developed. Furthermore, the catalytic combustion reaction supports with different structures were designed. It was found that the developed self-thermal methanol steam reforming microreactor had better reaction performance. Compared with A-type, the △Tmax of C-type porous reaction support was decreased by 24.4 °C under 1.3 mL/min methanol injection rate. Moreover, methanol conversion and H2 flow rate of the self-thermal methanol steam reforming microreactor with C-type porous reaction support were increased by 15.2% under 10 mL/h methanol-water mixture injection rate and 340 °C self-thermal temperature. Meanwhile, the CO selectivity was decreased by 4.1%. This work provides a new structural design of the self-thermal methanol steam reforming microreactor for hydrogen production for the fuel cell

Decisions of multinational firms : plant location and sourcing

The spatial distribution of economic activities is shaped by the way in which firms operate. I study firms' intra- and inter-national decisions and how their responses to policies affect the social welfare. First, I develop a quantitative model of multi-plant oligopolists where firms decide on plant locations, export and pricing, taking into account competition and cannibalization. I advance the existing trade literature on multinational firms by allowing for interdependent entry, oligopolistic rivalry and variable markups. Despite having a combinatorial discrete choice problem, I provide a toolkit to estimate the model in three steps. I present simulation-based evidence to show that neglecting interdependencies among plant locations within a multi-plant firm introduces quantitatively relevant differences in estimation. Second, I estimate the multi-plant firm model developed in the first essay focusing on the cement industry in the US and Canada. Estimates reveal important features of this industry, especially fixed costs as a key determinant of the market structure. I apply the estimated model to quantify firm-level responses to changes in environmental, trade and competition policies, and highlight the welfare implications of having multi-plant production. Results indicate that a carbon tax of $50/tCO2 in Canada will generate carbon leakage to the US which offsets 18% of the domestic emissions abatement, and also exacerbate the market distortion. A unilateral tariff increase of 20% reduces domestic welfare and does not lead to significant increase in number of plants in the US. I also analyze a hypothetical acquisition in which the efficiency gains of multi-plant firms offset the effects from competition-lessening. Last, I study firms' sourcing decisions through rules of origin (RoOs) in regional trade agreements. They are a set of criteria that define the origin of a product to qualify for preferential access. This chapter distinguishes trade diversion through RoOs from tariff reduction on intermediate goods, focusing on the automotive industry. I find that RoOs significantly distort where to source auto parts and the effect is nonlinear depending on the restrictiveness of rules. The shift from foreign to regional inputs increases before a stringent content rule backfires.Business, Sauder School ofManagement Information Systems, Division ofGraduat

Development of Quantitative Multi-spectral Fluorescence Endoscopic Imaging for Early Cancer Detection

Thesis (Ph.D.)--University of Washington, 2014The incidence of esophageal adenocarcinoma has been rising in the western world with a low 5 year survival rate of less than 15%, but it can be treated when diagnosed early. However, conventional white light endoscopy screening has significant limitations because early cancerous lesions usually lie flat on the tissue surface and do not differ in contrast relative to the surrounding area. In an effort to detect early stages of cancer, fluorescence molecular imaging was developed since it improves diagnostic sensitivity and specificity through targeted visualization of multiple biomarkers at the cellular and sub-cellular level. This dissertation presents technologies and algorithms developed for real-time wide-field and quantitative multispectral fluorescence endoscopy for image-guided biopsies and resections. The body of work includes a realistic Barrett's esophagus phantom model development with calibrated fluorescence targets, enhanced visualization and documentation of diagnostic endoscopic records through image mosaicking, real-time compensation for fluorophore emission cross-talk and mitigation of background tissue autofluorescence for high contrast and quantitative molecular endoscopy. Although the scanning fiber endoscope technology forms the basis of the multispectral imaging that was implemented, the concepts, algorithms, and techniques can be applied to a wide range of in vivo and in vitro imaging technologies. In the long term, this work provides the foundation and framework of computer-aided quantitative endoscopy for personalized diagnosis and treatment

Immunological checkpoints in the control of murine Salmonella enterica infection: IFN-γ pathways and early dendritic cell death

© 2017 Chenying YangSalmonella enterica is a Gram-negative intracellular pathogen, which can cause typhoid fever and non-typhoidal salmonellosis. Every year ~22 million cases and ~200,000 deaths are reported for typhoid fever and ~93 million cases and ~155,000 deaths for non-typhoidal salmonellosis. Innate immunity provides the very early protection against Salmonella, a better understanding of which may lead to a progress in treatment and prevention to Salmonella infection. Dendritic cells (DC) are one of the first cells to sense Salmonella in vivo, and play an important role in initiating a cascade of innate immune control, including phagocytising bacteria and the activation of inflammasomes, which further induces cell death and the production of pro- inflammatory cytokines, such as IFN-γ. In addition, dendritic cells are potent antigen presenting cells (APC) that induce the development of protective adaptive immunity against Salmonella. However, it has been reported that Salmonella possesses various mechanisms, including regulating phagolysosomal fusion and delaying vacuole acidification, and down-regulating flagellin expression to prevent antigen presentation, highlighting the dynamic and complex nature of DC-Salmonella interactions. In recent years, the critical role of DCs in immunity against Salmonella has gained increased attention, however the cellular and molecular mechanisms of the DC-Salmonella interactions are not fully understood. The first aim of this study was to study the survival and death in infected DCs during Salmonella infection, utilising murine bone marrow-derived DCs (BMDCs), which are sensitive to Salmonella-induced cell death within hours of infection. It is found that several virulence factors such as lipopolysaccharide (LPS), Type III secretion system 1 (SPI-1) and flagellin contribute to, and in combination maximise, death in BMDCs. Intriguingly, BMDCs that were not directly infected with Salmonella were killed upon infection of neighbouring cells in culture. An apparently similar ‘bystander’ cell death was induced by co-culturing with filtered supernatant from infected BMDCs, suggesting a role for contact-independent mechanisms. Infected BMDCs released several cytokines, including IL-6, MCP-1 and TNF-α. However, blockade of intracellular protein transport and secretion of cytokines by monesin did not alter Salmonella-induced cell death in uninfected bystanders, suggesting that the bystander effect is not dependent on mediators released from infected BMDCs. BMDCs from mice with gene knockouts in key pathways that are involved in DC immune responses against Salmonella were also tested, and decreased death was observed in ICE-/- BMDCs, suggesting that caspase-1/caspase-11-mediated pyroptosis could be responsible for direct as well as bystander BMDC death. The second aim was to determine the contribution of IFN-γ and the IFN-γ induction pathways in Salmonella infection. Previous studies in our lab have shown that flagellin-induced NLRC4 inflammasome activation in splenic DCs triggers non- cognate memory CD8+ T cells to produce IFN-γ, a critical mediator of innate immunity against Salmonella. It was shown in the present study that deletion of individual components of the NLRC4 inflammasome pathway, e.g. caspase-1 or IL- 18, can lead to a moderate reduction of IFN-γ production, but the impact on the control of Salmonella in infected mice is minimal, suggesting that NLRC4 pathway is not the only source of IFN-γ and that low level of IFN-γ may be sufficient for full protection against Salmonella. In the studies presented here, it was shown that LPS- induced activation of the TLR4 pathway is also an important source of IFN-γ and that mice deficient in components of TLR4 pathway has poor early control of bacterial load during S. Typhimurium BRD509 infection. Interestingly, deficiency in the TLR4 pathway led to an increase rather than reduction of IFN-γ, suggesting that IFN-γ is regulated by different pathways and that TLR4 pathway may be involved in other immune responses that are important for early control of Salmonella