Identifying Critical Genes for Cholesterol Metabolism in Macrophages Using CRISPR-Cas9 Whole-Genome Screens

Macrophage foam cells contribute to atherosclerotic plaque formation, a pathology that underlies heart disease, peripheral arterial disease and stroke. Foam cells form when macrophages take up excessive amounts of low-density lipoprotein (LDL) leading to elevated cellular levels of neutral lipids, which are packaged into lipid droplet organelles. Despite the high public health priority motivating research of cardiovascular disease processes, current understanding of macrophage cholesterol metabolism including mechanisms of LDL uptake, cholesterol trafficking, lipid droplet biogenesis, lipid droplet degradation and cholesterol efflux is limited. Here, we implemented a CRISPR-Cas9 whole genome screening strategy to identify critical genes regulating macrophage cholesterol metabolism. Murine bone marrow derived macrophages (BMDM) were derived from transgenic mice expressing Cas9 protein and transduced with a pooled library of guide RNAs (sgRNA) that contained ~80,000 sgRNA targeting the majority of protein-coding genes in the mouse genome. BMDM were transduced with low viral multiplicity of infection to produce a single sgRNA insertion per cell. To identify gene disruptions conferring gain-of-function or loss-of-function effects on LDL uptake, cholesterol trafficking, or lipid droplet biogenesis, we exposed the mutant populations of BMDM to acetylated or oxidized LDL for 24 h, stained for cellular neutral lipid content using BODIPY 493/503 and sorted low- and high-fluorescence cells by flow cytometry. Similarly, to identify genes critical for lipid droplet degradation, cholesterol metabolism and cholesterol efflux, we exposed the BMDM to modified LDLs for 24 h followed by LDL removal and a 48-h chase. DNA from the sorted cells was deep sequenced to quantify sgRNA inserts. Bioinformatics and statistical analyses identified sgRNA inserts that were enriched in the low neutral lipid or high neutral lipid pools and generated ranked gene lists containing genes regulating cholesterol metabolism in macrophages. To validate the screen results, we made targeted gene disruptions in BMDM followed by staining and fluorescence microscopy of BODIPY and perilipin to verify the neutral lipid phenotype and evaluate lipid droplet morphology and cellular distribution. Of note, we highlight the identification of novel genes regulating neutral lipid levels in macrophages. Emc3 encodes an endoplasmic reticulum membrane complex protein, and when disrupted led to lower cellular neutral lipid content than wildtype cells. Whereas, Atg9a, an autophagy-related protein, when disrupted leads to higher neutral lipid due to inefficient neutral lipid clearance. In addition, gene set enrichment analysis (GSEA) analysis of screen results suggest that Golgi-conserved oligomeric complex and endoplasmic reticulum membrane complex proteins are critical for LDL processing. sgRNA for genes in these gene sets were enriched in the low fluorescence population in the 24-h oxidized or acetylated LDL conditions. Further, our screens identified many genes previously characterized in modified-LDL processing such as genes regulating receptor mediated clathrin-dependent endocytosis, Golgi-toendoplasmic reticulum trafficking, and autophagy-associated proteins as critical regulators for neutral lipids in BMDMs. Overall, these results provide new avenues for research to determine the mechanisms of cholesterol metabolisms in macrophages

The Impact of Ligand Design on the Coordination Chemistry and Reactivity of Metal Pincer Complexes

Pincer ligands are uninegative tridentate metal-coordinating agents of the form [XZY]- where Z is the central, anchoring Lewis donor while X and Y are flanking Lewis donors. Ever since initial reports of transition metal pincer complexes were published in the late 1970\u27s, there has been burgeoning interest in such complexes because of their desirable robust nature, generally simple syntheses, and the spectacular chemical transformations that they can mediate. In this research project, two new sets of pincer ligands with a diarylamido anchor and either two pyrazolyl nitrogenous flanking donors (NNN pincer) or one pyrazolyl and one diphenylphosphine donor (NNP pincer) have been prepared and their late transition metal complexes have been studied. First, for tricarbonylrhenium(I) complexes it was demonstrated that the NNN pincers bind in bidentate or fac- tridentate modes. By increasing steric bulk at the 3-pyrazolyl position near the metal, the fac-Re(CO)3 moiety distorts the ligand to enhance ligand-centered reactivity. Second, for carbonylrhodium(I) complexes, (NNN)Rh(CO), substitution at the para-aryl positions predictably modulates the electronic properties and chemical reactivity. Oxidative addition reactions of the (NNN)Rh(CO) with iodoalkanes proceed about three orders of magnitude faster than those reported for the Monsanto catalyst, [Rh(CO)2I2]-. Third, there is also interest in metal complexes of redox-active ligands because it is hoped that one could use the ligand as an electron reservoir to help arbitrate difficult multi-electron processes. For (NNN)RhIIIXYZ, varying non-pincer ligands(X, Y, and Z) changes the (NNN)/(NNN)+ oxidation potential by 700 mV. An empirical ligand additivity model was discovered that predicts the half wave potential of the ligand-based redox couple. Such a model is envisioned to be important for future considerations when designing complexes for exothermic electron transfer reactions. Finally, a comparison of related (NNN)Rh(CO), (NNP)Rh(CO) and (PNP)Rh(CO) complexes revealed that substitutions of pyrazolyl for diphenylphosphine primarily impacts sterics (not electronics), thereby affecting kinetics of reactions. The PPh2 moiety permits the isolation of a coordinatively-unsaturated 16-electron rhodium(III) complex that showed metal ligand cooperativity in its reactions with HI. The hemilability of the (NNP)Rh fragment was also demonstrated by reactions with t-BuNC

Development Of Ruthenium/terpyridine Complexes For Water Oxidation

The work presented in the dissertation is focused on developing catalysts for water oxidation. In this regard, a series of unsysmmetrical ruthenium complexes of type [Ru(terpy-R)(phen-X)Cl]PF6 where terpy-R= 4\u27-(4-methylmercaptophenyl)-2,2\u27:6\u272 -terpyridine and phen-X=H (1), X= 5-nitro (2), X=5,6-dimethyl (3), and X= 3,4,7,8-tretramethyl (4) was synthesized as precursors for self-assembled monolayers. Water oxidation properties of these complexes were evaluated in the presence of (NH4)2[Ce(NO3)6] as the sacrificial oxidant by measuring catalytic turnover number (TON) after 24 h of reaction time and rate of dioxygen evolution in solution during early stages of catalysis. The results revealed that all complexes 1 - 4 are catalytic towards water oxidation. However, the presence of electron-withdrawing nitro substituent on phenanthroline dramatically decreased the water oxidation activity (TON 60, rate of dioxygen evolution 2.40 × 10-4 µmol O2/s) compared to the complex 1 (TON 410, rate 8.19 × 10-4 µmol O2/s). In contrast, complex 4 with electron-donating methyl substituents on phenanthroline indicated enhanced rate of dioxygen evolution (27.3 × 10-4 µmol O2/s) in solution than complex 1. Furthermore, reactivity of complexes 1 - 4 towards (NH4)2[Ce(NO3)6] followed a trend similar to rate of dioxygen evolution in solution and suggests chloride ligand was likely to retain in the coordination sphere during formation of high-valent ruthenium species. This is an important study as it challenges the accepted mechanism of water oxidation catalysts with halogens as axial ligands. In addition, 1H-NMR and ESI mass analysis of complex 1 isolated after catalysis indicated catalyst core remained intact during catalysis. However, the -PhSCH3 substituent on terpyridine was oxidized to -PhSO2CH3 in the presence of strong oxidizing environment. The need of a sacrificial oxidant such as CeIV to initiate the catalytic cycle can be avoided if a catalyst was deposited on to an electrode surface and activated electrochemically. In this regard, use of Langmuir-Blodgett technique to deposit catalyst onto a surface was studied. Amphiphilic nature essential for LB film formation was introduced to the procatalytic core of [Ru(terpyridine-X)(phenanthroline)Cl]PF6 by long alkane chain attached to the terpyridine(X = C6H6-O(CH2)8CH3). The LB film formation indicated that the film collapsed at 50 mN/m. UV-visible spectra and infrared reflection absorption spectra (IRRAS) of the complex deposited onto an ITO surface were in good agreement with the complex in solution confirming the catalytic core was intact after LB deposition process. The complex indicated 86 catalytic turnover cycles during a period of 24 h in solution in the presence of CeIV as the sacrificial oxidant. The catalytic wave for water oxidation appeared at 1.5 V vs. Ag/AgCl indicating 0.36 V overpotential compared standard potential of water oxidation. In addition, catalytic wave increased linearly with number of LB monolayers, suggesting catalytic properties were not confined to the outer most LB monolayer. In the next series of ruthenium/terpyridine complexes, capping ligand was changed to redox non-innocent 3,5-di-tert-butyl-2-(phenylamino)catechol (L1) and 3,5-di-tert-butyl-catechol (L2). Following complexes [Ru(TpyOC9)(L1)Cl]PF6, [Ru(TpyOC9)(L2)Cl], and [Ru(TpyOC9)(L2)DMSO]Cl were synthesized and characterized. The change of ligand architecture to from L1 to L2 and change in axial ligand from chloride to dimethyl sulfoxide resulted in dramatic changes in electronic and redox properties. LB isotherm and Brewster angle micrographs of complexes with L1 and L2 ligands indicated a uniform monolayer formation. However, UV-visible, redox, and IRRAS studies indicated only the complex with L1 ligand can be successfully transferred onto a substrate. Unfortunately, none of the above mentioned complexes with L1 and L2 ligands were catalytic towards water oxidation. In summary, studies on ruthenium/terpyridine complexes found that, (i) water oxidation properties depends on the nature and the position of electron-withdrawing/donating substituents on phenanthroline, (ii) LB technique can be used to deposit water oxidation catalysts onto a surface, (iii) the complexes with redox non innocent ligands indicated that the ligand architecture and the axial ligand coordinated to the ruthenium center played a significant role in regulating redox processes and electronic spectral properties

Application of CRISPR/Cas9 Whole Genome Screens to Advance Macrophage Biology

Advances in CRISPR/Cas9 gene editing technologies present opportunities to better understand the contribution of individual genes to complex eukaryotic cellular processes. I have applied CRISPR whole genome screen technologies to inform further mechanistic understanding of macrophage fitness and neutral lipid metabolism. This work is important because macrophages play critical roles in immune function and are implicated as causative agents in disease states. Chapter One of this dissertation provides insights into how CRISPR genome screening technologies have improved biological discovery and key findings from their application and discusses how this technology can be implemented to advance the discovery of novel macrophage biology. Chapter Two reports the results of CRISPR whole genome screens that identified novel positive and negative regulators of macrophage fitness. There is limited understanding of regulators of macrophage fitness, including mechanisms of survival, proliferation, and adhesion. The screens uncovered p21-activated kinase-2 (PAK2) as a novel suppressor of macrophage fitness. We show that Pak2 gene disruption led to those mutants becoming more proliferative via NF2/Merlin-mediated downregulation of cyclin-D1. In addition, this work indicates that PAK2 suppresses macropinocytic uptake by modulation of actin dynamics. In Chapter Three, I applied CRISPR/Cas9 screening technology to identify critical genes for modified LDL-derived neutral lipid metabolism in macrophages. This is important because macrophages are contributors to atherosclerotic plaque formation. We identified lysosomal nutrient sensing, mTOR signaling, and autophagy as critical pathways and processes regulating neutral lipid content in macrophages. This work provides evidence that lipophagy, the autophagy-mediated degradation of lipid droplets, is the dominant mechanism promoting lipid droplet clearance and supporting cholesterol efflux in macrophages. Chapter Four of this dissertation contextualizes how the findings from the CRISPR whole-genome screens from the preceding chapters contribute to the advancement of macrophage biology and how the findings of this dissertation inform current macrophage-based therapeutical approaches. Chapter Four provides examples of existing approaches where macrophages are therapeutically targeted, their effectiveness, and challenges. Overall, the work presented in this dissertation will serve as a valuable source to inform continued hypothesis generation from unexplored hits and continue to expand our understanding of macrophage biology

Classification and Identification of Volatile Organic Solvents based on Functional Groups using Electronic Nose

The Metal Oxide Semiconductor gas sensors based on SnO2 indicate cross sensitivity to many volatile organic compounds. Therefore, this study is focused on developing a methodology to distinguish organic solvents based on the functional groups present using an array of Metal Oxide Semiconductor gas sensors. Here, representative compounds for aliphatic, aromatic hydrocarbons, carbonyl groups, esters, alcohols and dichloromethane were used to evaluate gas sensors. Then data were analyzed using Principal Component Analysis and k-Nearest Neighbor methods. Finally, k-Nearest Neighbor best model was developed to predict the chemicals based on the sensor data. The overall results of this study sufficiently explain that the developed electronic nose system can distinguish the chemicals tested with Principal Component Analysis (96.6 percentage) and can predict with k-Nearest Neighbor (k=5) (90 percentage) the chemicals based on the sensor responses. These results demonstrate that the developed electronic nose can be used to classify and identify chemicals based in different functional groups

Politicizing ‘the virtual’: examining the internet on the intersections of gender and sexuality in Sri Lanka

This chapter provides a review of existing scholarship that lies on the intersections of sexuality, gender, and technology in relation to Sri Lanka. Key intellectual developments and debates on technology, especially the internet and social media, as a site of both violence and contestation for gendered and sexualized subjects in Sri Lanka will be examined. The chapters look at the ways in which majoritarian politics, patriarchy, and heteronormativity surface online and what this means for women and queer Sri Lankans. Additionally, it also examines the ways in which women and queer Sri Lankans appear to be working on and through these platforms to resist these exclusionary power structures. In doing so, the intention is to highlight the complex ways in which the virtual is characterized by violence, inequality, and injustice, as well as community, solidarity, resistance, joy, and pleasure

A Review on Evapotranspiration Estimation in Agricultural Water Management: Past, Present, and Future

Evapotranspiration (ET) is a major component of the water cycle and agricultural water balance. Estimation of water consumption over agricultural areas is important for agricultural water resources planning, management, and regulation. It leads to the establishment of a sustainable water balance, mitigates the impacts of water scarcity, as well as prevents the overusing and wasting of precious water resources. As evapotranspiration is a major consumptive use of irrigation water and rainwater on agricultural lands, improvements of water use efficiency and sustainable water management in agriculture must be based on the accurate estimation of ET. Applications of precision and digital agricultural technologies, the integration of advanced techniques including remote sensing and satellite technology, and usage of machine learning algorithms will be an advantage to enhance the accuracy of the ET estimation in agricultural water management. This paper reviews and summarizes the technical development of the available methodologies and explores the advanced techniques in the estimation of ET in agricultural water management and highlights the potential improvements to enhance the accuracy of the ET estimation to achieve precise agricultural water management

Chemical Switching Behaviour of Tricarbonylrhenium(I) Complexes of a New Redox Active ‘Pincer’ Ligand

The structures and optoelectronic properties of tricarbonylrhenium(I) complexes of di(2-pyrazolyl-p-tolyl)amine in its neutral and deprotonated (uninegative amido) form were investigated. Reactions of the complexes with Brønsted acids or bases result in distinctive changes of colour and electrochemical activity owing to the non-innocent nature of the ligand

E-marketing Tools for Food Businesses Amidst Covid-19 Pandemic: Advantages and Challenges

Many businesses are interested in searching for the latest technologies to secure the tie with existing customers and to prevent potential customers from abandoning their businesses during the COVID-19 pandemic. The food industry is one of them. Therefore, this review article is an attempt to identify various e-marketing tools adopted in the business world in the pre-covid era and to underpin their applicability in the food industry in the covid era by highlighting the advantages and challenges of their adoption. Research articles, conference proceedings, book chapters, theses, and dissertations regarding the topic of applications of e-marketing published from 2000 to 2019 available in the google scholar database, were considered in the review. The final search of the literature was carried out in February 2020. The study reveals that the benefits of applying e-marketing tools in food businesses could compensate for the pitfalls of adopting them. Authors suggest that the ability to compare prices of similar products from different sellers as the most promising benefit of e-marketing, from the customer perspective. Similarly, the cost-effectiveness experienced by food suppliers compared to traditional marketing, makes the e-marketing concept attractive, for food businesses. This work discovers the ability and the inclination of people to launch, manage and organize business ventures with e-marketing approaches in the present as well as the future world.

Using Sterics to Promote Reactivity in \u3cem\u3efac\u3c/em\u3e-Re(CO)\u3csub\u3e3\u3c/sub\u3e Complexes of Some ‘Non-Innocent’ NNN-Pincer Ligands

Two new redox active ligands based on di(2-(3-organopyrazolyl)-p-tolyl)amine have been prepared in order to investigate potential effects of steric bulk on the structures, electronic properties, or reactivity of tricarbonylrhenium(I) complexes. Replacing the hydrogens at the 3-pyrazolyl positions with alkyl groups causes significant distortion to the ligand framework due to potential interactions between these groups when bound to a fac-Re(CO)3 moiety. The distortions effectively increase the nucleophilic character of the central amino nitrogen and ligand-centered reactivity of the metal complexes