The role of E2F4 in the growth suppressive properties of the retinoblastoma protein

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.The growth suppressive functions of the retinoblastoma protein (pRB), the first identified tumor suppressor, are considerably mediated through the repression of the E2F transcription factors. Functional inactivation of pRB, and subsequent deregulation of E2F activity, is a critical event in the formation of most human cancers. pRB is a member of the pocket protein family, which includes p107 and p130. The pocket proteins have some functional redundancy; however, they have differential binding properties to the E2Fs and make very different contributions to the suppression of tumors. The E2F proteins that associate with the pocket proteins can be divided into two groups based on structural and functional similarities. The activating E2Fs, E2F1, E2F2 and E2F3a, are exclusively regulated by pRB and are primarily involved in activating the transcription of E2F-responsive genes that are required for cell cycle progression. The repressive E2Fs, E2F3b, E2F4 and E2F5, are regulated by the entire pocket protein family and are important for transcriptional repression. Mechanistically, the inappropriate proliferation promoted by the absence of pRB is, in large part, attributed to the activating E2Fs. This study investigates the contribution of a repressive E2F, E2F4, to the growth inhibitory properties of pRB during normal development and tumorigenesis. The characterization of mutant mice demonstrated that E2F4 loss significantly suppresses tumor formation in the Rb+/- animals. Molecular analyses suggest a novel mechanism in which p107 and p130 compensate for the loss of pRB by re-establishing the proper regulation of the activating E2Fs. The function of E2F4 in the developmental phenotypes arising from homozygous mutation of Rb was also assessed.(cont.) In contrast to the tumor setting, E2F4 loss aggravated the placental defect in the Rb-/- mice, resulting in earlier lethality. Furthermore, there was no cooperation between E2F4 and pRB within the developing embryo. These results indicate that E2F4 acts in a manner differing from the activating E2Fs in mediating the effects of pRB deficiency in development. Thus, this study has established that E2F4 is a key downstream effector of pRB and has reshaped our understanding of the roles of the activating and repressive E2Fs with respect to the pocket proteins.by Eunice Y. Lee.Ph.D

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Lightweight Reduction Unit

Harmonic gears, which consist of a fixed outer gear with a flexible inner gear that rotates around by a motor, causing the teeth of the gears to contact, can have many functions, but many of these functions cannot be done because the harmonic gears are too heavy to use for smaller machines and too expensive for consumers to regularly use. An inexpensive and lightweight gear could contribute to building a robotic actuator, while keeping the same structure and function as a harmonic gear. Additionally, if a part of the gear were to break, the whole harmonic gear has to be replaced while this gear would have interchangeable parts. In collaboration with Harmonic Drive Co., research is currently focused on finding a suitable control gear for smaller sized robots to compare how well our gear works. Using SolidWorks, we have made the design for an inexpensive, lightweight gear and eventually 3D printed our gear. Our gears worked just as well as the harmonic gear in the same size we used as a control. We decided to take things further and build a prototype of a SCARA robot arm with two of these gears

Session 2E: Lightweight Reduction Unit

The number of uses for the harmonic gear are endless: from aerospace devices, to medical equipment, to robotics; the harmonic drive is essential to these devices. Its advantages include having high torque, zero backlash, high reduction ratios and precision, but the harmonic drive is often times expensive and heavy. To improve the design of the harmonic gear, the goal of our study is to create an actuator with the same function and advantages as a harmonic drive with it being more affordable and lightweight. The implementation of such a prototype will contribute to making more affordable and lightweight actuators that could replace harmonic drives in the future. The proposed prototype will adopt the same function and structure of the harmonic drive (the wave generator, flexspline, and external spline). However, the prototype will be gearless in attempt to implement a unique flexspline. The process to ensure the completion and success of the prototype will follow a continuous cycle of research, design, and revision

Multifunctional Cellular Targeting, Molecular Delivery, and Imaging by Integrated Mesoporous-Silica with Optical Nanocrescent Antenna: MONA

Multifunctional nanoprobes have attracted significant attention in a wide range of disciplines such as nanomedicine, precision medicine, and cancer diagnosis and treatment. However, integrating multifunctional ability in a nanoscale structure to precisely target, image, and deliver with cellular spatial/temporal resolution is still challenging applications. This is because the development of such high-precision resolution needs to be carried out without labeling, photobleaching, and structurally segregating live cells. In this study, we present an integrated nanostructure of a mesoporous-silica nanosphere with an optical nanocrescent antenna (MONA) for multifunctional cellular targeting, drug delivery, and molecular imaging with spatiotemporal resolution. MONA comprises a systematically constructed Au nanocrescent (AuNC) antenna as a nanosensor and optical switch on a mesoporous-silica nanosphere as a cargo to molecular delivery. MONA made of antiepithelial cell adhesion molecules (anti-EpCAM)-conjugated AuNC facilitates the specific targeting of breast cancer cells, resulting in a highly focused photothermal gradient that functions as a molecular emitter. This light-driven molecular, doxorubicin (DOX) delivery function allows rapid apoptosis of breast cancer cells. Since MONA permits the tracking of quantum biological electron-transfer processes, in addition to its role as an on-demand optical switch, it enables the monitoring of the dynamic behavior of cellular cytochrome pivoting cell apoptosis in response to the DOX delivery. Owing to the integrated functions of molecular actuation and direct sensing at the precisely targeted spot afforded by MONA, we anticipate that this multifunctional optical nanoantenna structure will have an impact in the fields of nanomedicine, cancer theranostics, and basic life sciences

Intelligent Fusion Imaging Photonics for Real-Time Lighting Obstructions

Dynamic detection in challenging lighting environments is essential for advancing intelligent robots and autonomous vehicles. Traditional vision systems are prone to severe lighting conditions in which rapid increases or decreases in contrast or saturation obscures objects, resulting in a loss of visibility. By incorporating intelligent optimization of polarization into vision systems using the iNC (integrated nanoscopic correction), we introduce an intelligent real-time fusion algorithm to address challenging and changing lighting conditions. Through real-time iterative feedback, we rapidly select polarizations, which is difficult to achieve with traditional methods. Fusion images were also dynamically reconstructed using pixel-based weights calculated in the intelligent polarization selection process. We showed that fused images by intelligent polarization selection reduced the mean-square error by two orders of magnitude to uncover subtle features of occluded objects. Our intelligent real-time fusion algorithm also achieved two orders of magnitude increase in time performance without compromising image quality. We expect intelligent fusion imaging photonics to play increasingly vital roles in the fields of next generation intelligent robots and autonomous vehicles

An optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.Published versio

Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2R306C and MECP21155Δ32) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT.NRF (Natl Research Foundation, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Accepted versio