Blog Archives

Overview

Although advances made in health and safety have more than doubled life expectancy throughout much of the world since 1900, it hasn’t been without consequence. Disease, disability, and frailty have all impacted the quality of life associated with these later years. This unfortunate reality was recently illuminated by the COVID-19 pandemic, which severely affected this population, likely due to physiological changes and preexisting conditions. Fortunately, a primary goal of geroscience researchers is to attenuate age-related health issues so that older people not only enjoy an improved quality of life, but also maintain the resilience to survive severe diseases and infections.

While it’s irrefutable that we cannot avoid aging, it’s no longer within the realm of science fiction for us to temper and even reverse the aging process. On May 19, 2021, the New York Academy of Sciences hosted a virtual symposium that brought together geroscience experts spanning various disciplines, including genetics, endocrinology, gerontology, clinical psychology, and more. Speakers discussed targeting the key hallmarks of aging, developing biomarkers for geriatric therapies, and translating findings that extend healthspan and lifespan to the clinic.

Symposium Highlights

• The Target Aging with Metformin study uses the FDA approved anti-diabetic metformin, which targets the hallmarks of aging, to investigate the prevention of age-related diseases.
• Precluding the age-associated decline of chaperon-mediated autophagy restrains the aggregating effects of Alzheimer’s disease and extends lifespan in murine models.
• Lower IGF-1 levels in older adults are associated with decreased cognitive impairment, age-related diseases, and mortality.
• Epigenetic clocks can be applied to study biological aging differences, with accelerated epigenetic aging correlating with the prevalence and incidence of morbidity and mortality.
• The metabolome is a powerful locus of opportunity to bridge the gap between genotype and age.
• Alternative splicing is upregulated in response to declining mitochondrial function and increasing age.
• Senescent cells upregulate pro-survival pathways, and their elimination alleviates diverse age-related conditions.
• The mitochondrial-derived peptides humanin and MOTS-c are associated with increased longevity in animal models and humans.

Speakers

Targetable Aging Processes

Speakers

Keynote: Age Later: Translational Geroscience

Aging is the strongest risk factor for all age-related diseases, with diverse maladies accumulating during the later years of life. Hence, to abate or avert the relevant disorders, it’s critical to target the central driver—aging itself. Physician Nir Barzilai, the founding director of the Institute for Aging Research, investigates the genetics of longevity by studying centenarians and their offspring, interrogating the hypothesis that these individuals have genes that prolong aging and protect against age-related diseases.

Using Slow Off-Rate Modified Aptamer, Barzilai’s team assessed 5,000 proteins in a population of 1,000 individuals between the ages of 65-95, a period during which aging accelerates. Results demonstrated a significant change in the level of hundreds of proteins as a function of age. Among the top hits were proteins from collagen breakdown of tissue and cellular products, highlighting the pivotal role this process plays in aging, and suggesting that deterring disintegration may be a universal biomarker for geroprotection.

A predominant challenge to translating advances made in geroscience from animal models to humans is the FDA, which currently doesn’t consider aging a disease indication or preventable condition. Barzilai and others are utilizing metformin, an FDA-approved anti-diabetic, to refute this contention. Various groups have shown that metformin has substantial effects on human healthspan, including delaying type-2 diabetes mellitus (T2DM). In this patient subset, metformin also impedes cardiovascular disease, cognitive decline, and Alzheimer’s and is associated with decreased cancer incidence, with population effects approaching 30% in all cases.

Barzilai’s team designed the Target Aging with Metformin, or TAME, study to investigate whether or not there’s a shift in the timeline of disease occurrence between a cohort receiving metformin versus a control cohort. Various biomarkers of aging and age-related diseases will be used to provide convergent evidence of broad, age-related effects, while also establishing a resource for innovation and discovery of emergent biomarkers.

“The most important thing for us is to develop biomarkers that will change when we use a gerotherapuetic,” Barzilai asserted, as this will expedite therapeutic prospects.

Targeting Selective Autophagy in Aging and Age-related Diseases

Physician-scientist Ana Maria Cuervo’s research seeks to understand the molecular basis of autophagy dysfunction with age and the contribution of defects in this cellular pathway to diseases such as neurodegeneration, metabolic disorders, and cancer. Autophagy belongs to the proteostasis network, which regulates protein content and quality control.

Chaperon-mediated autophagy (CMA) is a subset of the mammalian autophagy program that directly targets proteins to the lysosome for degradation. CMA has been shown to decrease with age in human and animal models. Cuervo’s lab developed a fluorescent murine reporter construct to visualize CMA and track the kinetics of its activity in different organs.

Blocking this pathway in neurons resulted in the aggregation of proteins like α-synuclein (α-syn), tau, and others that are causal in Alzheimer’s Disease (AD). Additionally, CMA reporter mice crossed with a mouse model of AD revealed that CMA activity dramatically decreases in the neurons of AD mice.

Leveraging these findings, Cuervo’s group generated a mouse model to restore CMA activity conditionally. Mice with preserved CMA exhibited an extended median and maximal lifespan compared to controls. Evaluation of the proteostasis network in mice with and without CMA restoration revealed major changes in the proteome. Mice in which CMA was preserved more closely resembled younger animals than their age-matched controls.

“By acting in one of these pathways, we can have an impact in the other hallmarks of aging… because of this interconnection among [them],” Cuervo emphasized.

A compound to selectively activate CMA was developed and tested in an AD model, with results illustrating a reduction in tau pathology and microglial activation in the presence of this agent.

Further Readings

Biomarkers for Therapies

Speakers

Translational Geroscience: Role of IGF-1 in Human Healthspan and Lifespan

Physician Sofiya Milman conducts translational research to uncover the genomic mechanisms regulating the endocrine and metabolic pathways involved in age-related conditions like diabetes, cardiovascular disorders, and Alzheimer’s.

“The goal of geroscience is really to extend healthspan, and not necessarily lifespan,” Milman opened. “What we’re really trying to do is to compress the period of morbidity.”

To discover the biological pathways that allow humans to live long, healthy lives, Milman’s team focused on IGF-1: a reduction of this factor has been consistently shown to extend healthspan and lifespan in models. IGF-1 levels peak during the teenage years before gradually declining. If the reduction of IGF-1 protects from aging, Milman reasoned that lower IGF-1 levels would delay aging and prevent age-related diseases.

Examining a cohort of centenarians expressing lower levels of IGF-1 revealed a 50% reduction in cognitive impairment compared to higher IGF-1 level controls. Genetic studies demonstrated that centenarians were enriched for rare mutations in the IGF-1 receptor that diminished signaling. Additionally, individuals 65+ with low IGF-1 had less cognitive impairment, and delayed onset of cognitive impairment, multi-morbidities, and mortality.

Milman’s team also addressed the link between IGF-1 and age. Younger individuals with lower levels of IGF-1 were at an increased risk for mortality and age-related diseases compared to older individuals, while higher levels of IGF-1 in older adults were associated with increased risk. This suggests that the IGF-1 network aligns with the concept of antagonistic pleiotropy, wherein a factor that’s beneficial to individuals when they’re younger may become harmful when they’re older. It’s advantageous to maintain functionality of proteostasis and resilience as an individual gets older, but IFG-1 inhibits programs involved in these processes.

“So from this, we think it would be wise to maintain IGF-1 levels in youth, but to reduce them with aging,” Milman concluded.

Epigenetic Biomarker of Aging for Lifespan and Healthspan

Biological age is defined by changes or alterations in a living system that renders it more vulnerable to failure and is behind the age-related increase in susceptibility to chronic diseases. Unlike chronological age, it is very difficult to measure because it’s unobservable.

Morgan Levine integrates theories and methods from statistical genetics, computational biology, and mathematical demography to develop biomarkers of aging for humans and animal models. Among this work are efforts to establish systems-level outcome measures of aging to facilitate evaluation for gero-protective interventions.

“There’s some disagreement on how we actually quantify [biological age],” Levine started. “But I would argue that it’s really important to try and do so, because quantifying [this] will really help us in a number of endeavors in the field.”

Levin’s lab is particularly interested in epigenetic aging, as aging drastically remodels the DNA methylation landscape, with widespread increases and decreases as a function of age.

Epigenetic clocks estimate DNA methylation across the genome and combine supervised machine-learning approaches to develop predictors of biological age.

“We think people who have a predicted [epigenetic] age that’s younger than their chronological age should be actually aging slower, whereas the opposite is true for people that have a genetic age that is predicted higher,” said Levine.

Applying these measures to diseased states yielded several pertinent findings. For example, individuals who have pathologically diagnosed Alzheimer’s post-mortem show accelerated epigenetic aging in their brain relative to their chronological age. Tissue differences were also captured, revealing that tissues seem to age asynchronously, with highly proliferative tissues and tumor cells having accelerated aging compared to slower aging brain tissue.

Levine’s group also evaluated cellular senescence and energy disruption, with results revealing that near senescent, HRAS oncogene induced senescent, and replicative stress senescent cells have an acceleration in epigenetic age compared to early parental control cells. Additionally, deletion of mitochondrial DNA accelerated epigenetic aging, while caloric restriction in mice stalled their epigenetic clocks.

Further Readings

Omics for Therapies

Speakers

Metabolomics in the Search for Biomarkers and Mechanisms of Aging

Daniel Promislow applies metabolomics and systems biology approaches to study aging, with a focus on understanding the evolutionary and molecular traits that shape fitness in the natural human population. Although genome-wide association studies have allowed researchers to identify thousands of polymorphisms associated with the complement of measurable traits, including aging, the disparities identified explain less than half of 1% of the phenotypic variations.

Many genes interacting with each other ultimately influence phenotypes, and the biological distance between the two is astronomical. To bridge this gap, researchers use endophenotypes—from the epigenome, transcriptome, proteome, metabolome, and microbiome—along with various omics approaches. Promislow’s lab focuses on the metabolome, which integrates information from the environment and genotype to ultimately affect aging.

Promislow’s team utilizes translational metabolomics in various insect and animal models to understand and translate aging patterns to human populations. Applying this approach to Drosophila demonstrated that the metabolome could predict stress resistance, completely separating groups of sensitive or resistant flies to a metabolic stressor by principal component analysis. These effects could not be recapitulated with a whole fly genome sequence dataset. Evaluating response to diet restriction (DR) also revealed changes in metabolite levels with age. Among nearly 200 different inbred strains, roughly 75% showed a benefit to DR.

“Interestingly, the effect of specific genetic variants on the lifespan response was very weak,” Promislow began. “But we did find genes that were associated with metabolites, which were associated with the lifespan response, reinforcing this idea…that the metabolite profile can be a kind of bridge between genotype and phenotype.”

Promislow’s group also demonstrated that the metabolome could serve as a biological clock, revealing that shorter-lived genotypes appeared to have a higher biological age than expected for their chronological age.

Translational Potential of the Biology of Aging

As individuals age, the incidence of chronic disease increase, and disease progression quickens. Physician-scientist Luigi Ferrucci aims to interrogate the causal pathways that lead to progressive physical and cognitive decline in aging.

Cellular damage is accumulated during a person’s life, eventually reaching a pathology threshold that becomes clinically relevant when the damage presents as a disorder. Conventionally, the disease is often only addressed once it reaches this stage. The problem with this approach is that the present disease is often a marker of a more profound and invasive disorder to come.

“[Instead], we need to measure the underlying force that determines the emergence of diseases and their consequences,” Ferrucci argued.

By interfering with the basic mechanisms of aging to curtail it, broader effects of abating multiple chronic disorders can be achieved.

The rate of biological aging can be defined by the ratio of cellular damage accumulation to repair capacity. If the rate of damage accretion is fast, but the repair capacity is high, there won’t be an accumulation of damage, and aging will be slowed. However, when damage outpaces repair, aging accelerates.

Repair pathways require energy to operate effectively, and mitochondrial function declines dramatically with age. Ferrucci’s team discovered that this decline is associated with an upregulation of alternative splicing of mitochondrial proteins. Delving deeper into this mechanism, they applied gene set enrichment analysis to 5,325 RNAs with at least one splice variant significantly altered in response to changing mitochondrial function, as measured by AMPK and aging.

Among the top hits were GLUT4, VEGFA, IRS2, mTOR, PI3K, ULK1, ACC1, NRF2, and PGC1-α. Of note, the splice A variant of the topmost hit, VEGFA, appeared to be geronic, while the B variant appeared to be anti-geronic, with the ratio of these variants declining with age. Thus, alternative splicing is a method by which the body copes with energy decline due to mitochondrial dysfunction.

Further Readings

Translational Research for Healthspan and Lifespan

Speakers

Senolytics: The Path to Translation

Physician-scientist James Kirkland studies the impact of cellular aging, specifically senescence, on age-related dysfunction and chronic diseases to develop methods for removing these cells and attenuating their deleterious effects. Senescent cells accumulate with aging and diseases, eliminating cells around them due to their senescence-associated secretory phenotype (SASP), which 30%-70% of senescent cells exhibit under most conditions.

Kirkland’s team applied a bioinformatics-based approach to analyze SASP proteomic databases, revealing that pro-survival networks are upregulated, with diverse senescent cells relying on different pathways. Several agents, termed senolytics, were identified that could target multiple nodes of these cascades.

“We’re moving away from the one drug, one target, one disease approach here,” said Kirkland,  “to try and use agents that have multiple targets, or combinations of agents, to go after networks, and to go after senescent cells by doing this, and thereby improve…multiple conditions.”

Dasatinib (D), a SRC kinase inhibitor, preferentially killed senescent preadipocytes, which relied on survival pathways that signal through this kinase. Quercetin (Q) eliminated senescent human umbilical endothelial cells (HUVECs), which partly act through the Bcl-2 family and others that this cell type is susceptible to.

In an in vivo experiment, combining Dasatinib with Quercetin (D+Q) cleared transplanted luciferase-expressing senescent preadipocytes from mice, explicitly targeting those cells with a SASP. A single dose of senolytics also alleviated radiation-induced gait disturbance in mice, with the effects persisting long-term. Bi-weekly dosing reduced physical dysfunction in older mice, as measured by parameters of maximal speed, including treadmill and hanging endurance, grip strength, and daily activity, with D+Q significantly increasing performance across the board.

Many conditions have now been shown to be alleviated by various senolytics in a range of mouse models, with D+Q delaying death from all causes, and increasing healthspan and median lifespan.

Keynote: Mitochondrial-derived Peptides (MDPs) and the Regulation of Aging Processes

The discovery of mitochondrial peptides (MDPs), encoded from small genes less than 100 codons in length, established the birth and advancement of the microprotein subfield. Physician Pinchas Cohen works to understand mitochondrial biology and characterize MDPs, exploiting findings to target aging. MDPs are secreted from cells and circulate within the body.

“Overall, they serve as protective factors, or hormones if you will, that act in the brain, the heart, the liver, the muscle, and other organs,” Cohen stated.

Among these MDPs, Cohen’s lab identified humanin, encoded from the 16S region of mtDNA, and MOTS-c, encoded from the 12S region.

Humanin has a strong protective effect on neurons and against atherosclerosis, mitigates the side effects of chemotherapy while enhancing its benefit, and is related to longevity in model organisms and humans. Cohen’s lab employs mitochondrial-wide association studies (MiWAS) to link the dysfunction of MDPs to disease. MiWAS identified a single-nucleotide polymorphism (SNPs) in the humanin gene (rs2854128) associated with reduced levels and cognitive decline in humans and mice. Supplementing humanin in mice carrying this SNP improved their cognition.

MOTS-c is a novel exercise mimetic that has potential utility in numerous age-related diseases. Mice on a high fat diet receiving MOTS-c had dramatically lower weight compared to controls. MOTS-c treatment also improved exercise tolerance and performance in middle-aged and old mice, with older mice displaying the most dramatic improvement.

Cohen’s group also identified a link between a SNP in MOTS-c–K14Q–which nullifies MOTS-c activity and the risk of diabetes in males of the Asian population. Evaluating Japanese males from three cohorts revealed a 50% increase in the risk of diabetes for carriers, with almost double the risk seen exclusively in men who were sedentary. Like other MDPs, MOTS-c is reduced with age, and its administration to mice significantly extends lifespan.

“I think that everything we do in the aging field can be reduced to trying to simulate the beneficial effects of a healthy lifestyle, particularly diet…and exercise,” Cohen said. “We think that…mitochondria are the main source of action [here] by inducing the production of peptides such as MOTS-c, humanin, and others.”

Further Readings

Overview

According to the National Center for Education Statistics, white males made up 53% of all full-time professors in 2018. And while the “STEM pipeline” is becoming more diverse–more than 40% of women and roughly 15% of people of color receive their PhDs in STEM fields–colleges and universities need to implement inclusive policies to initiate change on a large scale.

On October 9, 2020, the New York Academy of Sciences hosted a webinar with Georgetown University Medical Center affiliates to share their progressive efforts to decrease systemic inequities and improve workplace culture at their institution. In 2019, the university launched the Bias Reduction and Improvement Coaching (BRIC) program to raise awareness of unconscious bias and attenuate systemic barriers at institutions with the hope of promoting diversity and inclusion in STEM.

Highlights

• Bias impacts application, hiring, and promotion processes, as people make decisions based on shortcuts, unconscious preferences, and assumptions.
• The Bias Reduction and Improvement Coaching (BRIC) program brings together a group of individuals from various demographic backgrounds for training in the skills and language needed to raise awareness of bias.
• This “train the trainer” model empowers people to feel confident starting conversations about prejudice and how to mitigate bias in their respective departments and workplaces.

Speakers

Reducing Systemic Inequities in Academia

Unconscious Bias in STEM

Search committees looking to fill a job should be as objective as possible, especially when studies have shown that teams made up of diverse people are more innovative and high-performing. However, people rely on mental shortcuts and assumptions when making hiring decisions. They often use reflexive habits and exhibit unconscious preferences without realizing it.

Caleb McKinney, who trained as a microbiologist, transitioned to science education, and is now an Assistant Professor and Assistant Dean for Graduate and Postdoctoral Training and Development, related this phenomenon of reflexive habits to a “hot pot.” You learn from previous experience to pull your hand away when a stove is hot. With the same mindset, you can use your prior knowledge to make quick assumptions and form preferences about someone. He urged everyone to take an Implicit Association Test online to learn more about unconscious bias.

But how does conscious and unconscious bias impact STEM community development? Assistant Professor and Senior Associate Dean for Diversity, Equity, and Inclusion, Susan Cheng, noted one example. STEM emphasizes innate intelligence over hard work, but in letters of recommendation, professors are more likely to refer to male scientists as “brilliant,” whereas female scientists are “productive.” The way a job description is written says a lot about what admissions may be looking for in a student or what faculty may desire during recruitment. Search committees may deem a person “not a good fit” for the institution. The only way to combat this is to use checkboxes to ensure job description criteria are followed systematically. “Implicit biases are always in the background, and you need to manage them actively,” said Cheng.

Kristi Graves, a clinical health psychologist and Associate Professor of Oncology, explained that bias also affects STEM professionals’ upward trajectories. For instance, scholarly productivity metrics are very numeric and usually include the number of papers published, impact factor for the journal in which you’ve published, and the amount of grant funding you’ve obtained. But faculty members don’t have access to the same opportunities. A male professor going to another male for a collaboration (because he is like him) is an example of similarity bias.

It’s critical to note that Black, Indigenous, People of Color (BIPOC) make up a small percentage of faculty members. BIPOC faculty members often act as the representative BIPOC for diversity panels and mentoring groups, which takes time away from work and research. And the amount of time spent on essential work and research affects prospects for promotions. Graves believes that hiring committees should have explicit discussions about implicit bias throughout the year to increase faculty diversity. “Everyone has bias,” said Graves. “The trick is to try to become aware of the bias, and then when you notice it, you do something about it so the negative impact that flows from that bias is not sustained or perpetuated.”

Although many colleges and universities have increased awareness and implemented more inclusive policies, the culture has not shifted enough to facilitate a more diverse institutional community. Even representative images on posters and brochures should indicate that a university values different types of people in STEM and that the depicted individuals can serve as role models for scientists who want to know what the institution values.

Understanding the BRIC Program

All three panelists have been heavily involved in the Bias Reduction and Improvement Coaching (BRIC) program at Georgetown University. The program leaders selected people from different backgrounds in various departments across the university for the program. Supervisor or departmental approval was required to participate since the program would take away hours spent on “numeric success metrics.” The inaugural group of 27 coaches—five of whom are coach leads—went through four, three-hour immersive sessions held quarterly. During these meetings, which covered the science of bias and its impact on hiring, promotions, retention, and overall workplace culture, participants learned evidence-based strategies to raise awareness and reduce discrimination. Participants led presentations and department talks on what they learned and received feedback from the coach leads.

The program’s goal is to have many people who can confidently initiate conversations about bias in their workplace. It is designed to establish training across the medical center by providing a faculty learning environment, explained Cheng. The messenger is so important because having the information come from a colleague you know and trust to understand the institutional context you work in is invaluable.

McKinney participated in the BRIC program and pinpointed three main attributes of the initiative. The training provided self-knowledge to reflect upon one’s personal bias, leadership skills to feel equipped to speak up about bias when necessary, and the ability to communicate these strategies when training others. Participants were asked to reflect on their time in the program and took surveys to assess its impact.  Additionally, audience members from BRIC coaches’ presentations were surveyed to see if it was scaled to the department, and follow-ups were conducted to see if departments made any significant changes.

Program Outcomes

Although the initial training was geared toward faculty and staff, post-docs and graduate students organized bias reduction workshops and helped create presentations for their departments. Cheng believes that many students have already developed these skills, while faculty and staff are trying to catch up by participating in the available programs. In addition, departments are asking for workshops on microaggressions, anti-racism, and bias. Graves explained that the program built tremendous confidence for those presenting the material in a safe and confidential space. She also shared a reflection from a participant in the program who felt BRIC was “an effective approach to raise awareness about unconscious beliefs and attitudes and to discover biases in a non-confrontational manner.”

By bringing in participants from different sections of the medical center, the BRIC program facilitated collaboration between faculty and staff from various departments who would not typically work together. This teamwork increased cognitive diversity and allowed participants to build on each other’s thoughts in a broad group discussion. McKinney emphasized that BRIC fostered peer mentorship and feedback, and helped strengthen a sense of community throughout Georgetown University Medical Center.

He was impressed with how much he learned about himself while participating in the program. “To be truly empathetic, you have to be self-aware. You have to recognize and challenge the assumptions you may have about people and situations,” McKinney said. “The BRIC programs allows you to put yourself in other people’s shoes. You learn from each other how people’s experiences and backgrounds shape their individual context, and that’s so important for building empathy.”

Metrics for Success

The panelists shared an essential set of standards that needed to be met for the BRIC program to be successful. Most importantly, improving diversity requires commitment from everyone. That commitment starts with raising awareness and then addressing the issues to create a sense of belonging in the workplace. “Once people are more aware of these biases and start to engage in mechanisms to reduce those biases, you can really [assess an] environment that’s hostile and not welcoming,” said Cheng.

Graves sought feedback from leadership at Georgetown before starting the program. “Until you enact specific behaviors and policy changes, it doesn’t mean a lot,” she said.  The institution is responsible for creating an inclusive environment for trainees, post-docs, and faculty and ensuring that people have equal opportunities to succeed. Establishing an inclusive environment doesn’t have to be a top-down method; valuable feedback can come from anyone.

Cheng also highlighted the importance of decisions and transparency. Critical decision making, which includes communicating news and defining expectations and limitations, should be collaborative. For example, department chairs should define a clear set of expectations, goals, and values before a selection process, and the selection committee should routinely review those criteria. “In STEM, we should be really good at creating objective definitions of how we know when we’ve made our goal,” said Graves.

In addition, all employees should periodically revisit training on inclusion and bias, not just at the onset of the job. McKinney advocated for structured mentorship for minority groups, including cross-cultural mentoring relationships. “Mentors from majority backgrounds have an opportunity to shape retention by fostering these welcoming environments for junior individuals to succeed,” he said. Mentorship also includes facilitating career identities for graduate students and post-docs.  While it is critical to have support and map out your trajectory on an academic career path, mentors can also highlight opportunities in industry and focus on broad career paths.

The Future of BRIC

Bias awareness is critical now, when many interviews and meetings are being held virtually due to the pandemic. Graves saw this as a positive because it allows institutions to create new networks and reach students early on in their academic trajectories. They can reach a wider talent pool, and recruit candidates from various backgrounds who were not previously reached. Companies should also reassess their open opportunities because the way job descriptions are written and where they are posted plays an essential role in who applies. Equal opportunity statements at the end of job descriptions show candidates that a company values diversity and inclusion.

Georgetown University is not the only institution spearheading programs for systemic inequity awareness. Cheng praised numerous universities, such as UCLA, UCSF, and The Ohio State University, who have been at the forefront of implicit bias research and training. All three panelists are eager to continue the BRIC program. They hope that by scaling this bias awareness across Georgetown, there will always be individuals on committees who have been trained and can challenge their colleagues’ assumptions.

Further Readings