Experiments

In this article, I have provided a brief history of my life. After tracing my family background and my early interest in physical sciences, I discuss how I entered biology under the influence of Robert Emerson. I have always enjoyed doing experiments and this led to new measurements and analyses of chlorophyll unit, efficiency of photosynthesis, excitation energy transfer, delayed light emission, thermoluminescence and electroluminescence in photosynthetic organisms. It is my view that discoveries are made because we follow our scientific curiosities.This article was written at the invitation of Govindjee.     

Reminiscences,collaborations and reflections

This is a personal account by a semi old-timer who completed his official term as a professor of plant biochemistry at Nagoya University in Japan in 1992. My university student life began soon after the World War II (1948). I shared the hardships of many in my age group, in that life was difficult during my college years. I was fortunate to have the opportunity of studying in the USA on a Fulbright scholarship first at Purdue University (1955–1956), and then at the University of California, Berkeley (1956–1957). My graduate study and postdoctoral training in the new world were vitally refreshing and stimulating, which gave me the impetus for becoming a natural scientist associated with academic institutions. Consciously and subconsciously I was impressed by the friendly and liberal atmosphere surrounding young students as well as senior scholars in the United States. But more importantly, I was inspired by the critical and competitive minds prevailing among these people.The appointment as a biochemist at the International Rice Research Institute (IRRI) in the Philippines (1962–1964) was the real start of my professional career. The work was continued upon my return to Nagoya to become a staff member of the Research Institute for Biochemical Regulation (1964–1992). Throughout the years, my major research interest has covered photosynthesis as a whole, involving photosynthetic CO₂-fixation (RuBisCO), carbohydrate metabolism, e.g. starch biosynthesis and breakdown (-amylase), and metabolic regulation, which are interrelated in the basic metabolism of plant cells.I shall briefly describe in this article highlights from my studies and discoveries made and I shall also discuss their possible significance in plant metabolism, with the hope that it does not contradict my sense of humility: They are (a) discovery of ADPG in plants and its role in starch biosynthesis; (b) structure-function relationship of RuBisCO proteins, in particular on heterologous recombination of their subunits of plant-type enzyme molecules derived from the prokaryotic photosynthetic bacteria; (c) molecular evolution of RuBisCO genes; (d) mode of actions (formation, intracellular transport and secretion) of rice seed -amylase and its structural characteristics (distinctive glycosylation), and (e) DNA methylation and regulatory mechanism of photosynthesis gene expression in plastids (amyloplasts). In each step of my research, I shared joy, excitement, disappointment, and agony with my colleagues, an experience that may be common to all researchers. Although it is now becoming well recognized among the scientific community in Japan, I want to point out that interaction of multinational scientific minds in the laboratory produces a vital and creative atmosphere for performance of successful research. I experienced and realized this important fact in my earlier days in the USA and the Philippines. Inasmuch as I believe that this is the most crucial element for any research laboratory to possess, I fondly remember the friendships gained with numerous overseas visitors and collaborators who have contributed immensely to our work.Written at the invitation of Govindjee.     

The story of science: successes,failures, luck,privilege, and bias

I am incredibly honored to receive the 2021 WICB Junior Award for Excellence in Research in WICB’s golden jubilee year. In this essay, I traverse my scientific journey starting with my PhD, highlighting the highs and the lows and how these intersect with luck, privilege, and bias.

V. AnanthanarayananMy pursuit for a PhD started with a hiccup—I had applied to several places in the United States, but barely got any offers due to the economic upheaval that happened that year (2008). I had to forgo any dreams of a PhD in the United States and remained in Bangalore, India to complete a project I had started with William (Bill) Thies at Microsoft Research India on a programming language for expressing biology protocols. Applying to U.S. schools was an expensive task, one which I was unwilling to put my family through again. So, a year later, when I recommenced my search for a PhD position, I set my sights on Europe. I had heard about the PhD program at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG ) at Dresden from a friend who had just joined the institute for her PhD. Fortunately, I received an interview call from MPI-CBG. At the end of a crucial interview week at Dresden, I “matched” with Iva Tolic´’s (now Institut Ruđer Boškovic´, Croatia) lab for my PhD. At the start of my PhD, I knew next to nothing about the cytoskeleton, motor proteins, or microscopy, but I found Iva and my lab members to be some of the warmest and most welcoming people. I made friends for life and graduated with a PhD in Biophysics, with a thesis focused on understanding the regulation of the motor protein cytoplasmic dynein. I was lucky to have been able to get a position at MPI-CBG and join Iva’s lab—of the other three places in Europe I had applied to for a PhD, only one other institute invited me for an interview, which also proved to be unsuccessful.On completing my PhD in 2014, I didn’t quite know what I wanted to do. Due to personal reasons, I had to return to India and was open to options in both industry and academia. But with my training in motor protein and cytoskeleton research, I had some ideas for exploring scientific questions related to dynein activation. However, most labs I approached for a postdoctoral position were not open to a project that was outside the realm of their research focus. Nonetheless, Iva, Nenad Pavin (University of Zagreb), and Jonathon (Joe) Howard (Yale University), who were members of my thesis advisory committee, gave me the courage to continue in academia. In my naïveté, I went ahead and applied for the INSPIRE Faculty Fellowship, which is targeted at fresh PhDs and junior postdoctoral fellows to establish their own independent group at an Indian institute. To my surprise, I ended up getting the fellowship. The next issue was finding a host institute that was preferably in Bangalore, where my partner was based. I applied at a few different places, but only after I attended IndiaBioscience’s Young Investigator Meeting in 2014 did I get the chance to meet representatives of potential host institutes, including the Indian Institute of Science (IISc). After a couple of research seminars at IISc, my application was assessed and I was offered the position of INSPIRE Faculty Fellow at the newly formed Centre for BioSystems Science and Engineering, IISc.While I did not have any additional start-up funding, I was given the infrastructure and the independence to pursue my research program. It was slow and frustrating at the start, not unlike most starting labs. I always wondered if it might have been easier if I had had a regular postdoctoral stint. During this time, I also started recognizing how hard it was to be a woman in Indian academia. As a woman principal investigator, one’s authority, expertise, and ability are constantly called into question. Justifying your presence in academia on a daily basis is an exhausting task. I had a great mentor in Sandhya Visweswariah (IISc) who helped me navigate the system. I also had an extremely supportive partner, who kept me going through some of the worst times. Eventually, my lab and I landed on our feet (more about this in “My INSPIRE’d Journey”). Our research has been recognized with grants and awards, but one of the most rewarding parts of the job is seeing other lab members discovering the joy of science (I wrote about my approach to mentorship recently [https://www.nature.com/articles/s41580-020-0256-6]).Three years into the faculty fellowship, I was able to transition to an Assistant Professor position in the same institute. However, this did not change my experience as a young woman in Indian science, and the implicit and explicit biases continued. In 2020, I accepted a fantastic opportunity to further my lab’s science as an EMBL Australia Group Leader at the Single Molecule Science Node at UNSW Sydney and made the move during a pandemic. My lab’s research focus is in understanding how stochastic and rare events pertaining to cytoskeleton and motor proteins give rise to complexity in intracellular organization. With this theme as the essence of our research, we ask specific questions about motor protein regulation to effect differential cellular trafficking, mitochondria-microtubule interactions, and their role in mitochondrial dynamics, and we aim to determine barcodes of global organelle positioning in health and disease.I have the privilege of being able-bodied, born in an upper middle-class family to college-educated parents who were extremely supportive of my choices. I have also inordinately benefitted from the fact that I was born to an Indian ‘upper caste’ family. I therefore had an undue head start in life. These were circumstances beyond my control and yet played a huge role in how my story turned out. I was embarrassingly ignorant of the rampant misogyny in academia until I had to contend with explicit and implicit gender-based biases myself when I started my independent research group in India. Women make up ∼40% of science PhDs awarded in India but represent only ∼13% of Indian academia (biaswatchindia.com), highlighting the stark gender biases at play in creating a leaky pipeline. While I tried my best to voice my discontent and affect changes to create an equitable environment within my department and institute, it was slow work. In 2020, when the pandemic hit and all conferences and meetings went virtual, conference posters advertised on social media made it immediately apparent just how much women were underrepresented in Indian STEM conferences. So, I teamed up with Shruti Muralidhar (now a scientist at Deep Genomics, Canada) to found BiasWatchIndia, an initiative to document women representation and combat gender-biased panels in Indian STEM conferences.BiasWatchIndia has been in existence for a little over a year now—we have achieved several milestones, but there’s still so much to do. “Manels” (conferences that feature only men) are still as rampant as they were when we first started—40% of all Indian STEM conferences are manels. And while we have just about started to tackle the underrepresentation of women in Indian STEM, we are conscious of the intersectionality of bias with gender, caste, ableism, and socioeconomic background and aim to understand how best we can advocate for all minorities.People who are in power in academia and who oppose equity, diversity, and inclusion initiatives and instead preach merit and equality as the gold standard need to introspect, because when options and opportunities are offered without consideration to the millennia of oppression based on gender, race, and background, it is not promoting equality but upholding values that will continue to oppress underrepresented groups. Still, I am optimistic and hope to see real changes that will result in equity in academia in my lifetime.     

Discussion: Reply to Hitchcock

Christopher Hitchcocks discussion of my use of screening-off in analyzing the causal process of natural selection raises some interesting issues to which I am pleased to reply. The bulk of his article is devoted to some fairly general points in the theory of explanation. In particular, he questions whether or not my point that phenotype screens off genotype from reproductive success (in cases of organismic selection) supports my claim that the explanation of differential reproductive success should be in terms of phenotypic differences, not genotypic differences. I will respond to this and show why the two supposed counter-examples to my position fail.     

Focal Adhesion: A Focal Point in Current Cell Biology and Molecular Medicine

Cell-extracellular matrix (ECM) is an important property of virtually all cells in multi-cellular organisms. Cell-ECM adhesion research, therefore, has broad impact on biology and medicine. Studies over the past three decades have resulted in tremendous advance in our understanding of the molecular basis and functions of cell-ECM adhesion. Here, I focus on some of the general lessons that we have learned from recent studies on cell-ECM adhesion. In addition, I highlight several topics in this rapidly advancing research area. These topics, which include assembly, disassembly and regulation of cell-ECM adhesion structures, the molecular mechanisms of bi-directional signaling through cell-ECM adhesions, and the tissue and organ pathobiology of cell-ECM adhesion, are pertinent to our understanding of cell-ECM adhesion and signaling.Key Words: Focal adhesion, integrins, extracellular matrix, cytoskeleton, cell migrationCell-ECM adhesion is a fundamental process through which cells interact and communicate with the environment. Cell-ECM adhesion is essential for organogenesis during embryonic development. In adult, it is vital for maintenance of tissue integrity and organ functions. Alterations of cell-ECM adhesion hence are frequently associated with human diseases. Because of the broad significance of cell-ECM adhesion in biology and pathology, understanding how cell-ECM adhesion is mediated and regulated and determining how cell-ECM adhesion influences cell behavior have been the subjects of numerous studies. In particular, studies over the past three decades have led to major breakthroughs in our understanding of cell-ECM adhesion. Many of the key discoveries, including identification of integrins as major transmembrane receptors for ECM proteins, demonstration of integrins as bi-directional (outside-in and inside-out) transmembrane signaling machines, identification of talin, focal adhesion kinase (FAK), integrin-linked kinase (ILK) and other cytoplasmic and membrane-associated proteins as key regulators and effectors of integrins, and delineation of multiple downstream signaling pathways that relay signals from cell surface integrins to diverse cytoplasmic and nuclear effectors, have been reviewed in refs. ^1–12. In this brief article, I will focus on some of the general features of cell-ECM adhesion and discuss from my personal perspective several key questions that remain to be answered in future studies.     

Ye Tian: Surveilling stress to live longer

Ye Tian investigates how mitochondrial stress signaling pathways regulate longevity using C. elegans as a model system.

An avid reader, Ye Tian used to save up her child allowance with the sole purpose of buying science fiction books. Reading and solving mathematical problems were her favorite hobbies; indeed, she liked mathematics so much that she was about to enroll herself as an architecture major but finally chose biotechnology. Ye moved from her hometown in the Northwest of China, Baoji—famous for housing the Zhou dynasty’s bronzeware and being close to the Terracotta Army—to Beijing for her college and graduate studies.Ye is proud of being among the earliest researchers working on Caenorhabditis elegans in her country; for her PhD studies, she joined the lab of Hong Zhang, who at that time has just established the first C. elegans lab in China at the National Institute of Biological Sciences in Beijing. Ye identified epg-2 as an adaptor for cargo recognition during autophagy. In 2010, she crossed the Pacific toward the U.S. West Coast for her postdoctoral training in the aging field with Andrew Dillin, first at the Salk Institute in San Diego and then at the University of California, Berkeley. There, she discovered that mild mitochondrial stress during development in worms rewires their chromatin landscape to establish specific gene expression patterns throughout the lifespan and promote longevity.Ye Tian. Photo courtesy of Ye Tian.Ye came back to China at the end of 2016 to start her own lab at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. Her research team studies mitochondrial stress signaling pathways and their interplay with aging. We chatted with her to learn more about her next scientific plans.What interested you about the interplay between mitochondria and aging?I became interested in mitochondrial biology during my postdoc in Andrew Dillin’s lab. Since the origin of eukaryotic cells, mitochondria have been a driving force of evolution. During reproduction, mitochondria are passed from the mother to the offspring through egg cells and they exhibit a unique inheritance pattern. As essential hubs that dictate cellular metabolism, it is clear now that mitochondria and the nucleus maintain a bidirectional communication. Early life “stressed” mitochondria communicate with the nucleus to induce gene expression changes that are beneficial on longevity and persist throughout the lifespan. The fact that mitochondrial function is crucial to aging fascinated me; I wanted to continue exploring that topic further, and that’s why I established my lab around the question of how mitochondrial surveillance mechanisms regulate the aging process.What are you currently working on? What is up next for you?My research team focuses on the interplay between mitochondrial stress signaling pathways and aging. The first work that my lab published was a project that I started during my postdoc. The Dillin lab reported a phenomenon in which perturbations of mitochondria in neurons induced a mitochondrial stress response in the peripheral tissues and hypothesized that a secreted signal molecule, named after mitokine, is required for the cell non-autonomous regulation (1). The identity of this molecular signal remained elusive for almost ten years until we found that a secreted Wnt ligand, EGL-20, functions as the mitokine to coordinate mitochondrial stress signaling across tissues and promote longevity of the organism (2). We are also interested in how the crosstalk between mitochondria and the nucleus influences lifespan. We found that mitochondrial perturbations alter the nuclear epigenome to induce longevity via the histone deacetylation complex NuRD in response to cellular acetyl-CoA levels, the key metabolite at the entry point of the Krebs cycle (3).Lab group picture; current lab members (2021). Photo courtesy of Ye Tian.Our latest work stemmed from a serendipitous observation that neuronal mitochondrial stress is sensed by and transmitted through the mitochondria in the germline. Intergenerational, maternal inheritance of elevated levels of mitochondrial DNA via the mitokine Wnt/EGL-20, which causes the activation of the mitochondrial unfolded protein response (UPR^mt), provides descendants with a greater tolerance to environmental stress. This makes the offspring live longer (4).Among our short-term scientific plans, we’re determining how mitochondria functions during the aging process at both the genetic and biochemical levels and searching for ways to apply our findings from C. elegans to neurodegenerative disease models in mammals.What kind of approach do you bring to your work?The curiosity about how things work drives me; what I enjoy the most is when I see things happening in front of my eyes and when I figure out why they occur that way. That enthusiasm is what I try to spread to my team every day. In the lab, we rely on C. elegans as our model system and on genetics to dissect complex biological processes like aging. We have also adapted modern biochemical and imaging techniques as well as bioinformatics to complement our genetic studies. I’m a geneticist at heart, and I like to initiate a project with a well-designed genetic screen. The best part is that the screen often leads me to answers I was not expecting, and that’s genuinely inspiring!What did you learn during your PhD and postdoc that helped prepare you for being a group leader? What were you unprepared for?Like most scientists, my research career has gone through ups and downs. I had to change my research project in the last year of my graduate school; that was nerve-racking, but I eventually managed to redirect my thesis and get exciting results under time pressure, thanks in large to the support of my parents, mentors, and lab mates. That helped me prepare to become a principal investigator; I gained confidence in problem solving, and since I’ve experienced the stress of dealing with last-minute scope changes firsthand, I connect better with my students.I guess, as many other non-native English speakers, I wasn’t prepared for writing grants and papers fluently in English. This issue wasn’t obvious during my graduate and postdoctoral studies, as my mentors were always there for me and proofread and edited my writing. Now I have to stand up for myself. I spend most of my time writing; I’ve improved my writing skills but it’s still an ongoing process.Reconstruction of the nerve system of C. elegans by confocal microscopy. Green corresponds to YFP-labeled neuronal specific marker Q40, and red labels germline specific mitochondrial outer membrane protein TOMM-20::mkate2. Image courtesy of Ye Tian’s lab.What has been the biggest accomplishment in your career so far?My very first PhD student, Qian Zhang, graduated with two first-author papers and decided to pursue a research career in academia. Being responsible for someone else’s career is challenging but also rewarding.What has been the biggest challenge in your career so far?I use the model organism C. elegans for my research in aging, so from time to time, peers criticize the relevance of my work to human health. I’m used to justifying my scientific approach to funding agencies and peers in other fields, but sometimes it’s exhausting or not pleasant.Who were your key influences early in your career?My PhD mentor, Hong Zhang. He is very passionate about the science he does, and he is courageous to shift his research directions to answer new biological questions.What is the best advice you have been given?I think the best advice I’ve gotten is that “tomorrow is another day.” It reminds me to keep going and be optimistic.What hobbies do you have?I love art and music. When I was in San Diego, I used to play in the Chinese Music Band; I miss my musician friends over there. In my teens, I used to hike mountainside trails along the river with my parents. Now, running has become my new favorite hobby. I enjoy the tranquility and peace of mind while running; it’s soothing.     

Charles Darwin's debt to malthus and Edward Blyth

Conclusion It is not justifiable to accuse Darwin of conscious or unconscious plagiarism. This charge is contrary to the historical evidence and to the extensive information that we have about his character. When Darwin listed the writers on the origin of species by natural selection before himself, he did not mention Blyth, and this omission did not disturb the cordial relations between Darwin and Blyth. Blyth continued to supply Darwin with information which Darwin used in his later publications with due acknowledgment to Blyth. For example, in The Descent of Man, Darwin cited Blyth: Mr. Blyth, as he informs me, saw Indian crows feeding two or three of their companions which were blind.⁶³ Blyth felt no resentment. If he did, he would have so informed Darwin. Blyth did not regard himself as in any sense a predecessor of Darwin and he certainly did not resent Darwin as a plagiarizer of himself. Moreover, Darwin went to a great deal of trouble to find his own predecessors and to give them proper credit.⁶⁴ After Darwin had completed his work on natural selection, he wrote a letter to the Reverend Baden Powell in which he clearly showed recognition of the contribution of others to his own work:No educated person, not even the most ignorant, could suppose I mean to arrogate to myself the origination of the doctrine that species had not been independently created. The only novelty in my work is the attempt to explain how species became modified, and to a certain extent how the theory of descent explains certain large classes of facts; and in these respects I received no assistance from my predecessors.⁶⁵ *** DIRECT SUPPORT *** A8402011 00002     

Decoding the genetics of rare disease: an interview with Monkol Lek

Monkol Lek, Assistant Professor at Yale University School of Medicine, and Associate Editor at Disease Models & Mechanisms, dedicates his research to finding a genetic diagnosis and improving treatments for rare disease patients. As he originally studied computer engineering at the University of New South Wales in Sydney, Australia, he now utilises computational methods to optimise large-scale genetic studies, provide globally accessible resources for genetic research communities and, importantly, resolve diagnostic odysseys for rare disease patients. Monkol completed his PhD in Prof. Kathryn North''s lab at the University of Sydney, studying the genetics of muscle strength and performance, and then continued his investigation of muscle disease in Prof. Daniel MacArthur''s lab at Massachusetts General Hospital and the Broad Institute. During his postdoc, he led several large-scale studies aimed at distinguishing pathogenic from benign variants, including the Exome Aggregation Consortium (ExAC) project ( Lek et al., 2016). Monkol established his own lab at Yale University School of Medicine, which continues to improve the diagnosis and treatment of rare muscle disease, and also focuses on underserved populations, whose genetic mutations are not as well characterised as those of European ancestry. In this interview, Monkol discusses how his own diagnosis with limb girdle muscular dystrophy has shaped his career and what he envisions for the future of genetic research in rare disease.

You have a very unique career path – could you tell us a little bit about that? My first degree was in computer engineering. When I first went to university, I studied the hardware and software of computers. I really liked the software aspect of the degree, and so I worked for IBM as a software developer when I finished university. However, during the last few years of university, I noticed that my muscles were getting weaker. My university was on a big hill, with classes at the bottom and top of the hill, and I had to stand up for about 3 h a day while commuting on public transport. It started becoming obvious that I had something wrong with my muscles because I felt totally exhausted at the end of the day. It was frustrating, because I felt that my performance at university was impacted by something that had nothing to do with my ability to think. So, I went from doctor to doctor to try to find out what was wrong with me. As a lot of doctors are not trained in rare diseases, they didn''t consider a rare disease diagnosis. Then one doctor did a blood test for creatine kinase (CK), which is leaked into the bloodstream when muscle is damaged. In healthy people, high levels of CK are detected in the bloodstream after they''ve done intensive exercise, like a marathon. If someone hasn''t done something like that, but they have high levels of circulating CK, it could be an indication that there''s something wrong with their muscles. As I had high levels of CK in my bloodstream, I then went to a neurologist, which was when I got a clinical diagnosis. At that point, they didn’t know the root cause of the problem, but they knew that I have a muscle disease based on several tests, including a nerve conduction test.I received this clinical diagnosis during my time in IBM, and that''s when I became dissatisfied with my job, because I felt that I was using all my talents to make a very big, international company richer. I was also becoming frustrated when visiting the neurologist every 6 months, as all they would tell me was that my muscles were getting weaker, which I already knew. I began to think that not much was happening in the neuromuscular disease field if that''s the best they could offer me. I wanted to know what the root cause of my disease was and if there were any treatment options. I came to the conclusion that no one would care about my disease more than I would, because I''m the one that has lived with it every day of my life.That''s when I decided to leave IBM and pursue a career in researching muscle disease. It didn''t go down well with my parents and friends, because I was leaving a well-paid job to go back to university to get paid nothing for an unknown number of years. If I had known my chances of success – completing a meaningful PhD, doing a meaningful postdoc and landing a faculty position – I wouldn''t have gone on this journey. I have been very fortunate, but I wasn''t always in the right place at the right time.When I finished my undergraduate degree in bioinformatics and physiology at the University of New South Wales, I started a PhD in Melbourne, but it didn''t work out, because not all supervisors are perfect. My wife and I then returned to Sydney, where my wife bumped into one of the professors from our undergraduate degree. She explained that we''d had a bad experience in Melbourne with our PhDs, but our passion was still to do muscle research. The professor''s daughter was researching muscle disease in Kathryn North''s lab at the University of Sydney, and she invited us to visit the lab. I was offered an opportunity to do my PhD in Kathryn''s lab, but I was initially reluctant as it was a diagnostic lab, and I was more interested in developing therapies for people with muscle disease. However, I thought I could still learn a lot about muscle physiology and, in the long term, I''m glad that I received training and mentorship from Kathy''s lab. Also, if I hadn''t done my PhD there, I wouldn''t have met Daniel MacArthur, my future boss. He was a very talented student in Kathy''s lab, who taught me a lot about scientific communication among other things, and I taught him some coding skills. He left to work on the 1000 Genomes Project in Cambridge, UK, but I kept in contact with him to get his advice on my project.When I was finishing my PhD, Daniel asked if I wanted to join the lab he was setting up in Massachusetts General Hospital and the Broad Institute. His lab was going to study common loss-of-function mutations in human populations using large datasets from the 1000 Genomes Project, but he offered me a project investigating neuromuscular diseases. As soon as I submitted my PhD thesis, I started working in his lab. This was perfect timing, because it was 2012, when exome sequencing had recently been published in the context of rare diseases (Ng et al., 2010) and, more importantly, it was becoming affordable, in terms of research. I waited over 10 years for a genetic diagnosis, so my goal was that no one should have to wait that long in the future.Through collaboration with our former PhD lab, Daniel and I used samples from undiagnosed patients to find answers for Australian families. The first family had two affected girls with undiagnosed nemaline myopathy, who had been on a diagnostic odyssey for about 9 years. It was amazing how quickly we progressed from receiving the samples to identifying the novel gene, LMOD3, associated with their disease (Yuen et al., 2014). This was part of my main project during my postdoc – working on gene discovery in neuromuscular diseases and finding answers for patients that have been waiting years and years to get a genetic diagnosis (Ghaoui et al., 2015; O''Grady et al., 2016).The project that most people know me for is the ExAC project, which was initially my ‘side’ project during my postdoc. The idea was to create a big database of all rare variants that we see in the general population, so we can better interpret the rare variants that we see in rare disease patients. When we were creating it, we thought that it may be useful to other researchers around the world. Therefore, we tried to ensure, through data-use agreements and consent processes, that we could share as many of our findings as possible. I''m happy to say my side project was quite successful. After that, I led other projects, including an analysis group in the Centre for Mendelian Genomics, to expand that framework and idea across all rare diseases, not just neuromuscular diseases (Baxter et al., 2022).I was having a lot of fun at the Broad Institute, and I was co-author on a lot of high-impact papers. However, the reason I left the Broad Institute was that I wanted to be involved in the full journey for the patients. Sometimes scientists don''t understand that getting a genetic diagnosis is not the end of the journey for a patient. After the diagnosis they want to know what treatment options are available. Yale gave me the opportunity to continue doing the gene discovery and analytical work that I was doing at the Broad Institute, plus the capability of doing experiments with mouse models to investigate gene replacement therapies and other therapeutic approaches.

“I waited over 10 years for a genetic diagnosis, so my goal was that no one should have to wait that long in the future.”

How has being both a researcher and a patient affected your career? When I was first diagnosed, there was a neurologist who discouraged me from researching my own disease and this became the basis of my TEDx talk, because I thought it was very condescending. I thought, “Just because I have this disease, it doesn''t mean that I have a low IQ”. However, this experience motivated me more. I discussed it with Kathy before starting my PhD, and her encouragement and enthusiasm was refreshing. At the time, in the early 2000s, people hadn''t accepted the idea of patients researching their own disease. Things have changed since then, mainly because there are more examples of it now (Branca, 2019), but at the time, it was really hard for me to progress in science. I always thought that people were looking at me with sympathy, and I felt like I had to achieve twice as much to get the same respect as someone else who wasn''t as talented or didn''t work as hard as me. It was frustrating, but in everyday life people still correlate physical disability with intellectual disability. For example, if my wife is pushing me in the wheelchair in public, no one ever directs a question to me because they assume that the physical disability comes with mental disabilities. There are well-known examples of scientists with physical disabilities, like Stephen Hawking, but it is still challenging in academia when you have a physical disability and people make certain assumptions about you.On the other hand, just before starting at Yale, my collaborators at the University of Massachusetts took a skin biopsy from me. With this skin biopsy, they created induced pluripotent stem cells, and, using CRISPR, they corrected my disease-associated gene variant in the cultured cells. They then published this in a Nature article, in which fig. 1 is the experiment in which they corrected my mutation (Iyer et al., 2019). Are there specific skills or knowledge you learned while working in computer engineering that have helped shape and develop your research today? When I started my PhD, there was an increase in how much genetics research, and biological research in general, relied upon big data. It can be very challenging to work with big data if you''re a biologist without a background in computer science. You can go online to teach yourself to an extent, but it gives you an advantage to learn the theory behind a lot of algorithms and other aspects of software engineering, in a formal setting. It makes the difference between building tools that take a week to analyse a set of data and building tools that take a few minutes to analyse the same data. If you can analyse the data more quickly, you can explore different possibilities and ideas much more quickly. You can''t learn everything online, and having a firm foundation of knowledge can enable you to work with big data in an efficient way.The other thing that you learn from computer science is a certain mindset when approaching problem solving. This is because you have to debug code frequently and, due to this fast pace, you learn quickly. This helped me to troubleshoot problems in biological research quickly.

“Getting a genetic diagnosis is not the end of the journey for a patient. After the diagnosis they want to know what treatment options are available.”

What do you think are the key challenges for rare disease research and diagnosis moving forward? I now have a greater appreciation of the challenges because I see it from two points of view: one as a researcher in a group and one as a PI, who leads the research. The diagnosis rate for rare disease is about 50%, so there are still 50% of patients with a disease that has an unknown genetic cause. The gold standard requirement for associating a new disease gene with a novel phenotype is that it presents in multiple unrelated families (MacArthur et al., 2014). However, when you work with rare diseases, there is the issue of small sample numbers. One challenge for basic scientists is creating good collaborations with physician scientists across the world to enable you to create a large enough dataset.The other challenge is the cost of research for these diseases with unknown genetic cause. The 50% of cases for which we know the genetic cause are no longer considered an area of research, as clinical genetic services can now diagnose these patients. To diagnose the remaining patients, you have to use more expensive technologies, such as long-read sequencing.The last thing is the interpretation of rare variants. Although the ExAC project helped with this, there is still a challenge. For example, if a patient has a rare genetic variant, this doesn''t necessarily mean it is the cause of their rare disease. This is because even healthy people have rare variants. So, we have a massive interpretation challenge in rare disease genetics, which can be overcome by creating a laboratory model system with that genetic variant to investigate it further. However, if you had 1000 variants to consider, it''s not going to scale as an animal model. So, an important question is how can we interpret these variants in a scalable manner? This is one of the main driving forces behind the new Subject Focus, ‘Genetic variance in human disease: decoding diversity to advance modern medicine’, that we are launching in DMM. You have led and coordinated several studies involving very large cohorts. From your experience what are the key components of a successful study? I think the key to a successful large cohort study with unsolved rare disease patients, is the amount of structured phenotype data you can collect. This requires a good collaborator, who has the time to prepare that data in a meaningful way, which makes it easier to find other families with the same rare disease. The other thing is to have the ability to recontact patients and collect different samples from them, because we''re moving to a more multi-omics world. Therefore, we need the ability to go beyond just collecting DNA samples. Also, we''re in a world where we''re starting to link data to electronic health records, which allows the collection of deeper and richer phenotype data that enable associations to be made between families.In addition, you can''t work in isolation. In order for us to make a meaningful impact, we need to work with groups that have specialties outside of our own. For instance, we collaborate with groups that specialise in the interpretation of non-coding variants. This is important as variants in these regions could hold the answers for some of those unsolved cases.Another key aspect to a successful study is collaboration with statistical geneticists because some of the more complicated questions are best asked by them. Some of these questions go beyond monogenic diseases. We are seeing convergence between genome-wide association studies, looking for many variants, each with very small contributions to a disease, and studies of Mendelian disease that are looking for one gene that causes disease. The field has to start looking at diseases in the middle of this spectrum, which requires statistical geneticists. This is because you need to make sure that your conclusions are correct. For instance, if you''re asking whether a rare disease is caused by a combination of two genes, then you must have a robust statistical model to show that these variants aren''t presenting together by chance. You have to prove that those two variants are acting in concert, instead of independently, to cause this disease. My colleagues at Yale published a great paper that demonstrated this concept (Timberlake et al., 2016).Lastly, it is important to forge meaningful collaborations beyond academia. A lot of my colleagues are being funded by industry collaboration, and a lot of these companies have access to more samples than we do in academia. You can also collaborate with large biobanks, such as the UK Biobank, which has a rich set of phenotype data and also the ability to recontact patients (Glynn and Greenland, 2020). The FinnGen project is a recent public–private collaboration that combines genetic data with electronic health records from Finnish biobank participants to improve disease diagnosis and treatment (Kurki et al., 2022 preprint). So, working with biobanks and industry is another way of increasing sample numbers, which is the biggest challenge in rare disease research.

“We don''t want to create disparity in terms of health, especially in the context of genetics, which will continue to become more prominent in modern medicine.”

You dedicate a lot of your research towards patients in underserved populations, such as East Asian populations, whose genetic mutations are not as well characterised as those of European ancestry. Can you explain the importance of this? One of the reasons that it took over 10 years for me to get a genetic diagnosis was because the gene that causes my disease was first reported as not commonly associated with disease in populations of European ancestry. The problem with biomedical research is that when people read that, they think it applies to everyone, even patients who have non-European ancestry. Although the gene that causes my disease aligned with my muscle disease phenotype, it wasn''t sequenced because of this assumption. They only decided to sequence this gene once they did linkage analysis of my family, and this was the only gene associated with neuromuscular disease in the linkage region they identified. This is the reason why we need to have good data on all populations. The ExAC and gnomAD studies that I worked on acknowledged that we need good allele frequency data for populations of East Asian, South Asian, Latino and African ancestry, because we don''t want to create disparity in terms of health, especially in the context of genetics, which will continue to become more prominent in modern medicine.If you want to deliver the best healthcare, you have to realise that some variants and diseases are more common in certain populations, such as Tay-Sachs disease, which is common amongst the Jewish community, and sickle cell anaemia, which is more prevalent in populations of African ancestry. By understanding these differences, we can actually find a genetic diagnosis a lot quicker. If it''s not a de novo variant, and is instead a variant inherited in the population, and if you''ve made the discovery in East Asians, there is a better chance of identifying more incidences of this variant in the population in which it was first discovered.I think it''s also good for validation of data, because if you had discovered a potential disease-causing variant and you find that this variant has a frequency of 1% or higher in a non-European population, then it''s impossible for it to be the cause of a rare disease, regardless of its frequency in a European population (Lek et al., 2016).     

(M,R)-systems, (P,M,C)-nets, hierarchical decay, and biological aging: reminiscences of Robert Rosen

I have the pleasure to present a number of personal experiences that I had with Robert Rosen, both as his student and as a research colleague, and I will describe how this affected my academic career over the past decades. As a matter of fact, Rosen's work with (M,R)-systems as well as his continuing mentorship guided me into my own research in gerontology and geriatrics. Amazingly, this still continues to affect my work in complexity theory after 30 years.     

A career pathway in protein folding: from model peptides to postreductionist protein science

This review discusses the inherent challenge of linking "reductionist" approaches to decipher the information encoded in protein sequences with burgeoning efforts to explore protein folding in native environments-"postreductionist" approaches. Because the invitation to write this article came as a result of my selection to receive the 2010 Dorothy Hodgkin Award of the Protein Society, I use examples from my own work to illustrate the evolution from the reductionist to the postreductionist perspective. I am incredibly honored to receive the Hodgkin Award, but I want to emphasize that it is the combined effort, creativity, and talent of many students, postdoctoral fellows, and collaborators over several years that has led to any accomplishments on which this selection is based. Moreover, I do not claim to have unique insight into the topics discussed here; but this writing opportunity allows me to illustrate some threads in the evolution of protein folding research with my own experiences and to point out to those embarking on careers how the twists and turns in anyone's scientific path are influenced and enriched by the scientific context of our research. The path my own career has taken thus far has been shaped by the timing of discoveries in the field of protein science; together with our contemporaries, we become part of a knowledge evolution. In my own case, this has been an epoch of great discovery in protein folding and I feel very fortunate to have participated in it.     

On ornamental maturation of two Philippine hornbill species with a note on physiological colour change

Two Philippine hornbill species, the Visayan writhed-billed hornbill (Aceros waldeni) and the Visayan tarictic hornbill (Penelopides panini panini), display on their heads multiple sexual ornaments in both sexes. An account is given of the maturation of these ornaments, except for the hood on the hind neck, from the nestling stage through age 5 years in the writhed-bill, and from fledgling stage through age 3+ years in the tarictic. Development proceeds in a staggered fashion, in that component traits of the compound ornament are added sequentially to a baseline already present in the nestling (bare facial skin) up until maturity. In same-aged pair mates of the writhed-bill, elements (dark grooves and wreaths in the red bill) were continually added until the reproductive age of 5 years, when observations ceased. Contrary to published accounts, only the writhed-bill passes through a male-like plumage in the nestling/fledgling stage, whilst the tarictic juveniles attain their sexually dimorphic appearance right away. Writhed-bill chicks exhibit a colour dimorphism of eye (= iris) colour irrespective of sex, in which the two phases pass through two stages, but in reverse order. At least part of the ornaments can be interpreted as honest indicators of condition. Based on the concept of honest signalling, the multiple nature of the ornamentation is functionally explained by the multiple message hypothesis. A physiological colour change between white and blue within seconds/minutes occurs in the bare facial skin patches of the tarictic and thus adds to the arsenal of hornbill multiple ornaments. This constitutes the first evidence for this type of colour change in a bird.Communicated by F. BairleinThis paper is publication No. 54 of the Philippine Endemic Species Conservation Project of the Frankfurt Zoological Society. A word of thanks to Ernst Mayr: It is both a pleasure and an honour for me to be invited to humbly present to him a little piece of research. This appears truly fitting since Ernst has always taken an active interest in my research. This started when I was introduced to him in my early student days in Berlin in 1951 by our joint mentor, Erwin Stresemann. In his ornithological career Ernst had, in his awe-inspiring breadth of research, shown an active interest in the avifauna of the Philippines, of that world record treasure of biodiversity, when writing a book, together with Jean Delacour, on the Birds of the Philippines. In his usual generous manner, Ernst donated his last copy to me when I embarked on research and conservation in the Philippines 11 years ago. Though Ernst had followed my doings in science with benevolence from early on, his generous material support of our Philippines project became unrivalled among private donors, and it spurred our motivation immensely to carry on in what first appeared to be an almost hopeless situation, in a sea of a burgeoning population. I thank Ernst wholeheartedly for all his inspiration and farsighted support and wish him still many years to come.     

Rational men and the explanatory model approach

The previous issue of Culture, Medicine and Psychiatry (Vol. 5, N. 4) included my article When Rational Men Fall Sick: An Inquiry into Some Assumptions Made by Medical Anthropologists together with a series of comments. This paper consists of my replies to some of the commentators and a case study illustrating my points.My collaborators in this research were two physicians, Dr. Robert Like, of the Department of Family Practice of Case Western Reserve University and Dr. Rivka Plotkin of the Ben-Gurion University of the Negev (Israel). Also, I want to thank Avraham Blidstein for his invaluable assistance.     

The μI skeletal muscle sodium channel: Mutation E403Q eliminates sensitivity to tetrodotoxin but not to μ-conotoxins GIIIA and GIIIB

Voltage-sensitive Na channels from nerve and muscle are blocked by the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX). Mutagenesis studies of brain RII channels have shown that glutamate 387 (E387) is essential for current block by these toxins. We demonstrate here that mutation of glutamate 403 (E403) of the adult skeletal muscle I channel (corresponding to E387 of RII) also prevents current blockade by TTX and STX, and by neo-saxitoxin. However, the mutation fails to prevent blockade by the peptide neurotoxins, -conotoxin GIIIA and GIIIB; these toxins are thought to bind to the same or overlapping sites with TTX and STX. The E403Q mutation may have utility as a marker for exogenous Na channels in transgenic expression studies, since there are no known native channels with the same pharmacological profile.     

A year since George Floyd

The murder of George Floyd sparked an awakening, long overdue, which reverberated throughout society. As science begins to acknowledge its role in perpetuating systematic racism, the voices of Black scientists, which have largely been absent, are now being called on. As we rightly begin to make space for diverse voices and perspectives in science, we all must think about what it is we are asking minoritized individuals to do.

It has been roughly 1 year since the murder of George Floyd, an unarmed Black man, who was killed over an alleged counterfeit 20 dollar bill in Minneapolis, Minnesota (Hill et al. 2020; Kaul, 2020; Levenson, 2021). In many ways, his murder was no different than the murders of thousands of other murders of Black people in this country (Thompson, 2020; Lett et al., 2021; Tate et al., 2021). However, what distinguishes George Floyd’s murder from many other high profile cases is that it was unambiguously captured on video (Alexander, 1994), an act of bravery by Darnella Frazier, a 17-year-old Black woman (Izadi, 2021), at a time when the world was mostly housebound by a raging global pandemic. As a result, his murder reverberated through society in a way that has not happened in my lifetime. While there have been other high profile cases of murders carried out by police (Treyvon Martin, Walter Scott, Breonna Taylor, and Philando Castile, among many others), these cases failed to fully sustain the attention of a national and international audience (Chan et al., 2020; Chughtai, 2021). The murder of George Floyd was fundamentally different, and for once, more than just Black people were paying attention. His murder sparked protests across the nation led by the Black Lives Matter (BLM) movement (Day, 2015; Taylor, 2016; Banks, 2018; Taylor, 2021), and the demands for change were so loud people could not help but hear.As a Black, gay man who is also a scientist, I was thrown into despair. All of my life I have thought if I just worked hard enough, if I am kind and unthreatening, if I play the game and keep my head down, maybe I can make it in academia. Maybe then I will be seen and accepted, not just by society, but by the scientific community. George Floyd’s murder reminded me, and many of my Black colleagues, that our degrees can’t protect us, that our privileged middle-class upbringing (if we had one) was not a shield. Our lives were not worth more than a counterfeit 20 dollar bill.Science, which has always been a product of society, was not impervious to these reverberations. By late June my inbox began to slowly fill with invitations to speak at several institutions for their seminar series, retreats, or special symposia. It felt as if the scientific community, for the first time, realized that there were Black scientists among them. In the throes of my own despair, and the feeling that I needed to be doing something for my community, I began to say “yes.” I was not going to participate in the nightly protests that occurred in my newly adopted hometown of Portland, Oregon. Aside from fearing I could be next to lose my life at the hands of the police (Edwards et al., 2019), these protests were happening in the backdrop of a global pandemic. I came to the conclusion that by accepting these invitations to speak, this could be my activism, my way of sparking change, increasing visibility, and being seen not only for my own sake but also for other Black scientists.Before I write anything else, I want to be clear: I am extremely thankful to all the institutions and organizations that invited me and gave me a platform. I am extremely proud of my students’ work and of the research we produce. I am sharing my experiences with the hope that they can be instructive to the greater scientific community, but if I am being frank, there is a bit of anger.I received over 15 invitations and gave an additional three or four interviews over the course of the year. Most of these came with the expectation that I would also talk about my work in Diversity, Equity, and Inclusion. But here’s the lowdown: prior to this year, I did not view myself as someone who did Diversity, Equity, and Inclusion work. I am co-chair of the LGBTQ+ committee of the American Society of Cell Biology and a member of the Diversity, Equity, and Inclusion committee of the Genetics Society of America. I volunteer for both of these committees because they speak to something I care deeply about, the advocacy for minoritized ¹ scientists. I also embody both of these axes of diversity; so, in some way, I am only looking out for myself. This is far from being a scholar or doing “Diversity work.” I fully recognize that there are individuals who have dedicated their lives to this type of work with entire academic fields populated with accomplished scholars. So, I started this year of talks being invited because I am a Black, gay scientist at a time when science was grappling with its own systematic racism, under the guise of my nonexistent Diversity, Equity, and Inclusion work.What has this year actually taught me? The first thing it taught me is that I have been missing out. Prior to George Floyd’s murder, I had only received three seminar invitations from major research institutions and unfortunately all within a year of being posttenure. That is after nearly 6 years in my current position.In giving these talks I got the opportunity to meet with some of the giants in my field, people I have looked up to for years. I received reagents, offers to collaborate, and a litany of great ideas that will help drive my research program for years to come. I left some of these meetings truly inspired and excited to start experiments. These opportunities would have been invaluable to me, pretenure. One could argue, I did not need it. I made it even without this networking and the advantages these visits bring. Before you applaud my ability to persist and be resilient, we should take a deep look at the systems that have forced people who look like me to be doubly resilient. If George Floyd had not been murdered, would any of these invitations have happened? If the previous 6 years are any indication of a trend, I would have to say most certainly not. Why did it take a murder and the reignition of a Civil Rights movement for me to have the type of interactions I now know many of my straight, white counterparts have had from the very beginning of their independent careers? Let me be clear: this is a form of systematic racism, plain and simple.As I began to make the rounds, I was often asked to either share a bit of my journey or include my Diversity, Equity, and Inclusion work in my talks. This sometimes came at the expense of sharing my lab’s work. While I was very happy to do so, this was very much implicit in the invitations I received. At times it did feel that my inclusion was only checking a box, placating the graduate students so that they could see that their department or institution was responding to their demands. This also had the consequence of making me feel as though my science was merely performative. I was being invited to do the Diversity work institutions did not want to do. This is the tension I, and many other minoritized scientists, face. I want to share my experiences with the hopes that the next generation will have it better; but, my scholarly work is not in Diversity, Equity, and Inclusion. I fully recognize that it is my embodied diversity that is bringing me to the table; but, it is the science I want to share.On the first invitation to give a seminar, I promised myself that I was going to be honest. This meant that I would tell the truth about my experience and bare my soul over and over again. What I had not counted on was the emotional toll this would take on me. Reliving my own trauma, on a regular basis, left me emotionally drained after these visits. In one of my “stops” (I use quotes here because these “visits” were all virtual), I met with the queer, person of color (POC), graduate students. This session quickly turned into an emotional support group where I heard stories of mistreatment, racism, and discrimination. It was nearly impossible to maintain my composure. Diversity, Equity, and Inclusion work is clearly extremely important, but, maybe, we could just start by listening to the needs of the students and having a bit of humanity.The trial of Derek Chauvin has come and passed, and much to my surprise, and to the surprise of many other Black people nationwide, he was found guilty and was sentenced to prison (Arango, 2021; Cooper and Fiegel, 2021). This, of course, is not justice, not even close. Justice would mean that George Floyd is still alive and would get to live out his life in the way he chose. We are also at the beginning of the end of the pandemic. In 6 months or less, we may all be returning to life, more or less, as it was before George Floyd, before COVID-19. Does this mean we stop fighting? Does this mean that I, and many other Black scientists, suddenly disappear? For George Floyd, for countless other faceless Black people before him, I sincerely hope not. We need to continue to give Black scientists a platform. We need to ensure that they, too, are given the opportunity to network, collaborate, and interact with the larger scientific community. This means the invitations cannot stop. To further this, we need to ensure that Black scientists are included in every grant review panel, are included on speaker lists at every national and international meeting, are funded, and are in the room where funding, tenure, and other critical decisions are being made. We need to recognize that systematic racism has not gone away with Derek Chauvin’s conviction and sentencing. We need to continue to push forward. And, for all of you young, minoritized scientists (and allies) reading this, demand change and do not take "no" for an answer. I am truly sorry this has fallen on your shoulders, but enough is enough. The next generation of minoritized scientists should be recognized for their science without the additional burden of creating their own space.About the AuthorI am currently an Associate Professor of Biology at Reed College (https://www.reed.edu/biology/applewhite/index.html), which is located in Portland, Oregon. I arrived at Reed in 2014; prior to that, I was a postdoctoral fellow at the University of North Carolina, Chapel Hill. I received my PhD from Northwestern University in Cellular and Molecular Biology and a BS in Biology from the University of Michigan where I was also a 4-year letter winner in track and field. My research focuses on the cytoskeleton where I study cell motility and morphogenesis using Drosophila and Drosophila derived in tissue culture cells to explore actin, microtubules, and molecular motors. My current lab is composed of fierce, determined undergraduate students. I am a member of the American Society of Cell Biology (ASCB) and the current chair of the LGBTQ+ Committee (https://www.ascb.org/committee/lgbtq/). I am also a member of the Diversity, Equity, and Inclusion Committee for the Genetics Society of America (https://genetics-gsa.org/committees/). I also serve as an editor for MBoC’s Voices series.     

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.Recently, it has emerged that mitochondrial dysfunctions play an important role in the pathogenesis of degenerative diseases of the central nervous system (1–3). The processes underlying neuronal degeneration are complex, and some authors suggest that several genetic alterations are involved (4). However, another level of complexity may be derived from the fact that virtually all cellular activities depend upon energy metabolism in the cell (5). Alterations in energy metabolism processes within cells may also contribute to pathogenic mechanisms underlying neurodegenerative disease.A large body of evidence suggests that increased oxidative stress is an important pathogenic mechanism that promotes neurodegeneration (6). Because neurons have a long life span, and most neurodegenerative diseases have a clear association with age (7), it is important to understand mechanisms underlying reactive oxygen species (ROS)² production in neurons. Recently, Kudin et al. (8) analyzed the contribution of mitochondria to the total ROS production in brain tissue. They concluded that mitochondria are the major source of ROS and that at least 50% of ROS generated by brain mitochondria was associated with succinate-supported reverse electron transport (RET). Under conditions of normoxia, about 1% of the respiratory chain electron flow was redirected to form superoxide (8).Recently, we suggested that the organization of the respiratory chain complexes into supercomplexes that occurs in brain mitochondria (BM) (9) may represent one of the intrinsic mechanisms to prevent excessive ROS generation (10). In this paper, we put forward the hypothesis that inhibition of Complex II by oxaloacetate (OAA) represents another important intrinsic mechanism to prevent oxidative stress. We provide evidence that glutamate and pyruvate specifically exert control over the production of ROS at the level of Complex II. Below we present a brief account of published theoretical and experimental evidence that underlie our hypothesis.The neural processing of information is metabolically expensive (11). More than 80% of energy is spent postsynaptically to restore the ionic composition of neurons (11). When neurons are activated, reuptake of glutamate stimulates aerobic glycolysis in astroglial cells (12), thereby making lactate the major substrate for neuronal mitochondria (4, 13). However, rapid conversion of lactate to pyruvate in neurons requires activation of the malate-aspartate shuttle (MAS). The shuttle is the major pathway for cytosolic reducing equivalents from NADH to enter the mitochondria and be oxidized (14, 15). The key component of MAS is the mitochondrial aspartate/glutamate carrier (AGC) (16), and recent data suggest that the AGC is expressed mainly in neurons (14). Absence of the AGC from astrocytes in the brain implies a compartmentation of intermediary metabolism, with glycolysis taking place in astrocytes and lactate oxidation in neurons (13, 14, 17). Active operation of MAS requires that a certain amount of glutamate must be transported from synaptic clefts into activated neurons. In isolated BM, it has been shown that besides pyruvate, glutamate is also a good respiratory substrate (5, 18). In the presynaptic elements, the concentration of cytosolic glutamate is ∼10 mm at all times (19). Yudkoff et al. (18) have shown that synaptosomal mitochondria utilize glutamate and pyruvate as mitochondrial respiratory substrates. Glutamate is also oxidized by the astroglial mitochondria (13).Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 and EAAT2 located on presynaptic termini and glial cells (20–24). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (20, 25–28). At the climbing fiber to Purkinje cell synapses in the cerebellum, about 17% (28) or more than 50% (29) of synaptically released glutamate may be removed by postsynaptic transporters. Besides the cerebellum, EAAT4 protein was found to be omnipresent throughout the fore- and midbrain regions (30). Moreover, it was shown that although most of the EAAT2 protein is astroglial, around 15% is distributed in nerve terminals and axons in hippocampal slices and that this protein may be responsible for more than half of the total uptake of glutamate from synaptic clefts (24). These data suggest that postsynaptic transport of glutamate into nerve terminals where mitochondria are located (31) may occur in all brain regions. According to calculations of Brasnjo and Otis (28), in a single synapse, EAAT4 (excitatory amino acid transporter 4) binds and transports postsynaptically about 1.3 ± 0.1 × 10⁶ glutamate molecules. In the brain, on average, 1 mm³ of tissue contains 1 × 10⁸ synapses (32, 33). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus, in a narrow space of spines and dendrites, several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in the low millimolar range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions, may, in addition to metabolizing pyruvate, temporarily metabolize glutamate and succinate formed during mitochondrial catabolism of γ-aminobutyric acid in postsynaptic cells (34).The purpose of this study was to examine how the neuromediator glutamate affects respiratory activity and ROS generation in nonsynaptic BM when combined with pyruvate and the tricarboxylic acid cycle intermediates succinate and malate. We show that with pyruvate + glutamate + malate, the rate of oxidative phosphorylation increased more than 50%, and in resting mitochondria the rate of ROS generation associated with the reverse electron transport increased severalfold. These effects were observed only with brain and spinal cord mitochondria, not with liver or heart mitochondria, suggesting that they may be restricted to neuronal cells.Taken together, the data presented support the hypothesis that in activated neurons, the neuromediator glutamate stimulates mitochondrial ATP production when energy demand is increased. However, in the absence of energy consumption, glutamate + pyruvate may increase the generation of ROS severalfold. We suggest that intrinsic inhibition of Complex II by oxaloacetate is an important natural protective mechanism against ROS associated with reverse electron transport.     

Discovering the Mechanisms of Neurodegeneration in Prion Diseases

The purpose of my chapter in this issue of Neuroscience Reviews dedicated to Dr. Lawrence Eng is to summarize my contributions to understanding the mechanisms of neurodegeneration in prion diseases. I explain that I was able to advance the field of prion disease neuropathology largely because of the foundation of neurochemistry and immunohistochemistry that I learned while working 5 years in Dr. Engs laboratory. In my review, I relate how my Neuropathology Research Laboratory began as a collaboration with Dr. Stanley Prusiner 20 years ago that led from immunohistochemical staining of amyloid plaques in rodent and human brains using prion protein-specific antibodies to molecular evidence that the abnormal prion protein, PrP^Sc, is the cause of the clinically relevant neuropathological changes in animal and human prion diseases.Special issue dedicated to Dr. Lawrence F. Eng.