Center for Healthy Minds

In 2006, renewed interest in the project led the team to begin pilot work on thermal imaging to assess tukdam cases since one of the primary signs of tukdam recognized by the Buddhist tradition is a remnant warmth around the heart region where the subtle consciousness is understood to remain inside the body reliant upon subtle physiological activity still present there. The project formally began in 2007 as a collaboration with the Office of His Holiness the Dalai Lama, Men-Tsee-Khang — the Tibetan Medical and Astro-Science Institute under the auspices of the Dalai Lama, its biomedical partner Delek Hospital and the Center for Healthy Minds of University of Wisconsin-Madison.

The Principal Investigators comprised Dr. Richard Davidson from CHM and two of the Dalai Lama’s personal physicians – his Tibetan medical physician Dr. Tsewang Tamdin and his biomedical personal physician Dr. Tsetan Dorji Sadutshang. In 2007, CHM set up equipment in India to begin formal brain recordings and psychophysiological assessments for tukdam cases.

The first attempt of data collection occurred in September 2008 with the passing of the 100th Gaden Tripa, Lobsang Nyima Rinpoche, who remained in the tukdam state for 18 days. The aim was to assess whether remaining brainstem or heart activity via EEG and EKG, respectively, or other physiological signs of biological activity via thermal imaging, oximetry, and so forth, was present.

For an ethnographic account of this case, including the recognition of the reincarnation, see the doctoral work of Dr. Tenzin Namdul (2019), who was Director of Clinical Research at Men-Tsee-Khang at the time and the Tukdam team member leading the assessments in the field. He subsequently pursued graduate training in medical anthropology at Emory.

In 2011, the team re-designed the research protocol to include senior advanced practitioners as living healthy controls to get baseline data before passing and potentially entering the tukdam state. The practices that simulate entry into the tukdam state were not yet investigated at this time. Only psychophysiological measures of a resting concentrative state were assessed at this time. Pilot cases were conducted from 2011 to 2013, protocol formalized in 2012, and from 2013 to 2019, the team assessed 23 cases, evaluating the following:

• EEG (auditory brainstem response/mismatch negativity/resting state), EKG, blood oxygen levels, oxygen consumption, carbon dioxide production, skin temperature

• Environmental temperature and humidity

• Video and photographic documentation

• Thermal imaging

• Medical and meditation practice histories

The field team comprised Tibetan medical physicians from Men-Tsee-Khang, biomedically trained Tibetan physicians from Delek hospital, monastics at the host monastic institutions, and neuroscientific and anthropological staff from CHM. Of these cases, 13 are described in the first paper published by the study describing a null finding in Frontiers in Psychology.

The initial priorities of the investigations and field team training at this time was assessing neurophysiology; peripheral biology focused on blood oxygen levels, carbon dioxide consumption (measured via a capnography nasal cannula), and thermal imaging. Initial forensic assessments via imagery and video documentation were also beginning to develop.

Some of the challenges the team experienced included operational logistics in the demands of traveling to sites of tukdam case notifications and our physicians on the team pausing their clinic operations to assess cases. Developing trust with monastic, institutional and community members also took time as the study was just gaining public awareness. Likewise, tukdam cases are often not announced until 72 hours post clinical death so assessing the practitioner when there might still be remnant brain or measurable peripheral biological activity was challenging. The earliest the team was able to reach a case was 26 hours post clinical death.

Despite challenges, the study experienced many successes. Particularly, the team gained greater experience in practical knowledge and implementation of the study operations, building monastic and community partnerships, and publishing the first null finding paper.

In 2019, documentary filmmaker and Berkeley anthropology doctoral candidate Donagh Coleman joined CHM project lead Dylan Lott in the field to film tukdam cases. In the documentary, Coleman chronicles the efforts of the field team to record healthy living practitioners and tukdam cases, and the challenge of two intellectual traditions in investigating the phenomena. The feature length version of the documentary Tukdam: Between Worlds was released in 2023. The TV version provides further impressions from the attending physicians to the 16th Karmapa during his passing in Zion, Illinois outside of Chicago, neither of whom had seen a tukdam case and were surprised to each detect heat at the center of the Karmapa’s chest. In the context of the study, however, heat signatures still have not been recorded using high-end thermal imaging devices.

One of the public tukdam cases that gained greater attention on the global stage is that of Tenga Rinpoche, a revered Karma Kagyu lama and founder of Benchen monastery in Nepal. He had served the 16th Karmapa for seventeen years. His case became well-known due to the impressions of Vanessa Lopez, a mortician from New York who happened to be in Nepal at the time and was requested by the monastery to embalm Tenga Rinpoche’s body after his 3-day tukdam ceased. Her impressions are presented in the Tukdam: Between Worlds documentary as well, where she describes the blood and other body fluids she had to remove before embalming being indicative of someone who had passed hours previously not three days prior.

In November 2020, the Dalai Lama requested the Tukdam study team to collaborate with neuroscientists and physiologists from the Russian Academy of Sciences’ Institute of the Human Brain (IHB RAS) and Moscow State University.

The Russian team had recruited monastic field researchers, some of whom had trained in science at Emory University.

The Russian team trained their recruited monastic field researchers in further neuroscience methods through their program called "Middle-Wav Approach in Neuroscience" for which the principal trainings were held in Moscow and St. Petersburg. They opened three Russian Research centers located in Bylakuppe (Tashi Lhunpo Monastery), Mundgod (Drepung Gomang Monastery), and near Dharamsala (Gyutö Monastery) to facilitate tukdam and meditation field research. These research centers are now used by the joint collaboration as well.

The Russian team had a Russian pathologist as part of their team, and CHM recruited a forensic anthropologist as part of their team. The cross-disciplinary team initiated investigations together aiming to more comprehensively assess the physiological correlates of tukdam as well as the biological and neural mechanisms that may contribute to the physical signs observed, however, practically the focus targeted forensic measures primarily.

The teams also sought to explore the practices that facilitate the potential entry into the state vis-à-vis meditation practice and life history interviews with disciples of the tukdam practitioners as well as through the continued healthy living practitioner investigations. The Russian team published their initial findings.

Russian team conversation with the Dalai Lama, May 2021 “How to Study Meditation by Scientific Methods”

Prof. Medvedev leads the conversation from beginning of event asking His Holiness how the tukdam research might be valuable for the greater human community. His Holiness provides a response about its significance in the Buddhist world. He describes where tukdam might occur in the phenomenological experience of the practitioner and how the Guhyasāmaja Tantric Cycle provides particularly detailed explanations related to tukdam. He mentions the 18-day tukdam of his late tutor Ling Rinpoche and how to discern tukdam-like states (e.g., characterized by excess attachment) from real tukdam states.

Russian team discussing the study of tukdam with the Dalai Lama, March 2022*

(*Note: Tibetan-only recording available for first 11 minutes (for our Tibetan-speaking viewers), after which English is included.)

His Holiness describes tukdam as it relates to subtle consciousness and the experiences the practitioner will encounter upon entry into the state. He asks the scientists if there are any current understandings in science that could explain what is observed in tukdam. He encourages the research team to begin investigations as soon as clinical death is declared even prior to confirmation of the tukdam state. He describes that tukdam is difficult to declare in the first days after clinical death.

*Data is collected either sequentially as a full cycle long term study, or in parallel as partial studies of different phases with different subjects

Phase III. Post-mortem study.

This phase aims to observe the dynamics of biological processes in the brain and body of meditation practitioners days after diagnosed biological death. Frequently the team is not notified till 3 or more days after clinical death due to the time period commonly observed before a tukdam state is declared. Though the team attempts to study any meditation practitioner or community member immediately entering a postmortem phase to collect control cases, as well.

Phase I. Baseline intravital study.

This phase aims to examine psychophysiological, physiological and biological features of practitioners that are associated with meditation practice as well as identify potential factors that contribute to speed of decomposition postmortem.

Phase II. Perimortem study.

The aim of this phase is to investigate the transition process shortly before and immediately after biological death in meditation practitioners. This phase studies features of attenuation of psychophysiological, physiological and biological function in the brain and body as well as identifies potential factors that affect the dynamics of postmortem biological processes. To date, the project has not been able to observe any practitioners transitioning through phase II as participants approach death. However, the study design would facilitate regular intervals of data collection on these measures through the perimortem period. Following phase II, participants would be divided into those who are later deemed tukdam cases and those who are not.

All phases include:

Questionnaires: used to collect participant information on medical history, demographics, meditation experiences, experience of attentional qualities, history of their personal meditation practice and a general life history. This information is collected from the practitioner in phases I or II and from the practitioner’s family or attendants in phase III, if it is known. Any available medical records are collected from the participant or relevant medical institution.

Baseline physiological assessment: used to assess physiological condition of experienced monastic and layperson practitioners during several data collection sessions.

Data is collected during resting state, mismatch negativity task and auditory brainstem response task and may involve the following measures given consent and comfortability of the participant:

• Electroencephalography (EEG) using 6 flat active electrodes on the head

• Electrocardiography (ECG) using electrodes secured below the right collarbone and left lowest floating rib

• Respiration rate using respiration belt monitor

• Skin temperature and skin temperature distribution collected through Infra-Red Temperature Imaging System (IRTIS)

• Oxygen partial pressure of the skin and blood oxygen saturation using pulse oximetry

• Carbon dioxide production using capnography nasal cannula

• Blood tests (phase I and II only)

• Photographs and videos to document changes in skin color and hue variations, quality/position, etc.

Additional data collection in phase III: the postmortem period phase III data collection uses the same measures as phases I and II with the addition of the following measures for forensic analysis by forensic experts:

• Body temperature assessments across pre-specified locations

• Skin turgor analyses

• Photos (grayscale/color calibration cards allow for image comparisons across lighting conditions and cameras)

• Standardized Munsell color hue assessments across body regions

• Continual video footage

• Observational notes on presence of insect activity, emitted fluids such as purge fluids or other fluid emissions important to the tradition

• Humidity and temperature recordings of the room and outdoor ambient environment

Measures in development:

• Oral microbiome analysis via buccal mucosa swab

• Volatile organic compounds (VOCs) in air proximal to body via sorbent tube

  
  

Death, as the moment when coarse body and subtle mind disassociate, offers the greatest possibility for comprehending this moment and thus, actualizing spiritual realization. The ultimate aim of the various practices applied at this moment is to realize the nature of reality, the nature of mind, and often involves extensive methods to cultivate wisdom and compassion that benefit other beings. Those who have achieved this are considered community exemplars of realization. Further investigation among living practitioners could provide scientists a unique insight into the quality of mind that these practitioners possess.

The study is motivated by advancements in contemplative neuroscience in the last several decades as well as energized by the support of the XIV Dalai Lama. His Holiness actively supports and engages in collaborations with scientists to investigate the changes in the body and mind that contemplative practices create in order to provide evidence of their benefits and encourage incorporation of these practices supporting happiness and well-being globally.

The Dalai Lama on the Nature of Mind in Context of Tukdam

Excerpt on tukdam from conversation with His Holiness the Dalai Lama in the virtual book launch of "Science and Philosophy in the Indian Buddhist Classics, Volume 2: The Mind" from his residence in Dharamsala, HP, India on November 13, 2020. Brief comments from co-editors CHM Tukdam Study Scientist Dr. John Dunne, Distinguished Chair in Contemplative Humanities and Geshe Thupten Jinpa, His Holiness' principal translator, Compassion Institute Founder. Recording marked at beginning of comments and continues approx. 40 minutes.

Over the last four decades, the field of forensic sciences and, particularly the subfields of forensic anthropology, medicine, and pathology, have made significant progress in the ability to accurately estimate time since death through access to and assessment of actualistic studies of human remains throughout the postmortem period (e.g. The University of Tennessee Anthropological Research Facility in Knoxville, Tennessee; Forensic Anthropology Center at Texas State University in San Marcos, Texas; The University of Quebec at Trois-Rivières site for Research in Experimental and Social Thanatology in Quebec, Canada).

Such research studies allow for unique opportunities to investigate differential decomposition in various settings across this critical interval of physiologic change. Until these developments, the systematic study of long-term body decomposition had apparently not advanced substantially since the 13th century when Song Ci, a Chinese physician in China known as the world’s first forensic entomologist, made his postmortem assessments that informed publication of his coroner’s manual that became the official standard for generations in China.

Where differential decomposition can be visually appreciated and scientifically measured, observing and explaining the non-random distribution of those external and internal changes in specific climate zones, and the variables that may help explain their presence and patterning, is key to understanding if a body may have been moved from one location to another, from one position to another, and to highlight areas of the body that may exhibit ante- and perimortem trauma.

Within the fields of forensic anthropology and forensic medicine in general, the ability to provide accurate time since death estimates that minimize error, maximize precision, and consider a multitude of variables is a critical component of all forensic and medico-legal investigations that focus on identification of human remains and the circumstances surrounding death.

Contrary to many forensic cases where date of death is often unknown, the tukdam study begins observations with that critical information and progressively assesses, through external testing measures (temperature, skin elasticity) followed by photography and videography, decompositional changes over time. The study observes and records any decompositional changes associated with the states of pallor, algor, rigor, and livor mortis, as well as progressive changes in skin color and skin breakdown in an environment where variability in climate and ecological factors may vary from case to case.

The potential contributions to forensic science:

• Exceptional circumstances where human putrefactive changes do not follow their predictable general pattern and demonstrate postmortem plasticity of time, environment, and position, as well as potentially the decedent’s psychophysiological engagement with the dying context itself.

• Documentation of deviations from the anticipated expected rate and pattern of decomposition.

• Explore variation in the normal progression of the postmortem process, in odors emitted, and in other biological markers such as insect and microbial activity.

• Document the forensic significance of both intrinsic and extrinsic taphonomic variables that may contribute to differential and attenuated decomposition.

• Contribute to evaluating documented inconsistencies in the postmortem interval in an underappreciated and underreported part of the world.

• Investigates the possible relationship between a life of meditative practice and/or cultivation of specific qualities of mind, and the entrance into a state of tukdam and the postmortem phenomena observed.

The study of tukdam provides an opportunity to observe the possible persistence of brain activity following cardiopulmonary cessation as observed in a small sample of intensive care unit patients taken off life support and in various animal models (Norton et al., 2017; Auyong et al. 2010; Grigg et al., 1987; van Rijn et al., 2011; Borjigin et al. 2013), as well as potential systemic activity such as in the hypothalamo-pituitary axis (Arita et al., 1993).

We know from animal research and some limited human case studies (Norton et al., 2017) that when cardiac function ceases (Auyong et al., 2010; Grigg et al., 1987), a remnant brain signal measured by EEG persists for up to 30 minutes, until arterial blood pressure ceases (van Rijn et al., 2011). There has also been significant variation in the EEG oscillations emitted for such cases – both human and animal (Xu et al., 2023). Research at George Washington University (Chawla et al., 2017) shows about half of patients who pass in the ICU display end-of-life electrical surges (ELES), with waveforms at higher frequencies than those observed in animals. A high amplitude slow wave or “delta blip” has been reported within 1 minute of clinical death for rats where decapitation in rats has been thought to be indicative of synchronous neuronal death (Borjigin et al., 2013). Likewise, high-frequency synchronous gamma oscillations (25-100 Hz) have been reported in the rat brain for up to 30 seconds following cardiac arrest (Borjigin et al., 2013). Although no well-defined EEG states have been observed following early cardiac arrest for humans, most often, EEG activity declines dramatically well before the last heartbeat.

Yet, some show power in higher frequency ranges before arterial blood pressure cessation alongside an increase in 95% spectral edge frequency immediately before death (Norton et al., 2017). The significance of heterogeneous brain activity in the peri- and (early) postmortem period is unknown and is the source of significant controversy.

The tukdam state is a way to observe possible persistence of peripheral biology activity and understanding potential sustained responses vis-à-vis glucagon effects on glycogen cycling, remnant metabolic activity in specific cell groups, etc.; and similarities and differences with mechanisms of estivation and brown adipose tissue activation (see, for instance, Staple 2016).

Such understandings about the processes the body and mind are undergoing at end-of-life, are critical for medical teams, as well as for families, as they face difficult ethical and moral decisions around end-of-life care, including decisions about organ donation. Increasing our knowledge about the internal experiences of an individual just prior to and after medical death can further our ability to facilitate an ideal death whenever possible, regardless of the medical, cultural, or religious context in which the person is living and dying.

Culture shapes our biology and our biology is deeply shaped by our cultural practices. Burgeoning research on contemplative practices over the last several decades, on which CHM has had the fortunate opportunity to contribute, shows beneficial outcomes to wellbeing across numerous psychophysiological measures. We see the reduction of distractibility related to self-reports of greater happiness and greater well-being through cultivated focused attention and present-centered awareness. Reductions in depression and anxiety have been shown to enhance specific cognitive capacities, such as self- and other-perception.

Various contemplative practices have been shown to ameliorate physiological and inflammatory markers of stress, such as cortisol, blood pressure, heart rate, and triglycerides, and other lipids, C-reactive protein and numerous dimensions of peripheral cytokine expressions (see, for instance, Pascoe et al., 2017; Sanada et al., 2016; Zeidan et al., 2011). We also see the slowed progression of chronic inflammatory disease, particularly those with mental health co-morbidities, strengthening immune response, increasing vagal tone, slowing cellular aging and elongating telomeres, and modulating the inflamma-aging pathways (see, for instance, Davidson et al., 2003; Carlson et al., 2013; Epel et al., 2009).

Maria Kozhevnikov’s work has also shown several practices specific to Tibetan Buddhism (vajrayāna practices) create heightened arousal -- heightened sympathetic activation and phasic alertness, while others can intentionally elicit heightened parasympathetic activity and tonic alertness (such as a calm and alert state of mind).

Yet today we have very little work that has focused on how individuals vary across different outcomes of contemplative practices and the implications for a life of meditation lived on the outcomes of both well-being and dying phases. What we do know is that expert meditators compared to age-matched novice meditators, demonstrate significantly different activity in the brain – both structural and functional (Brefczynski-Lewis et al., 2007). We know that there is a wide degree of individual variation across outcomes, even among age-matched novices and meditation-naïve.

We also know that there's a lot of individual variation in response to different practice types, whether it be mindfulness (focused attention), lovingkindness/compassion, open monitoring, and so forth. We also know that the expert-level meditators, those who we might call ‘Olympic level’ practitioners, who have achieved more than 60,000 hours of meditation over the course of their lifetime, also show distinct physiologies compared to the meditation-naïve, such as particular sustained brainwave forms. (Brefczynski-Lewis et al., 2007)

We’ve learned that expert meditators have altered structure and function in the brain – sustained gamma waves irrespective of state/activity and heightened emotional regulation. They show differentiated immune response and inflammatory reactivity ameliorated to a given stimulus. And we also know that a select few of the expert meditators seem to present a different physiology in the dying process (Brefczynski-Lewis et al., 2007).

Tukdam has been widely recognized historically in the Tibetan cultural world as one of the unexcelled final states of realization.

However, its specific externally recognized signs related to it as a psychophysiologic state is minimally mentioned in the Buddhist canon, including the Kangyur, or scriptures of the Buddha’s word; the Tengyur, or commentaries on those scriptures; the Abhidharma, or the compilation of Buddhist psychology, philosophy, and metaphysics; the Revealed Treasures, or texts hidden to be revealed for applicability in later times; and later commentaries.

The earliest written historical account of the external signs of tukdam derive from the biography of Je Tsongkhapa’s passing into paranirvana in 1419, documented by his disciple Khedrup Je in Entryway of Faith (Rnam thar dad pa’i ’jug ngogs). The most detailed account of the different external signs demarking tukdam and their condition-dependent variation are from Karma Chakmé (1613-1678), an accomplished scholar-yogin who wrote one of the most highly regarded meditation retreat manuals called Mountain Dharma (Ri chos mtshams kyi zhal gdams). He warns us that an individual who enters tukdam, as the meditative concentration or samadhi at the time of death, may have more subtle signs than one might think and that many of the signs associated with tukdam, such as an upright meditation posture, fragrant odor, warmth at the heart, and attenuated decomposition, depend on the physical condition of the individual before they died.

Thus, the Tukdam Study is the first investigation to document the external signs related to this phenomena across individuals in different physiological states and geographic regions to explore its plasticity of physical presentation and duration. Likewise, the Tukdam Study is recording orally transmitted accounts of tukdam as they relate to the historical (hagiographic biographical) textual accounts.

Burgeoning research on meditation shows beneficial outcomes to wellbeing – across numerous psychophysiological measures:

1. Reduces distractibility, stress reactivity, depression and anxiety
2. Enhances cognitive capacities such as attention and perception
3. Modulates pain responses
4. Ameliorates physiological & inflammatory markers of stress
   a. E.g., cortisol, blood pressure, heart rate, triglycerides, C-reactive protein, and peripheral cytokine expression
   b. Slows progression of chronic inflammatory disease with mental health co-morbidities
5. Strengthens immune response
   
6. Differential regulation of parasympathetic (e.g, vagal) & sympathetic activity
   a. Intentionally cultivated heightened arousal vs. calm, alert state of mind
7. Elongates telomeres and slows cellular aging

References

1. Segal et al., 2016; Goldberg et al., 2018, 2021

2. Lutz et al., 2009; Fucci et al., 2018

3. Zeidan et al., 2011

4. Pascoe et al., 2017; Sanada et al., 2016

5. Davidson et al., 2003; Carlson et al., 2013

6. Amihai & Kozhevnikov 2015; Bornemann et al., 2019

7. Epel et al., 2009

This would include sending a team of our monastic researchers to travel to Dharamsala and do a residency at the Dalai Lama Archives and research literature and teachings in Tibetan on tukdam, the accompanying translation of the reviewed works, an editorial compilation and the independent publication.

The team is focused on publishing forensic findings and will begin designing a longitudinal healthy aging study (all monks age 75 and older) that aims to target the perimortem period as well as understand life history and practice history relationships to tukdam states (as well as normalize studying aging and dying processes to minimize ethical concerns).

Our monastic colleagues will convene training for our science teams on the doctrinal and philosophical understandings of tukdam, including the theoretical foundations in the commentarial literature, related practices, forms of tukdam, and tukdam-like states.

• The Guardian: The new science of death: ‘There’s something happening in the brain that makes no sense’
• Center for Healthy Minds: Center for Healthy Minds Makes New Partnerships, Progress on Post-Death Tukdam Research While in India
• Seven Pounds: Study: Brain Activity Can Surge at Time of Death
• Tricycle: Tukdam Project
• Tricycle: Tukdam: Between Two Worlds (Documentary Review)
• Center for Healthy Minds: New Science Explores process of Death With Cultural, Medical, Forensic and Ethical Implications

| DOWNLOAD NOW |

Donagh Coleman, documentary filmmaker and Berkeley doctoral student, follows the Tukdam Study 2019-2020 before the collaboration expanded to its current size. The film captures the tension still present for the study today of two intellectual traditions investigating a phenomena using distinct methods of inquiry and understandings of consciousness, life, and death. A television version of the documentary provides further interviews including observations from attending physicians during the 16th Karmapa's passing in Zion, Illinois. Since the filming, the team has integrated more extensive forensic measures and and collaborators, as well as inquiry into the meditation practices themselves.

Aria 1993 The Function of the Hypothalamo-Pituitary Axis in Brain Dead Patients. Acta Neurochirurgica 123(1-2): 64-75. https://pubmed.ncbi.nlm.nih.gov/8213281

Auyong et al. 2010. Processed Electroencephalogram during Donation after Cardiac Death. Anesthesia and Analgesia 110(5): 1428-1432. https://doi.org/10.1213/ANE.0b013e3181d27067

Borjigin et al. 2013. Surge of Neurophysiological Coherence and Connectivity in the Dying Brain. PNAS 110(35): 14432-14437. https://www.pnas.org/doi/10.1073/pnas.1308285110

Brefczynski-Lewis et al. 2007. “Neural correlates of attentional expertise in long-term meditation practitioners.” PNAS 104(27) https://www.pnas.org/doi/full/...

Chawla et al. 2017. Characterization of end-of-life electroencephalographic surges in critically ill patients. Death Studies 41(6): 385-392. https://doi.org/10.1080/07481187.2017.1287138

Grigg et al. 1987. Electroencephalographic Activity after Brain Death. Archives of Neurology, 44(9): 948-954. https://pubmed.ncbi.nlm.nih.gov/3619714

Norton et al. 2017. Electroencephalographic Recordings During Withdrawal of Life-Sustaining Therapy Until 30 Minutes After Declaration of Death. Canadian Journal of Neurological Sciences 44(2): 139-145. https://www.cambridge.org/core...

van Rijn et al. 2011. Decapitation in Rats: Latency to Unconsciousness and the "Wave of Death." PloS One 6(1): e16514. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3029360

Xu et al. 2023. Surge of neurophysiological coupling and connectivity of gamma oscillations in the dying human brain. PNAS 120(19):e2216268120. https://www.pnas.org/doi/10.1073/pnas.2216268120