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cryonics directory


“Nematodes Can Survive in a Suspended Form of Life for Indefinite Time”, Shatilovich et al 2022

“Nematodes can survive in a suspended form of life for indefinite time”⁠, Anastasia Shatilovich, Vamshidhar R. Gade, Martin Pippel, Tarja T. Hoffmeyer, Alexei V. Tchesunov, Lewis Stevens et al (2022-01-28; similar):

When environmental conditions are unfavorable, such as the complete absence of water or oxygen, high temperature, freezing or extreme salinity, some organisms can enter suspended animation (cryptobiosis)1. This reversible transition is preceded by execution of complex genetic and biochemical programs (preconditioning)2,3,4. Under laboratory conditions, however, animals have only been maintained in a viable cryptobiotic state for a short time. Here we show that desiccation followed by freezing allows C. elegans dauer larvae to retain full viability over very long periods (around 500 days). Consistent with this finding, recently nematode individuals have been reanimated from the Siberian permafrost5, that according to precise radiocarbon dating shows that they remained in cryptobiosis since the late Pleistocene, for about 46,000 years. Phylogenomic inference based on our high-quality genome assembly and morphological analysis demonstrate that these nematodes belong to a novel parthenogenetic species, which we named Panagrolaimus kolymaensis. Genome analysis revealed that the core of the molecular toolkit for cryptobiosis in P. kolymaensis and C. elegans is orthologous. To survive desiccation and freezing under laboratory conditions these two species display similar biochemical responses. Thus, nematodes possess extraordinarily robust adaptive mechanisms that potentially allow them to remain in suspended animation over geological time scales.

“A Connectomic Study of a Petascale Fragment of Human Cerebral Cortex”, Shapson-Coe et al 2021

“A connectomic study of a petascale fragment of human cerebral cortex”⁠, Alexander Shapson-Coe, Michal Januszewski, Daniel R. Berger, Art Pope, Yuelong Wu, Tim Blakely, Richard L. Schalek et al (2021-05-30; ; similar):

[Blog] We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex⁠. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at ~30 nm and imaged these sections using a high-speed multibeam scanning electron microscope⁠. We used computational methods to render the 3-dimensional structure of 50,000 cells, hundreds of millions of neurites and 130 million synaptic connections. The 1.3 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 100 manually proofread cells are available to peruse online⁠.

Despite the incompleteness of the automated segmentation caused by split and merge errors, many interesting features were evident. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. The E:I balance of neurons was 69:31%, as was the ratio of excitatory versus inhibitory synapses in the volume. The E:I ratio of synapses was statistically-significantly higher on pyramidal neurons than inhibitory interneurons.

We found that deep layer excitatory cell types can be classified into subsets based on structural and connectivity differences, that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others’ initial segments, and that among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to ~20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

…This “digital tissue” (Morgan & Lichtman 2017) is a ~660,000-fold scale up of an earlier saturated reconstruction from a small region of mouse cortex, published in 2015 (Kasthuri et al 2015). Although this scaleup was difficult, it was not hundreds of thousands of times more difficult and took about the same amount of time as the previous data set (~4 years). This means that many of the technical hurdles with imaging and computer-based analysis have improved dramatically over the past few years. This improvement was in large part due to two noteworthy advances: fast imaging owing to multibeam scanning electron microscopy (Eberle et al 2015) and the profound effect of AI on image processing and analysis (Januszewski et al 2018). The rapid improvements over the past few years (Briggman et al 2011⁠; Bock et al 2011⁠; Helmstaedter et al 2013⁠; Takemura et al 2013⁠; Lee et al 2016⁠; Motta et al 2019⁠; Scheffer et al 2020⁠; Dorkenwald et al 2020⁠; Yin et al 2020⁠; Gour et al 2021) argues that analyzing volumes that are even 3 orders of magnitude larger, such as an exascale whole mouse brain connectome, will likely be in reach within a decade (Abbott et al 2020).

Figure 1: Image acquisition for the human brain sample. A fresh surgical cerebral cortex sample was rapidly preserved, then stained, embedded in resin, and sectioned. More than 5000 sequential ~30 nm sections were collected on tape using an ATUM [Automatic Tape-collecting Ultra-Microtome] (upper left panel). Yellow box shows the site where the brain sample is cut with the diamond knife and thin sections are collected onto the tape. The tape was then cut into strips and imaged in a multibeam scanning electron microscope (mSEM). This large machine (see middle panel with person on chair as reference) uses 61 beams that image a hexagonal area of about ~10,000 μm2 simultaneously (see upper right). For each thin section, all the resulting tiles are then stitched together. One such stitched section is shown (bottom). This section is about 4 mm2 in area and was imaged with 4×4 nm2 pixels. Given the necessity of some overlap between the stitched tiles, this single section required collection of more than 300 GB of data.

“Accelerating Progress in Brain Recording Tech”, Mineault 2021

“Accelerating progress in brain recording tech”⁠, Patrick Mineault (2021-04-27; ; similar):

In Stevenson & Kording 2011⁠, the authors estimated that every 7.4 years, the number of neurons we can record with doubles. Think of it as Moore’s law for brain recordings. Since then, Stevenson has updated the estimate, which now stands at 6 years. Could it be that progress itself is accelerating?

Data from Urai et al 2021 (paper)

…We can do one better—fit a double-exponential model. This is only a few lines of code in PyMC3—a miracle of automatic differentiation and Hamiltonian Monte Carlo⁠. Here’s what that looks like:

Capability of electrophysiology, past, present and future

You can see visually this is a much better fit, and it implies something pretty dramatic: progress itself is accelerating. That means that doubling time itself has changed over time—and it currently stands at 3.6 years under this model [95% CI 3.5–3.7]…These results project a 1M neuron average recording capability by 2045—of course, this discounts ceiling effects and potential paradigm shifts, which could adjust these bounds far upward or downward.

“Isochoric Freezing and Its Emerging Applications in Food Preservation”, Nida et al 2021

2021-nida.pdf: “Isochoric Freezing and Its Emerging Applications in Food Preservation”⁠, Sundus Nida, J. A. Moses, C. Anandharamakrishnan (2021-03-31; ; similar):

The preservation of foods at low temperatures is a well-established concept. While conventional methods of food freezing rely on the isobaric (constant pressure) approach, they often result in a series of irreversible changes that can seriously hamper the quality of frozen foods.

In recent years, taking its roots from the biomedical industry, isochoric (constant volume) freezing is gaining both research and commercial interest as an effective method of food preservation.

The focus of this review is to present the state of the art of isochoric freezing of foods, highlighting the underlying mechanisms that make it unique, and understanding its impact on food quality, considering reports published in the past decade. An exclusive section is dedicated to its non-food applications, and this work also provides insights into the costs and economics of the process.

Importantly, as this is an emerging area, the review concludes by highlighting the challenges and provides perspectives on the directions for future research.

[Keywords: isochoric freezing, food preservation, food freezing, food quality, low-temperature preservation]

Thermodynamics of Isochoric Systems: The thermodynamic principles of isochoric preservation were first studied in the year 2005 by Rubinsky and his fellow researchers.3 During isochoric freezing, the volume of the system remains constant while variables like pressure and temperature vary in tandem. The phase diagram of pure water in Figure 1 shows that isochoric freezing is followed by a liquidus path that lies between ice I⁠, ice III⁠, and liquid water. The system exhibits equilibrium pressure until the triple point at the given sub-zero temperature. For pure water, the triple point is a temperature of 21.985 °C and a pressure of 209.9 MPa. Importantly, unique conventional freezing process, ice growth cannot occur due to the constant volume, which in turn generates a hydrostatic pressure in the isochoric system.4 Theoretical and experimental data confirm that 45% of the volume remains unfrozen at the triple point in a constant volume freezing process3, 5. This effect takes the benefit of the Le Chatelier’s principle which explains that the high pressure developed inside a system upon freezing would restrict any further development of ice 6.

Isochoric Freezing and System Designs: In a typical isochoric process, the food material is immersed in an isotonic solution inside a rigid container that is capable of withstanding elevated pressures. Depending on the pressure and temperature, materials such as stainless steel cylinders, carbon fiber composites, and hard phenolic thermosets with pressure transducers and rupture disks are employed for isochoric processes. Sugar or salt solutions are used for preservation. Then, ice crystals are introduced in the container as the nucleation site and the chamber is tightly packed. To preserve food materials in their aqueous phase without the formation of ice crystals, it is important to insert this nucleator. The chamber is then sealed with a metal screw to restrict any passage of air in and out of the container.3 This preserves the food material in a 2-phase thermodynamic condition, without the risk of cellular injury (Figure 2). The two-phase isochoric system is achievable only if the system is tightly packed and no air or liquid can escape out of it. A temperature bath is used to cool the system. Most systems also have pressure transducers and thermistors linked to the data acquisition card and connected to a computer for data processing.6

…The impact of high pressure in isochoric freezing seems advantageous in terms of microbial destruction. Isochoric preservation completely exterminated E. coli at −15 °C because bacterial suspension at this temperature is in a metastable and amorphous liquid state, not conducive for the bacteria to survive.26 It was observed that partial destruction of E. coli occurs at −20 °C and −30 °C in the isochoric freezing process due to the ice III and ice Ih formations where some E. coli try to shelter inside ice crystals and replicate after the freezing process.26

Combined Techniques (Spontaneous): Isochoric cryopreservation can tolerate liquid nitrogen temperatures and pressures ranging up to 413 MPa, explaining that pressure measurement is crucial for the control of vitrification and devitrification in aqueous solutions.35 Such ideas further helped in the experimental validation of the isochoric vitrification process. Vitrification in isochoric freezing can be facilitated using additives such as propanediol and dimethyl sulfoxide (Me2SO) at concentrations ranging from 0 to 49% (w/​v), and this has been proven for cryopreservation of organs and tissues.36 The concentration of cryoprotective additives for isochoric vitrification is substantially less in isochoric freezing than in the case of isobaric vitrification at 1 ATM and a hyperbaric process at 1000 ATM. Therefore, isochoric techniques promote vitrification more effectively than hyperbaric systems.36

Super-cooling in isochoric conditions improves the stability of the system when exposed to various mechanical stimuli such as drop impact, vibration, ultrasonication, and thermal fluctuations. This effect is achieved by combined thermodynamic and kinetic factors that reduce the microscopic density fluctuation, eliminate the air-water interface, and provide resistance to cavitation.37 Another combined technique is the modification of the existing isochoric system in which multiple aqueous phases are employed, separated by a membrane impermeable to mass transfer but transmit heat and pressure. This multiphase isochoric freezing model can be used for the complete removal of hypertonicity and ice crystal formation in cryopreservation protocols.38

Energy and Cost Comparisons: Slow freezing processes employed in industries for freezing of food items involve the use of deep cryogenic temperatures to reduce the size of ice crystals and then storing foods under freezing temperature, accounting to be an energy-intensive process. The consumption of energy in an isochoric system is substantially lesser than an isobaric system of equal mass. This is because of the reduction in total frozen mass and the temperature dependence of the latent heat of fusion of water. In an isochoric system, only a portion of the mass is frozen at a given sub-freezing temperature higher than the triple point, decreasing the total energy for ice fusion. However, in an isobaric system, phase transition takes place at the atmospheric freezing point and the latent heat of fusion decreases with temperature, consequently requiring more energy to freeze. Thermodynamic analyses showed that fish or meat when stored in an isochoric system at −5 °C consume 70% less energy than the conventional freezing process. Further, more energy savings can be achieved when foods like fruits and berries with high sugar contents are preserved. Isochoric storage can reduce energy expenditure at an industrial level as no ice formation takes place inside the food.4 Such systems aim to increase efficiency and can be designed by altering existing industry-scale freezers. This can be achieved without major infrastructural alterations and appliance wastages. Further, the simple design of isochoric systems makes them convenient and relatively economic in terms of usage.6

“Meet Elizabeth Ann, the First Cloned Black-Footed Ferret: Her Birth Represents the First Cloning of an Endangered Species Native to North America, and May Bring Needed Genetic Diversity to the Species”, Imbler 2021

“Meet Elizabeth Ann, the First Cloned Black-Footed Ferret: Her birth represents the first cloning of an endangered species native to North America, and may bring needed genetic diversity to the species”⁠, Sabrina Imbler (2021-02-18; ; similar):

Her successful cloning is the culmination of a years-long collaboration with the U.S. Fish and Wildlife Service, Revive & Restore⁠, the for-profit company ViaGen Pets & Equine, San Diego Zoo Global and the Association of Zoos and Aquariums⁠.

Cloned siblings are on the way, and potential (cloned) mates are already being lined up. If successful, the project could bring needed genetic diversity to the endangered species. And it marks another promising advance in the wider effort to use cloning to retrieve an ever-growing number of species from the brink of extinction…“Pinch me”, joked Oliver Ryder, the director of conservation genetics at San Diego Zoo Global, over a Zoom call. “The cells of this animal banked in 1988 have become an animal.”

…In 2013, the Fish and Wildlife Service approached Revive & Restore to explore how biotechnology, which the nonprofit develops in pursuit of the de-extinction of species, could help increase the genetic diversity of black-footed ferrets⁠. The following year, Revive & Restore sequenced the genomes of four black-footed ferrets. First there was Balboa, who was born by means of artificial insemination using cryopreserved, genetically diverse sperm. Second was Cheerio, who was born naturally and shares ancestry from all seven founders; Novak calls him an “every ferret.” The last two ferrets came from tissue samples at the Frozen Zoo, one male called “Studbook Number 2” and one female named Willa. “When we looked at Balboa, we saw from an empirical standpoint that a great deal of genetic diversity had been rescued by reaching back into the past”, Mr. Novak said.

Revive & Restore designed a proposal and submitted it to Fish and Wildlife. In 2018, the nonprofit received the first-ever permit to research cloning an endangered species. Revive & Restore partnered with the commercial cloning company ViaGen Pets & Equine to design the cloning process.

The first trial began around Halloween. The Frozen Zoo sent Willa’s cryogenically preserved cell line to ViaGen’s lab in New York. ViaGen created embryos and implanted them into a domestic ferret surrogate. At day 14, an ultrasound confirmed heartbeats. The surrogate was shipped to the conservation center and was watched 24 hours a day for signs of labor. On Dec. 10, Elizabeth Ann was delivered via C-section. “Our beautiful little clone”, Mr. Novak said. On Elizabeth Ann’s 65th day of life the technicians drew her blood, swabbed her cheek and sent the samples to Samantha Wisely, a conservation geneticist at the University of Florida, who confirmed that Elizabeth Ann was, in fact, a black-footed ferret.

…When the clones reach sexual maturity, they will breed, and then their offspring will be bred back with wild black-footed ferrets to ensure there is no mitochondrial DNA left over from the surrogate mother.

“The Przewalski’s Horse Project”, Restore 2020

“The Przewalski’s Horse Project”⁠, Revive & Restore (2020-09; ; similar):

The world’s first successfully cloned endangered Przewalski’s horse (Equus przewalskii) was born on August 6, 2020. Revive & Restore, San Diego Zoo Global (SDZG), and ViaGen Equine collaborated to clone from a cell line of a genetically important stallion that had been cryopreserved since 1980 at the SDZG Frozen Zoo. This groundbreaking achievement was conceived as a new strategy to help restore genetic diversity to the Przewalski’s horse species.

Cloning For Conservation: Now a portion of this lost genetic diversity may be recovered by cloning historic Przewalski’s horse from frozen cells. Successful breeding can increase genetic diversity by reintroducing lost variants to the surviving population. This is the hope for the new foal, Kurt, who was cloned from cells that had been cryopreserved at the SDZG Frozen Zoo in 1980. These were cells from a stallion that was born in 1975 in the UK, was transferred to the US in 1978, and lived until 1998. He was recorded as Stud Book number 615 (SB615) and known as “Kuporovic” by his zookeepers. Learn more about this cloning process.

The SB615 cell line was chosen for genetic rescue cloning because an analysis of the captive breeding pedigree revealed that the genome offers substantially more genetic variation than any living Przewalski’s horse. Now that the genetic variation from Kuporovic “lives” again in Kurt, Kurt may become the most important horse in the North American captive breeding population. He may also become the first cloned animal to restore lost genetic variation to its species.

“The Connectome of the Adult Drosophila Mushroom Body: Implications for Function”, Li et al 2020

“The connectome of the adult Drosophila mushroom body: implications for function”⁠, Feng Li, Jack Lindsey, Elizabeth C. Marin, Nils Otto, Marisa Dreher, Georgia Dempsey, Ildiko Stark, Alexander Shakeel Bates et al (2020-08-29; ; similar):

Making inferences about the computations performed by neuronal circuits from synapse-level connectivity maps is an emerging opportunity in neuroscience. The mushroom body (MB) is well positioned for developing and testing such an approach due to its conserved neuronal architecture, recently completed dense connectome, and extensive prior experimental studies of its roles in learning, memory and activity regulation.

Here we identify new components of the MB circuit in Drosophila, including extensive visual input and MB output neurons (MBONs) with direct connections to descending neurons. We find unexpected structure in sensory inputs, in the transfer of information about different sensory modalities to MBONs, and in the modulation of that transfer by dopaminergic neurons (DANs). We provide insights into the circuitry used to integrate MB outputs, connectivity between the MB and the central complex and inputs to DANs, including feedback from MBONs.

Our results provide a foundation for further theoretical and experimental work.

“Cryonics for All?”, Thau 2020

2020-thau.pdf: “Cryonics for all?”⁠, Tena Thau (2020-01-31; ⁠, ; backlinks)

“A Connectome of the Adult Drosophila Central Brain”, Xu et al 2020

“A Connectome of the Adult Drosophila Central Brain”⁠, C. Shan Xu, Michal Januszewski, Zhiyuan Lu, Shin-ya Takemura, Kenneth J. Hayworth, Gary Huang, Kazunori Shinomiya et al (2020-01-21; ; backlinks; similar):

The neural circuits responsible for behavior remain largely unknown. Previous efforts have reconstructed the complete circuits of small animals, with hundreds of neurons, and selected circuits for larger animals. Here we (the FlyEM project at Janelia and collaborators at Google) summarize new methods and present the complete circuitry of a large fraction of the brain of a much more complex animal, the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses, and proofread such large data sets; new methods that define cell types based on connectivity in addition to morphology; and new methods to simplify access to a large and evolving data set. From the resulting data we derive a better definition of computational compartments and their connections; an exhaustive atlas of cell examples and types, many of them novel; detailed circuits for most of the central brain; and exploration of the statistics and structure of different brain compartments, and the brain as a whole. We make the data public, with a web site and resources specifically designed to make it easy to explore, for all levels of expertise from the expert to the merely curious. The public availability of these data, and the simplified means to access it, dramatically reduces the effort needed to answer typical circuit questions, such as the identity of upstream and downstream neural partners, the circuitry of brain regions, and to link the neurons defined by our analysis with genetic reagents that can be used to study their functions.

Note: In the next few weeks, we will release a series of papers with more involved discussions. One paper will detail the hemibrain reconstruction with more extensive analysis and interpretation made possible by this dense connectome. Another paper will explore the central complex, a brain region involved in navigation, motor control, and sleep. A final paper will present insights from the mushroom body, a center of multimodal associative learning in the fly brain.

“Scientists Are Giving Dead Brains New Life. What Could Go Wrong? In Experiments on Pig Organs, Scientists at Yale Made a Discovery That Could Someday Challenge Our Understanding of What It Means to Die.”, Shaer 2019

“Scientists Are Giving Dead Brains New Life. What Could Go Wrong? In experiments on pig organs, scientists at Yale made a discovery that could someday challenge our understanding of what it means to die.”⁠, Matthew Shaer (2019-07-02; ⁠, ; similar):

In the course of his research, Sestan, an expert in developmental neurobiology, regularly ordered slices of animal and human brain tissue from various brain banks, which shipped the specimens to Yale in coolers full of ice. Sometimes the tissue arrived within three or four hours of the donor’s death. Sometimes it took more than a day. Still, Sestan and his team were able to culture, or grow, active cells from that tissue—tissue that was, for all practical purposes, entirely dead. In the right circumstances, they could actually keep the cells alive for several weeks at a stretch.

When I met with Sestan this spring, at his lab in New Haven, he took great care to stress that he was far from the only scientist to have noticed the phenomenon. “Lots of people knew this”, he said. “Lots and lots.” And yet he seems to have been one of the few to take these findings and push them forward: If you could restore activity to individual post-mortem brain cells, he reasoned to himself, what was to stop you from restoring activity to entire slices of post-mortem brain?

…The technical hurdles were immense: To perfuse a post-mortem brain, you would have to somehow run fluid through a maze of tiny capillaries that start to clot minutes after death. Everything, from the composition of the blood substitute to the speed of the fluid flow, would have to be calibrated perfectly. In 2015, Sestan struck up an email correspondence with John L. Robertson, a veterinarian and research professor in the department of biomedical engineering at Virginia Tech. For years, Robertson had been collaborating with a North Carolina company, BioMedInnovations, or BMI⁠, on a system known as a CaVESWave—a perfusion machine capable of keeping kidneys, hearts and livers alive outside the body for long stretches. Eventually, Robertson and BMI hoped, the machine would replace cold storage as a way to preserve organs designated for transplants.

…By any measure, the contents of the paper Sestan and his team published in Nature this April were astonishing: Not only were Sestan and his team eventually able to maintain perfusion for six hours in the organs, but they managed to restore full metabolic function in most of the brain—the cells in the dead pig brains took oxygen and glucose and converted them into metabolites like carbon dioxide that are essential to life. “These findings”, the scientists write, “show that, with the appropriate interventions, the large mammalian brain retains an underappreciated capacity for normothermic restoration of microcirculation and certain molecular and cellular functions multiple hours after circulatory arrest.”

…“What’s happened, I’d argue”, says Christof Koch, the president and chief scientist at the Allen Institute for Brain Science, “is that a lot of things about the brain that we once thought were irreversible have turned out not necessarily to be so.”

“Persistence of Long-Term Memory in Vitrified and Revived Caenorhabditis Elegans”, Vita-More & Barranco 2015

“Persistence of Long-Term Memory in Vitrified and Revived Caenorhabditis elegans⁠, Natasha Vita-More, Daniel Barranco (2015-10-19; ):

Can memory be retained after cryopreservation? Our research has attempted to answer this long-standing question by using the nematode worm Caenorhabditis elegans⁠, a well-known model organism for biological research that has generated revolutionary findings but has not been tested for memory retention after cryopreservation.

Our study’s goal was to test C. elegans’ memory recall after vitrification and reviving. Using a method of sensory imprinting in the young C. elegans, we establish that learning acquired through olfactory cues shapes the animal’s behavior and the learning is retained at the adult stage after vitrification.

Our research method included olfactory imprinting with the chemical benzaldehyde (C6H5CHO) for phase-sense olfactory imprinting at the L1 stage, the fast-cooling SafeSpeed method for vitrification at the L2 stage, reviving, and a chemotaxis assay for testing memory retention of learning at the adult stage.

Our results in testing memory retention after cryopreservation show that the mechanisms that regulate the odorant imprinting (a form of long-term memory) in C. elegans have not been modified by the process of vitrification or by slow freezing.

“Saturated Reconstruction of a Volume of Neocortex”, Kasthuri et al 2015

“Saturated Reconstruction of a Volume of Neocortex”⁠, Narayanan Kasthuri, Kenneth Jeffrey Hayworth, Daniel RaimundBerger, Richard Lee Schalek, José Angel Conchello et al (2015-07-30; ; backlinks; similar):

  • Tape-based pipeline for electron microscopic reconstruction of brain tissue
  • Annotated database of 1,700 synapses from a saturated reconstruction of cortex
  • Excitatory axon proximity to dendritic spines not sufficient to predict synapses

We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and mitochondria) are rendered and itemized in a database.

We explore these data to study physical properties of brain tissue. For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters’ rule).

This online minable database provides general access to the intrinsic complexity of the neocortex and enables further data-driven inquiries.

“LWer Effective Altruism Donations, 2013–2014”, Branwen 2015

EA-donations: “LWer Effective Altruism donations, 2013–2014”⁠, Gwern Branwen (2015-05-12; ⁠, ⁠, ; backlinks; similar):

Analysis of 2013-2014 LessWrong survey results on how much more self-identified EAers donate suggests low median donation rates due to current youth and low incomes.

A LW critic noted that the annual LW survey reported a median donation for “effective altruists” of $0, though the EA movement encourages strongly donations. I look closer at the 2013-2014 LW surveys and find in multiple regression that identifying as an EA does predict more donations after controlling for age and income, suggesting that the low EA median donation may be due to EAers having low income and youth (eg. being a student) rather than being unusually or even averagely selfish.

“High-resolution Whole-brain Staining for Electron Microscopic Circuit Reconstruction”, Mikula & Denk 2015

2015-mikula.pdf: “High-resolution whole-brain staining for electron microscopic circuit reconstruction”⁠, Shawn Mikula, Winfried Denk (2015-04-13; ):

Currently only electron microscopy provides the resolution necessary to reconstruct neuronal circuits completely and with single-synapse resolution. Because almost all behaviors rely on neural computations widely distributed throughout the brain, a reconstruction of brain-wide circuits—and, ultimately, the entire brain—is highly desirable. However, these reconstructions require the undivided brain to be prepared for electron microscopic observation.

Here we describe a preparation, BROPA (brain-wide reduced-osmium staining with pyrogallol-mediated amplification), that results in the preservation and staining of ultrastructural details throughout the brain at a resolution necessary for tracing neuronal processes and identifying synaptic contacts between them.

Using serial block-face electron microscopy (SBEM), we tested human annotator ability to follow neural ‘wires’ reliably and over long distances as well as the ability to detect synaptic contacts.

Our results suggest that the BROPA method can produce a preparation suitable for the reconstruction of neural circuits spanning an entire mouse brain.

“Principles of Cryopreservation by Vitrification”, Fahy & Wowk 2015

2015-fahy.pdf: “Principles of Cryopreservation by Vitrification”⁠, Gregory M. Fahy, Brian Wowk (2015; ; similar):

Vitrification is an alternative approach to cryopreservation that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice.

Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and enables cooling to be rapid enough to “outrun” chilling injury, but it complicates the osmotic effects of adding and removing cryoprotective agents and introduces a greater risk of cryoprotectant toxicity during the addition and removal of cryoprotectants.

Fortunately, a large number of remedies for the latter problem have been discovered over the past 30+ years, and the former problem can in most cases be eliminated or adequately controlled by careful attention to technique. Vitrification is therefore beginning to realize its potential for enabling the superior and convenient cryopreservation of most types of biological systems (including molecules, cells, tissues, organs, and even some whole organisms), and vitrification is even beginning to be recognized as a successful strategy of nature for surviving harsh environmental conditions.

However, many investigators who employ vitrification or what they incorrectly imagine to be vitrification have only a rudimentary understanding of the basic principles of this relatively new and emerging approach to cryopreservation, and this often limits the practical results that can be achieved. A better understanding may therefore help to improve present results while pointing the way to new strategies that may be yet more successful in the future.

To assist this understanding, this chapter describes the basic principles of vitrification and indicates the broad potential biological relevance of vitrification.

[Keywords: vitrification, freezing, intracellular ice formation, devitrification, recrystallization, chilling injury, cryoprotective agents, cryoprotectant toxicity, osmotic limits, protein denaturation⁠, biobanking⁠, glass transition⁠, glassy state, optimal cooling rate, organ preservation]

“Genome Sequence of a 45,000-year-old Modern Human from Western Siberia”, Fu et al 2014

2014-fu.pdf: “Genome sequence of a 45,000-year-old modern human from western Siberia”⁠, Qiaomei Fu, Heng Li, Priya Moorjani, Flora Jay, Sergey M. Slepchenko, Aleksei A. Bondarev, Philip L. F. Johnson et al (2014-01-01; ; backlinks)

“Recalibrating Equus Evolution Using the Genome Sequence of an Early Middle Pleistocene Horse”, Orlando et al 2013

2013-orlando.pdf: “Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse”⁠, Ludovic Orlando, Aurélien Ginolhac, Guojie Zhang, Duane Froese, Anders Albrechtsen, Mathias Stiller, Mikkel Schubert et al (2013-06-26; ; backlinks; similar):

The rich fossil record of equids has made them a model for evolutionary processes1. Here we present a 1.12× coverage draft genome from a horse bone recovered from permafrost dated to ~560–780 thousand years before present (kyr BP)2,3. Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr BP), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski’s horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr BP), twice the conventionally accepted time to the most recent common ancestor of the genus Equus4,5. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski’s and domestic horse populations diverged 38–72 kyr BP, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski’s horse investigated. This supports the contention that Przewalski’s horses represent the last surviving wild horse population6. We find similar levels of genetic variation among Przewalski’s and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski’s horse. Such regions could correspond to loci selected early during domestication.



“Physical and Biological Aspects of Renal Vitrification”, Fahy et al 2009

“Physical and biological aspects of renal vitrification”⁠, Gregory M. Fahy, Brian Wowk, Roberto Pagotan, Alice Chang, John Phan, Bruce Thomson, Laura Phan (2009; similar):

Cryopreservation would potentially very much facilitate the inventory control and distribution of laboratory-produced organs and tissues. Although simple freezing methods are effective for many simple tissues, bioartificial organs and complex tissue constructs may be unacceptably altered by ice formation and dissolution. Vitrification, in which the liquids in a living system are converted into the glassy state at low temperatures, provides a potential alternative to freezing that can in principle avoid ice formation altogether. The present report provides a brief overview of the problem of renal vitrification. We report here the detailed case history of a rabbit kidney that survived vitrification and subsequent transplantation, a case that demonstrates both the fundamental feasibility of complex system vitrification and the obstacles that must still be overcome, of which the chief one in the case of the kidney is adequate distribution of cryoprotectant to the renal medulla. Medullary equilibration can be monitored by monitoring urine concentrations of cryoprotectant, and urine flow rate correlates with vitrification solution viscosity and the speed of equilibration. By taking these factors into account and by using higher perfusion pressures as per the case of the kidney that survived vitrification, it is becoming possible to design protocols for equilibrating kidneys that protect against both devitrification and excessive cryoprotectant toxicity.

“Sequencing and Analysis of Neanderthal Genomic DNA”, Noonan et al 2006

“Sequencing and analysis of Neanderthal genomic DNA”⁠, James P. Noonan, Graham Coop, Sridhar Kudaravalli, Doug Smith, Johannes Krause, Joe Alessi, Feng Chen et al (2006; ; backlinks; similar):

Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library are of Neanderthal origin, the strongest being the ascertainment of sequence identities between Neanderthal and chimpanzee at sites where the human genomic sequence is different. These results enabled us to calculate the human-Neanderthal divergence time based on multiple randomly distributed autosomal loci. Our analyses suggest that on average the Neanderthal genomic sequence we obtained and the reference human genome sequence share a most recent common ancestor ~706,000 years ago, and that the human and Neanderthal ancestral populations split ~370,000 years ago, before the emergence of anatomically modern humans. Our finding that the Neanderthal and human genomes are at least 99.5% identical led us to develop and successfully implement a targeted method for recovering specific ancient DNA sequences from metagenomic libraries. This initial analysis of the Neanderthal genome advances our understanding of the evolutionary relationship of Homo sapiens and Homo neanderthalensis and signifies the dawn of Neanderthal genomics.

“Isolation of a 250 Million-year-old Halotolerant Bacterium from a Primary Salt Crystal”, Vreeland et al 2000

2000-vreeland.pdf: “Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal”⁠, Russell H. Vreeland, William D. Rosenzweig, Dennis W. Powers (2000-10-19; ):

Bacteria have been found associated with a variety of ancient samples⁠, however few studies are generally accepted due to questions about sample quality and contamination. When Cano & Borucki 1995 isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25–30 million-year-old amber⁠, careful sample selection and stringent sterilization techniques were the keys to acceptance.

Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2–9–3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation⁠.

Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus. Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation.

Samples were rejected if brine inclusions showed physical signs of possible contamination. Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions. Sterilization procedures reduce the probability of contamination to less than 1 in 109.

“Revival and Identification of Bacterial Spores in 25 to 40-Million-Year-Old Dominican Amber”, Cano & Borucki 1995

1995-cano.pdf: “Revival and Identification of Bacterial Spores in 25 to 40-Million-Year-Old Dominican Amber”⁠, Raúl J. Cano, Monica K. Borucki (1995-05-19; ; backlinks):

A bacterial spore was revived, cultured, and identified from the abdominal contents of extinct bees preserved for 25 to 40 million years in buried Dominican amber⁠. Rigorous surface decontamination of the amber and aseptic procedures were used during the recovery of the bacterium.

Several lines of evidence indicated that the isolated bacterium was of ancient origin and not an extant contaminant. The characteristic enzymatic, biochemical, and 16S ribosomal DNA profiles indicated that the ancient bacterium is most closely related to extant Bacillus sphaericus⁠.

“Ability of the Ground Squirrel, Citellus Lateralis, to Be Habituated to Stimuli While in Hibernation”, Pengelley & Fisher 1968

1968-pengelley.pdf: “Ability of the Ground Squirrel, Citellus lateralis, to Be Habituated to Stimuli While in Hibernation”⁠, Eric T. Pengelley, K. C. Fisher (1968-08-20; ; similar):

The animals used were golden-mantled ground squirrels⁠, Citellus lateralis, whose hibernating behavior had previously been well studied…

…After the regular 12–14-day pattern of continuous hibernation had been established, several hibernating animals were removed from their nests, tossed once 2 to 3 ft in the air, caught, returned to their nests and the sawdust replaced in a pyramid on their dorsal surface. Such a stimulus invariably caused the animals to arouse as evidenced by observing them in the state of arousal some hours later or by the absence on the following day of the sawdust, which was then replaced on the animal that had re-entered hibernation.

Using this procedure animals were stimulated daily until it was found that they responded to such a stimulus by not arousing until the next normal arousal was due. This clearly indicated that the animals were being habituated to the tactile stimulus while in hibernation, and subsequently the daily “tossings” were increased gradually until it became possible to repeat this procedure 100× without causing the animal to arouse from hibernation.

Admittedly this experiment is “crude”, and in future studies the more accurately controllable electrical stimulus will be used. Nevertheless, the results are conclusive enough to establish the fact that with a body temperature as low as 1℃, the nervous system of these hibernating mammals is fully functional even to the point of establishing a habituated response to stimuli.



Royal Humane Society


Robert Ettinger


René Antoine Ferchault de Réaumur




Cryonics Institute


Alcor Life Extension Foundation