Notes: males are tiny compared to females. In many species of anglerfish, mating occurs through males attaching to and then fusing with females such that their circulatory systems join together. The male provides sperm and is eventually absorbed into the female.
Notes: characterized by transparent flaps connecting the dorsal and dorsolateral tentacles in adult females. In a case of extreme sexual dimorphism, the females grow to around 2 m in size while the males are only about 2.4 cm.
Notes: depending on the type crinoid adults can either swim freely or be tethered to the sea floor by a stalk. The former are called feather stars and the latter are called sea lillies. Crinoids can also crawl using rootlike structures called cirri as legs. They consume plankton and detritus by filtering through their featherlike arms and then propelling it towards a mouth. They reproduce sexually, releasing sperm and eggs into the water. Fertilized eggs hatch into freely swimming larvae that settle on the sea floor and transition into a stalked juvenile state before eventually breaking away (in the case of feather stars) as adults to swim freely once more.
Notes: cuttlefish are among the most intelligent invertebrates. They can rapidly change color using their chromatophore cells as a mode of communication and camouflage as well as to warn off predators (called a deimatic display).
Notes: garden eels are distinguished by their behavior of living in burrows on the sea floor and poking their heads out of the burrows to eat prey and sliding back into the burrows to avoid predators. Colonies of them can resemble grasses, hence their name.
Notes: a type of small (up to 3 cm) sea slug that floats upside down using a gas-filled stomach to stay adrift. They feed on Portuguese man o’ war jellyfish and similar organisms. After consuming venomous nematocysts from their prey, they store the stinging cells in sacs (called cnidosacs) located in their own extremities. This concentrates the nematocysts, making the sting of Glaucus atlanticus potentially more potent even than that of the man o’ war jellyfish itself.
Notes: a category of animals that exist as flat sheets a few cells thick with a ciliated epithelium on their undersides. They use these cilia to move along the seafloor and most of them reproduce asexually by budding or fragmenting into smaller individuals (though one subtype does also reproduce sexually).
Notes: large sea stars which can reach diameters of 1 m. Sunflower sea stars are predatory and consume various prey such as sea urchins, other sea stars, clams, sea cucumbers, and more. They move at a speed of about a meter per minute using thousands of tube feet located on their undersides.
Notes: a deep sea cephalopod of about 30 cm length that lives in the ocean’s aphotic zone. Vampire squid (Vampyroteuthis infernalis) have flaps of tissue connecting their tentacles, each of which is lined by fleshy spines. It is covered in photophores that produce disorientating flashes of light to confuse predators. Rather than ink, they can eject a sticky cloud of bioluminescent mucus when highly agitated by predators.
Humongous Fungus, a specimen of Armillaria ostoyae, has claimed the title of world’s largest single organism. Though it features honey mushrooms above ground, the bulk of this creature’s mass arises from its vast subterranean mycelial network of filamentous tendrils. It has spread across more than 2,000 acres of soil and weighs over 30,000 metric tons. Yet I would contend that Humongous Fungus represents a mere microcosm of the world’s true largest organism, a creature that I will call Cyborg Earth. What is Cyborg Earth? Eastern religions have suggested that all life is fundamentally interconnected. Cyborg Earth represents an extension of this concept.
All across the globe, biological life thrives. Quintillions upon quintillions of biomolecular computations happen every second, powering all life. Mycoplasma bacteria. Communities of leafcutter ants. The Humongous Fungus. Beloved beagles. Seasonal influenza viruses. Parasitic roundworms. Families of Canadian elk. Vast blooms of cyanobacteria. Humanity. Life works because of complexity that arises from simplicity that in turn arises from whatever inscrutable quantum mechanical rules lay beneath the molecular scale.
All creatures rearrange atoms in various ways. Termites and beavers rearrange larger bunches of atoms than most organisms. As humans progressed from paleolithic to metalwork to industrialization and then to the space age, information revolution, and era of artificial intelligence, they learned to converse with the atoms around them in an ever more complex fashion. We are actors in an operatic performance, we are subroutines of evolution, we are interwoven matryoshka patterns, an epic chemistry.
Thermodynamics and memetic natural selection juggle our civilization while riding a unicycle along a path to an as-yet unknown destiny. Because of humanity, technology represents a fundamental part of the biosphere. Computers, airplanes, factories, shipping routes. All of it is natural. This does not mean it is good or bad. Simply that it comprises part of the great biological conversation. This is Cyborg Earth. The world’s largest organism is the biosphere itself, including all of the remarkable ways that its constituents have reshaped its body.
Cyborg Earth has always been a uniquely gleaming gem in the cold vastness of the cosmos. But I believe that Cyborg Earth still resides in an embryonic state. As we hurtle into the expanse of the infinite future, Cyborg Earth will grow and change. We must realize that we are all connected by the electric dance of atoms. As a constituent species within Cyborg Earth, we currently possess an enormous power and a tremendous responsibility to properly steward our world’s embryogenesis.
Cover image modified from “Confocal 3D-image of a fungal network with reproductive spores containing nuclei” by Vasilis Kokkoris, source.
Combining synchrotron x-ray microscopy with expansion microscopy may represent a feasible approach for whole human brain connectomics at the nanoscale. Synchrotron x-ray microtomography on its own provides extremely fast imaging at high resolution, yet necessary tradeoffs between imaging throughput and resolution mean that the imaging an entire human brain with voxel sizes of less than 100 nm may still take much too long with current synchrotron technology. Fast imaging with voxel sizes of 300-1000 nm is much more readily achievable in the near future. Furthermore, expansion microscopy isotropically enlarges tissue by infusion of a swellable hydrogel, facilitating resolution increases. The combination of x-ray microtomography and expansion microscopy (hereafter referred to as ExxRM) could thus push the effective voxel size down to the level needed for dense connectomics. However, because tissue volume and imaging time scale cubically with expansion factor, careful balance between design of the synchrotron x-ray optical setup and the degree of expansion will be vital. In this perspective, I will explore balances between synchrotron optical engineering choices and expansion factor, propose methods to successfully implement ExxRM in the context of human brains, and estimate how much it would cost to image the human brain in this way. Imaging brains via ExxRM may represent a crucial paradigm shift in connectomics which paves the way for holistic understanding of human brain function.
Nanoscale connectomic imaging of the entire human brain represents a long sought-after goal that could provide the foundation for dramatic advances in neurobiology, neurotechnology, and artificial intelligence.1,2 Currently, the leading method for nanoscale connectomics is volume electron microscopy (EM). But imaging a 1 mm3 volume of mouse cortex over a period of 6 months required a tremendous collaborative effort by Yin et al. to develop a parallelized and fully automated transmission electron microscopy (TEM) system consisting of six instruments working in parallel.3 Each of these six instruments cost $125,000. The mouse brain has a volume of roughly 500 mm3, meaning that if these numbers were directly scaled, the process would take 250 years.4 That said, Yin et al.’s TEM dataset had high resolution with 4×4×40 nm voxels, so throughput might be increased by imaging at somewhat lower resolution. As such, it is conceivable to argue that advances in EM technology may enable imaging of an entire mouse brain at sub-100 nm voxel size over the course of a few years at a cost of around $10M. EM therefore represents a viable option for mouse brain connectomics. But the human brain’s volume is roughly 1200 cm3, around 2400-fold larger than the mouse brain.5 Even if EM technology somehow advances to the point where an entire mouse brain can be imaged at 100 nm3 voxel size in a single year for $10M, mapping a human brain with comparable parameters would take thousands of years. In my view, this provides a strong argument for the idea that a radically different approach is necessary for human brain connectomics.
Expansion light-sheet fluorescence microscopy represents a promising alternative to EM, yet this modality also falls short when considering the volume of the human brain (particularly after expansion). For instance, Lillvis et al. utilized 8-fold expansion and lattice light-sheet microscopy (ExLLSM) to image the Drosophila central complex in three colors with effective 30×30×100 nm voxels over the course of 5 days.6 But even accounting for the 8-fold expansion (512-fold volume increase), this amounts to a volume of less than 0.5 mm3. Imaging even a 4-fold expanded mouse brain assuming these numbers would take 876 years. Lattice light-sheet microscopes are relatively inexpensive at a few hundred thousand dollars each7 and thus might be parallelized enough to image an entire mouse brain within a year. However, the 2400-fold larger volume of the human brain relative to the mouse brain indicates that it is probably not reasonable to expect success in human connectomics through ExLLSM.
Based on these calculations, I suggest that a radically different strategy is needed for to put the goal of human brain connectomics within reach. Uniting synchrotron x-ray microtomography (XRM) (Figure 1A-B), expansion microscopy (ExM), a recent staining method known as Unclearing Microscopy8 may facilitate “ExxRM” imaging of the human brain at sub-100 nm voxel size on timescales of around 1 year for a cost of around $10M. Success of this approach will necessitate overcoming some technical hurdles, yet I am optimistic that these particular challenges can be conquered. ExxRM may represent a feasible platform to acquire images suitable for dense connectomics across entire human brains.
Figure 1 Principles of synchrotron XRM. (A) Synchrotrons generate electrons from a source, propel them through a linear accelerator (linac), raise their energy in a booster ring, and then keep the electrons circulating for long periods of time at relativistic speeds in the storage ring. As relativistic electrons move along a curved path controlled by bending magnets, they emit brilliant x-ray beams in the direction tangent to the curve in the direction of the electron movement. Insertion devices that inject the x-rays into beamlines are placed at straight sections of the synchrotron ring. These insertion devices stimulate emission of bright and coherent beams into the experimental stations. (B) Microtomography beamlines receive an x-ray beam from an insertion device and filter out a narrow band of wavelengths using a monochromator. The beam passes through a sample on a rotating tomography stage that can be positionally adjusted to change the location of the field of view within the sample. Projection images are taken across 360° of rotation. X-rays passing through the sample are converted to visible light using a scintillator, then directed by mirrors to a lens system that magnifies the image. This light hits a detector camera and data is recorded for 3D reconstruction.
Recommended methodologies for ExxRM
ExxRM’s success will require developing methodological strategies to mitigate technical challenges. Obtaining sufficient contrast to resolve subcellular features will be vital. ExM cubically dilutes the amount of cellular material per unit volume, so creative staining techniques will be needed. Approaches that ensure stability of expanded tissues under brilliant x-ray illumination for long durations will also be crucial. Multicolor imaging would greatly benefit the usefulness of whole brain connectomics data, so ways of efficiently obtaining tomograms in multiple colors are needed. These challenges must be conquered to translate ExxRM.
A central engineering hurdle for ExxRM is attaining sufficient feature contrast for capture of clear images. Because expanded tissues experience cubic dilution of target biomolecules, contrast from traditional stains such as osmium tetroxide almost entirely vanishes during x-ray microtomographic imaging (Collins et al., unpublished data). An elegant solution to this problem has come in the form of a recently developed technique called Unclearing Microscopy.8 For this technique, M’Saad et al. biotinylated primary amines (found on proteins, phosphatidylethanolamine lipids, etc.) throughout expanded samples, treated the sample with streptavidin horseradish peroxidase (streptavidin-HRP) fusion protein, and then stained with ionic silver reagents (from the EnzMet™ HRP Detection Kit) or with 3’3-diaminobenzidine (DAB). This triggered enzymatic deposition of enough chromogenic silver or DAB to make 20-fold expanded HeLa cells visible to the naked eye despite their 8000-fold increase in volume relative to the unexpanded state. Phase contrast light microscopy subsequently revealed subcellular features such as mitochondrial cristae, nuclear pore complexes, and nuclear membrane. Unclearing thus facilitates physical reconstruction of the structures that are pulled apart by ExM, filling in the gaps left by the expansion process (Figure 2A-B). Silver stain Unclearing could enable either absorption XRM or phase contrast XRM of expanded tissues since silver has excellent x-ray attenuation index β at relevant beam energies as well as excellent x-ray phase decrement δ. Focusing on phase contrast XRM may represent a better option since it can decrease the necessary dose of radiation per unit time by using x-ray wavelengths which are not absorbed as strongly by the tissue. Furthermore, phase contrast XRM is most sensitive to differences in sample density9 and silver staining forms dense precipitates of metallic silver (which in pure form has a high density of 10.49 g/cm3), so this approach might provide superior contrast in the context of ExxRM. Here, ExM’s sample dilution might prove advantageous because it could generate strongly distinct densities between silver-stained cellular features and the rest of the stabilized hydrogel. It should be noted that, for phase contrast XRM, the previously mentioned stabilization approach would need to fill the space between cellular features with a substance that differs substantially in density from the silver (or similar stain). Combining Unclearing Microscopy with phase contrast XRM could provide excellent feature contrast for ExxRM connectomics.
Figure 2 Proposed sample preparation technique for ExxRM. (A) Macroscale view of a human brain undergoing expansion, Unclearing, and stabilization. Slicing into centimeter-scale subvolumes is not shown but may also represent a beneficial strategy. (B) Nanoscale view of a neuron within the brain undergoing expansion, Unclearing, and stabilization. Considerable amplification of signal density after Unclearing should occur.
Expanded tissues are known for their fragility and synchrotron x-rays are known for their harshness, so strategies for solving the problem of sample degradation are needed. Newer expansion recipes which use high monomer concentrations have displayed substantially greater physical sturdiness than earlier generations of ExM hydrogels,10,11 so this may aid in stabilization to some degree. Yet additional advances in ExM sample preparation may still be necessary due to high required fluxes and long imaging times. Cutting human brain samples into smaller volumes on the order of a few centimeters may improve the situation by decreasing imaging times per sample. Keeping the sample at cryogenic temperatures with the help of cryoprotectants to prevent ice crystals damaging the tissue may also help since heat generation from the x-rays is particularly problematic for hydrated samples.12,13 That said, low temperatures would not fix the issue of radiation damage from ionization, so this issue should still be considered.14 Further stabilization might come in the form of infusion of a rapidly crosslinkable or crystalizing substance which irreversibly locks all biomolecules into place (Figure 2A-B). The chosen substance would need to undergo an inducible crosslinking or crystallization reaction that does not cause distortion of the expanded tissue, to not shrink or distort the expanded tissue while it diffuses into the hydrogel, and to minimally absorb x-rays at the energy range used for imaging, and to contrast sharply with the chosen stain material. The phase decrement of the stabilizing substance should be similar or lower compared to amorphous ice. These methods may enable sufficient stabilization for preventing nanoscale tissue degradation even with exposure to extremely brilliant synchrotron x-rays.
Multicolor ExxRM would greatly enhance the value of acquired image data since synapses and key biomolecules could then be tagged within the 3D reconstruction.15,16 Fortunately, multicolor absorption x-ray microscopy represents an established technique wherein two or more staining materials undergo two or more rounds of imaging at beam energies corresponding to the absorption edges of the chosen materials. The absorption edges in question must be sufficiently distinct that minimal overlap in detection occurs across the different beam energy imaging rounds. As an example, Depannemaecker et al. employed this approach by using antibody-linked gold nanoparticles to mark neuronal nuclei in mouse brains while also staining neurons with silver via Golgi’s method.15 They imaged at a beam energy corresponding to an absorption edge of silver and at a beam energy corresponding to an absorption of gold. In addition, multicolor phase contrast x-ray imaging might be accomplished by using molecularly targeted tags with distinct densities relative to the Unclearing silver stain. For instance, gold has a density about twice that of silver, so it might be possible to segment gold nanoparticles after data acquisition. A potential challenge for these methods comes from the difficulty of inducing reliable diffusion of large metallic nanoparticles into tissue. That said, post-expansion pre-stabilization staining could help overcome the problem since expanded gels are typically more porous than pre-expansion tissue. Multicolor ExxRM in 2-3 colors should be achievable, opening doors to more useful whole-brain image datasets.
Though some trial and error will doubtless prove necessary for ExxRM optimization, the required technologies lay within fairly close reach. High contrast could be achieved by applying Unclearing Microscopy to counter signal dilution from expansion. Stability under brilliant x-rays may be possible by leveraging stabler expansion recipes with higher monomer concentrations, by cutting the brain into centimeter-scale subvolumes to decrease radiation dose per sample, by employing cryogenic temperatures during imaging, and by developing rigidity-enhancing materials to infuse into expanded gels. Multicolor ExxRM could be achieved by staining with metallic nanoparticles linked to affinity reagents and performing additional scans tuned to the absorption edges of the chosen metals. These directions may provide a path towards successful ExxRM.
How fast can synchrotrons image expanded brains?
Synchrotron facilities offer bright and coherent x-rays that can rapidly image large volumes of tissue at high resolutions (Figure 1A-B). For example, Bosch et al. employed synchrotron XRM at the Swiss Light Source (SLS) and the Diamond Light Source (DLS) to acquire 3D reconstructions of an entire mouse olfactory bulb with 325 nm voxel size.17 In their study, tomograms of approximately 1 mm3 volume took around 20 minutes to acquire. Another investigation by Dyer et al. used the Advanced Photon Source (APS) synchrotron to image a region of mouse cortex at 650 nm voxel size and 1.47 mm3 volume. Imaging this volume took only 6 minutes.18 Walsh et al. leveraged the European Synchrotron Radiation Facility Extremely Brilliant Source (ESRF-EBS) to image a variety of whole human organs at low resolution and subvolumes within human organs at higher resolution.19 Notably, they reconstructed a cylinder with ~57 mm3 volume (5.4 mm diameter and 2.5 mm height) within human spleen at 1290 nm voxel size over the course of 2 hours. Ding et al. used the APS to image larval zebrafish with 743 nm voxels and 1.5 mm3 volumes per tomogram in 20 minutes with monochromatic x-ray scans and in just 20 seconds with polychromatic “pink beam” x-ray scans.20 Despite their much higher flux and correspondingly much shorter imaging times, the polychromatic scans unfortunately showed lower resolution due to poor SNR. It should be noted that resolution represents a distinct concept from voxel size and that contrast and SNR play major roles in the final resolution. That said, voxel size can act as a rough proxy for comparisons across different imaging setups assuming that contrast and SNR are consistent. In another key example, Foxley et al. utilized the APS to image whole mouse brains (11.7×11.7×17.7 mm) over the course of 7 hours of acquisition time with 1117 nm voxels.21 Finally, Rodgers et al. used the SOLEIL synchrotron and an alternative technique for tomographic acquisition (discussed in more detail later) to image an entire mouse brain a 650 nm voxel size in 8 hours.22 Though the parameters for image acquisition vary considerably across different biological samples and synchrotron hardware setups (table 1), these examples illustrate the power of synchrotron imaging for mapping biological tissues.
Table 1 Specifications of synchrotron tissue imaging experiments from selected references.
Smallest voxel size
Single tomogram volume
Total reconstructed volume
Imaging time per tomogram
Total imaging time
Bosch et al.
~40 min. to ~4 hours
Dyer et al.
Walsh et al.
Ding et al.
20 min. or 20 s*
1 hour or* 1 min.
Foxley et al.
Rodgers et al.
*with pink beam acquisition
While the voxel sizes described above are not small enough for nanoscale connectomics, incorporating ExM into the sample preparation pipeline presents the possibility of attaining sufficient resolutions for tracing neurites. Expansion factors of around 4x, 10x, and 20x are respectively possible with standard ExM,23,24 some of the newer enhanced ExM protocols,10,25,26 and iterative ExM methods.11,27 Expansion factor can also be intentionally decreased somewhat by adjusting salt concentration. It is important to note that not all ExM protocols retain lipid membranes, so selecting a recipe that preserves such cellular boundaries10,11,28 will be vital for morphology reconstrction. Balance between expansion factor and imaging time will represent a key determinant of the success of ExxRM for whole brain imaging. Higher expansion factors increase resolution linearly while increasing imaging time cubically. Synchrotron imaging’s extreme speed has the potential to keep up with such volumetric increases, though even this has certain limits.
Du et al. offer a helpful mathematical model to estimate the number of photons needed for imaging a sample with sufficient contrast to distinguish between voxels of desired size.12 The model starts by considering how many photons n̄pixel are needed to image a single pixel on a 2D tomographic slice. The pixel is assumed to contain a “feature” material with a phase decrement δf that must be distinguished against a background with a phase decrement δb. Distinctions can be made between adjacent background pixels b, overlaying background pixels bo’ and underlaying background pixels bu’. The SNR is assumed to equal 5 as based on the Rose criterion, which qualitatively represents an acceptable SNR for visually useful images. Background attenuation coefficient is given by µb’ and the thicknesses of the overlaying and underlaying background material are tb’o and tb’u respectively. After the value of n̄pixel is determined, the value is multiplied by the square N2 of the number of pixels N on the detector’s horizontal axis to find the number of photons needed per tomogram. As Du et al. explain, the number of slices is not used as a multiplicative factor since the photons are distributed across all of the rotation angles for each pixel. These calculations act as a guide to what sample properties and beam properties are needed for ExxRM.
The Du et al. model can help determine the feasibility of ExxRM by estimating the necessary synchrotron flux for imaging the brain at a desired voxel size in a reasonable time frame. Consider 1 cm3 of brain tissue expanded 4-fold to a block with dimensions of 4×4×4 cm. Based on this, assume that the average thickness of overlaying and underlaying background material is 2 cm. Also assume that the background material is made of amorphous water ice with a density of 0.92 g/cm3. Since the widths of the smallest neuronal features are about 20 nm wide,29 assume that the 4-fold expansion provides a minimum feature thickness of 80 nm. By using the Unclearing Microscopy technique, such features might be “filled in” after expansion with a metallic silver stain.8 Thus, I will assume that the 4-fold expanded membrane features are made up of metallic silver with a density of 5.25 g/cm3, half the density of pure metallic silver (to account for imperfect staining). Feature phase decrements δf, background phase decrements δb, and background attenuation coefficients µb’ across x-ray energies ranging from 10 keV to 30 keV can be obtained using the calculator available at (https://henke.lbl.gov/optical_constants/pert_form.html) which is based on the Henke dataset.30 This calculator does not directly give µb’, but it does give attenuation index β, for which µb’ = 4πβ/λ. I will furthermore assume that a 3×3 mm beam and detector are possible to employ and that the pixel size is 300 nm (75 nm effective post-expansion). According to the resulting model, the total number of photons needed to image a single tomogram (volume of 21.21 mm3) within the 4-fold expanded block at an energy of 30 keV is 1.47×1013. By examining this model across a range of x-ray energies (Figure 3A) and across a range of feature sizes (Figure 3A), one can obtain a better understanding of how the different variables might play out in an experimental setting.
Figure 3 (A) Total photons needed to image a single tomogram of 4-fold expanded tissue with volume of 21.21 mm3 at 300 nm voxel size and two different silver stain densities while assuming feature thickness of 80 nm, plotted against x-ray energy in keV. (B) Total photons needed to image a single tomogram of 4-fold expanded tissue with volume of 21.21 mm3 at 300 nm voxel size and two different silver stain densities while assuming 30 keV x-ray energy, plotted against desired feature thickness necessary to resolve.
How might this translate to imaging time for a single tomogram, the 4×4×4 cm expanded block, and the whole human brain? Contemporary beamlines which employ multilayer monochromators can reach fluxes of more than 1012 photons/s.31–35 As such, the single tomogram under the assumptions of the model might be acquired in around 10 seconds. Comparing the tomogram volume of 21.21 mm3 and the expanded block volume of 64000 mm3, this means that the whole block would require about 3000 tomograms and take 8.3 hours to image. Since the entire human brain should contain approximately 1200 of these blocks, whole-brain acquisition might take around 1.14 years of continuous imaging. All of this assumes the post-expansion effective voxel size of 75 nm (4-fold expansion and actual voxel size of 300 nm), 30 keV beam energy, and Unclearing of neuronal features to a density of 5.25 g/cm3 metallic silver. If these theoretical projections successfully align with experimental outcomes, ExxRM could facilitate nanoscale connectomics for the entire human brain in a time frame of around 1-2 years.
Cost estimates for whole brain ExxRM
While the power of the synchrotron facility comes with a high price tag, ExxRM may still represent the most overall cost-effective option for human brain connectomics. Consider the costs associated with the DLS as an illustrative example. The DLS is a third-generation facility and is currently one of the better synchrotrons in terms of its ability to produce bright and coherent x-rays. Building the DLS and its first seven beamlines from 2003-2007 cost about $316M, its later upgrades cost $144M and $134M, and its yearly operational costs have increased from $28M in 2007-2008 to $81M in 2019-2020 (table 2). Based on these data points, construction of a new synchrotron beamline costs approximately $10M and yearly maintenance may cost roughly $500K. This provides a framework for estimating the cost of a dedicated human brain ExxRM connectomics beamline.
Table 2 Costs associated with the Diamond Light Source36 as a case study on how much money is needed to build and maintain a state-of-the-art synchrotron facility.
Diamond Light Source
$316M: synchrotron, first seven beamlines, surrounding buildings, construction started in 2003 and completed in 2007
Upgrade 1 costs
$144M: fifteen more beamlines, detector development program, construction started in 2004 and completed in 2012
Upgrade 2 costs
$134M: ten more beamlines, not stated when construction started but was completed in 2021
Maintenance and operational costs
$28M in 2007-2008, $48M in 2012-2013, $81M in 2019-2020
Compact Light Source (CLS) technology should be considered as well before continuing. CLS instruments produce x-ray beams that fall somewhere between laboratory x-ray microscopes and synchrotrons in terms of brightness and coherence.37 CLS instruments are furthermore small enough to fit into a single room and are inexpensive enough that a large number of them could potentially be constructed in parallel. At first glance, CLS technology seems a more economically viable alternative to synchrotron beamlines, yet it still probably is not a good option at this time. Existing CLS instruments are not likely suitable for human brain (or even mouse brain) ExxRM connectomics in the foreseeable future because their optical engineering requirements and mediocre level of x-ray flux preclude rapid tomography at submicron resolution, particularly when a large field of view is desired.38 There is a small possibility that future advances in CLS technology could change this situation, yet this seems fairly unlikely, so synchrotron-based imaging remains the best route.
Building an entire synchrotron solely for ExxRM connectomics is probably less efficient than establishing an agreement with an existing synchrotron to construct a connectomics beamline. While one might envision additional parallelization through custom design of the beamline to split the beam to land in multiple sample chambers, splitting the beam would divide the photon flux and therefore increase imaging times for no net gain in speed. As such, parallelization would likely require an additional insertion device and thus an entire new beamline for each new sample chamber. Insertion devices consist of a series of precisely engineered magnets built into straight sections of the synchrotron’s ring. These magnets, known as undulators or wigglers depending on the specifics of the insertion device, stimulate directed emission of a brilliant x-ray beam out from the storage ring.39 Yet even if we assume $10M total plus yearly maintenance costs for each connectomics beamline, imaging multiple human brains over the course of a year or a single human brain in just a few months remains a reasonable proposition. For a project as important as mapping the human brain at sufficient resolution for dense neuronal reconstruction, price tags in the range of tens of millions of dollars may not be out of reach.
Conclusion and outlook
Connectomics needs a technological paradigm shift if it is to feasibilize dense mapping of one or more human brains. Though it comes with some technical challenges, ExxRM may represent a key strategic shift that drastically reduces human brain connectomics timelines from centuries down to 1-2 years for the image acquisition step. Data storage and early processing steps (e.g. tomographic reconstruction) will of course require data centers and high-performance computing, but this field is rapidly advancing and may indeed be capable of handling the challenge. Assuming 1 byte per voxel, the amount of storage needed for a 4-fold expanded human brain with 300 nm physical voxel size (75 nm effective voxel size) is about 2.84 exabytes. Segmentation of the human brain dataset will probably represent a vastly more difficult problem as well as require substantially more compute resources, so further advances in this area will need to occur in parallel with ExxRM development. Realizing the benefits of connectomics in the form of complete computational models of the brain will take additional extensive research. Precisely correlating gene expression and electrophysiological properties with neuronal morphology (i.e. “cell type”) may represent a major step towards bridging the divide between structural data and functional activity. Nonetheless, the allure of having a holistic anatomical picture of the brain may serve as a driving force in the meantime. ExxRM has the potential to transform the dense connectomics field, enabling anatomical imaging of the entire human brain with sub-100 nm voxel size and high contrast in around 1-2 years for a price of roughly $10M.
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24. Tillberg, P. W. et al. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol.34, 987–992 (2016).
25. Damstra, H. G. J. et al. Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). Elife11, e73775 (2022).
26. Park, H.-E. et al. Scalable and Isotropic Expansion of Tissues with Simply Tunable Expansion Ratio. Adv. Sci.6, 1901673 (2019).
30. Henke, B. L., Gullikson, E. M. & Davis, J. C. X-Ray Interactions: Photoabsorption, Scattering, Transmission, and Reflection at E = 50-30,000 eV, Z = 1-92. At. Data Nucl. Data Tables54, 181–342 (1993).
31. Albrahim, M. et al. Reduction and Agglomeration of Supported Metal Clusters Induced by High-Flux X-ray Absorption Spectroscopy Measurements. J. Phys. Chem. C125, 11048–11057 (2021).
32. Owen, R. L., Holton, J. M., Schulze-Briese, C. & Garman, E. F. Determination of X-ray flux using silicon pin diodes. J. Synchrotron Radiat.16, 143–151 (2009).
33. Weitkamp, T. et al. Parallel‐beam imaging at the ESRF beamline ID19: current status and plans for the future. AIP Conf. Proc.1234, 83–86 (2010).
34. Baussens, O. et al. Characterization of High-Flux CdZnTe with optimized electrodes for 4th generation synchrotrons. J. Instrum.17, C11008 (2022).
35. Moosmann, J. et al. X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation. Nature497, 374–377 (2013).
As a scientist, I am driven by the power of technological breakthroughs to make positive change for humanity. While I also take immense pleasure in the artistic/creative aspects of technology design, my motivation is centered on helping people and on protecting the future of the human species. For this reason, I am interested in a wide array of contemporary challenges as described in this outline. Because I am a synthetic biologist and synthetic biology has many applications, I have the ability to explore solutions to such diverse challenges despite their highly multidisciplinary nature.
That said, one of the tools in any good researcher’s repertoire is collaboration. Since I am just one person, my knowledge can only go so deep in so many areas. Interdisciplinary projects are much more likely to succeed when experts from multiple areas work together. So, I leverage collaboration extensively when carrying out my projects and will continue to do so in the future.
It should be noted that, though I am publicly presenting a number of conceptual explanations of possible solutions to important problems via this list, I have deliberately stated them in somewhat vague language to prevent their public disclosure from precluding outside investment.
I am sharing this list as a way of increasing my likelihood of connecting with potential collaborators over time and as a method of inspiring others to consider how they too might contribute to building a bright future.
I chose the featured image of a Martian-hued rendering of the Anasazi Cliff Palace at Mesa Verde National Park as a symbolic tribute to the creative and culturally rich spirit of humanity, an homage to where we have come from, and a hopeful indication of where we might go in the future.
Infectious Disease Burden in Developing Countries
Especially malaria, tuberculosis, and HIV.
Translational strategies for dissemination are key.
Some possible solutions:
Gene drives to prevent mosquitos from carrying pathogens
Inexpensive home diagnostics
Rapid inexpensive biomanufacturing of treatments
Thermostable treatments and vaccines
Inexpensive immune enhancement gene therapies
Rapid inexpensive biomanufacturing of vaccines
Especially highly concerning pathogens such as carbapenem-resistant Enterobacterales, carbapenem-resistant Acinetobacter, Clostridioides difficile, and drug-resistant Neisseria gonorrhoeae.
Adaptability and rapid generation of new solutions are key.
Some possible solutions:
Adaptable probiotic treatments
Rational design of phage therapies
Diversified phage therapy production platforms
Lysogenic phage therapies for widespread resistance shutdown and/or bactericide
Sentinel bacteria for detection and elimination of resistance in environment (esp. livestock and wastewater)
Bacterial conjugation for elimination of resistance in environment
Immune enhancement gene therapies
Rapid inexpensive biomanufacturing of vaccines
Rapid vaccine discovery platforms
Inexpensive home diagnostics
Affordable Gene Therapy Manufacturing
Many existing gene therapy delivery vehicles are extremely expensive to produce, so treatments are very costly for patients and not enough can be made to reach large populations.
Novel manufacturing solutions are vital to scalably make existing vectors (esp. AAVs).
New vectors which retain the benefits of existing vectors yet can be made inexpensively are also important.
Some possible solutions:
Synthetic biology methods for radically redesigning cellular platforms of virus production
Novel inexpensively manufacturable viral vectors
Hybrid nanoparticle-viral vectors that are easier to produce yet retain benefits of viruses and perhaps bring extra advantages as well
Methods for production of DNA origami nanorobots
Use of DNA origami to support viral capsid assembly and genome packaging
Gene Therapy for Radiation Resistance in Space
Extended periods of time in space, on the moon, on Mars, etc. expose people to large amounts of radiation.
Future space colonization efforts could be severely jeopardized by human radiation exposure, especially since this can cause problems with reproduction.
Enhance human DNA repair pathways by adding new genetic circuits and/or altering gene regulation
Polygenic gene therapy delivery vectors
Develop ways of safely delivering genes to most or all cells in the human body
Gene Therapy for Aging
Aging affects everyone and is marked by a deterioration of health and eventual death.
Treatments for aging would greatly improve the human condition by making people both much healthier and longer lived.
Life extension only linearly affects population growth, whereas reproduction exponentially affects population growth, so concerns about life extension causing overpopulation are often exaggerated.
As human longevity increases, its small contribution to population growth will likely be mitigated by parallel growth of technologies that improve human sustainability (e.g. vertical farms, cultured meat, renewable energy, space habitation, etc.)
Gene therapy has great potential for extending human longevity and simultaneously improving overall global health.
Some possible genetic approaches:
Polygenic gene therapy delivery vectors
Identify multiple genes that synergistically improve healthspan and deliver them together
Engineer regulatory pathways and insert genetic circuits that optimally modulate gene expression for improving healthspan
Biological Carbon Capture for Climate Change
The climate crisis threatens human and nonhuman life since is leading to desertification, flooding, extreme weather events, ecosystem collapse, etc. and will cause many millions of deaths if it continues unchecked.
Addressing climate change will necessitate both policy solutions and technological solutions.
Carbon capture is a particularly promising route towards mitigating climate change yet is difficult to scale.
Self-replicating biological carbon capture approaches would come with risks but demonstrate much greater scalability.
Some possible biological carbon capture solutions:
Genetically enhanced trees that more rapidly capture carbon
Genetically enhanced cyanobacteria (or algae) that more rapidly capture carbon
Bacteriophages that propagate genes in ocean cyanobacteria to enhance their carbon uptake
Use bacterial conjugation in ocean cyanobacteria to propagate genes that enhance their resistance to bacteriophages and thus rapidly increase cyanobacterial population size and carbon capture capacity
Develop computational models and experimental model systems to explore possible negative side effects of all of the above, find ways to counterbalance those side effects
Strong nanorobots with capabilities resembling those in science fiction would revolutionize every human activity.
Strong nanorobots should specifically possess (1) inexpensive mass producibility or self-replication capabilities, (2) ways of programming them to alter their functionality and environmental responses, (3) automated locomotion oriented towards accomplishing programmed tasks.
Only relatively simple nanorobots have been created so far.
Some possible routes towards strong nanorobotics:
Develop minimal cells derived from Mycoplasma bacteria, equip them with extensive optogenetic equipment for external programming, keep the “programming mode” turned off except when a special chemical switch is flipped to prevent light from scrambling their instructions
Synthesize complex dynamic DNA origami nanostructures that include optically programmable logic systems inspired by digital logic circuits as well as automated locomotion modules, also devise scalable manufacturing methods
Horizontal Gene Drives to Repair Pollinator Insect Networks
Bees and other insect pollinators form a crucial part of global ecosystems, yet populations of these insects are declining.
Decline of insect pollinators is negatively affecting many crops, limiting food production across the world.
Some of the most prominent reasons for bee decline are the spread of invasive Varroa mites that carry deformed wing virus (DWV) and the prevalence of toxic pesticides in the environment.
Horizontal gene transfer via seeding donor bacteria into insect gut microbiota may help protect insect pollinators in a scalable fashion.
Some possible approaches involving horizontal gene drives:
Give bees gut bacteria that spread conjugative plasmids carrying anti-Varroa genes.
Give bees gut bacteria that spread conjugative plasmids carrying anti-DWV genes.
Give bees gut bacteria that spread conjugative plasmids carrying genes that facilitate breakdown of toxic pesticides.
Connectomics Towards Whole-Brain Emulation
Mapping the brain and simulating it in a computer would provide an unparalleled holistic understanding of how our minds work.
Even partial connectomes and/or animal connectomes could give remarkable insights into neurobiology.
The convergence of connectomics and computational neuroscience has applications in bioinspired artificial intelligence, bioinspired robotics, neural prostheses, medicine for brain disease, and more.
Some possible routes for connectomics:
Expansion microscopy with genetically encoded synaptic and neuronal barcodes coupled with spatial transcriptomics
Massively parallel electron microscopy approaches
Improved x-ray nanotomography coupled with special sample treatment methods to improve tissue stability, allow multicolor imaging (and possibly barcodes), and enhance resolution
Construction of many compact light source devices to rapidly map tissue in parallel
Construction of extremely bright next-generation synchrotrons coupled with sample treatment methods to greatly improve stability
Nanobiotechnology for Neural Interfaces and Neural Prostheses
Existing neural interfaces and neural prostheses utilize microelectrode-based technologies for communicating with brain tissue.
Nanobiotechnology approaches may enable more precise, less invasive, and more powerful neural interfaces and prostheses.
Some possible approaches:
Polymersome nanocompartments that mimic neurons in their electrical response properties via embedded transmembrane proteins and ion gradients, link to nanowires that transmit electrical potential to external devices or other parts of the brain
Leverage gene therapy for modifying human neurons to express optogenetic channels, design nanomachines that incorporate upconversion nanoparticles for targeted stimulation
The future of humankind depends on our ability to colonize other planets, moons, etc.
Earth is the only planet in the solar system where humans can survive unaided.
Terraforming the moon or Mars would provide humankind with a new habitable world, yet this represents an enormously challenging task.
Seeding the moon or Mars with heavily engineered microorganisms may provide a first step towards transforming these celestial bodies into habitable worlds.
Some possible microorganism-based terraforming methods:
Perform extensive metabolic engineering on extremophiles that metabolize substances similar to those found on the moon or Mars, make them convert regolith to Earthlike atmospheric gases
Borrow genetic pathways from Deinococcus radiodurans to provide radiation resistance
Extensively engineer microorganisms to metabolize regolith and form seaweed-like colonies that bud into edible fruits
HIV’s genome is a 9.7 kb linear positive-sense ssRNA.1 There is a m7G-cap (specifically the standard eukaryotic m7GpppG as added by the host’s enzymes) at the 5’ end of the genome and a poly-A tail at the 3’ end of the genome.2 The genome also has a 5’-LTR and 3’-LTR (long terminal repeats) that aid its integration into the host genome after reverse transcription, that facilitate HIV genetic regulation, and that play a variety of other important functional roles. In particular, it should be noted that the integrated 5’UTR contains the HIV promoter called U3.3,4
HIV’s genome translates three polyproteins (as well as several accessory proteins). The Gag polyprotein contains the HIV structural proteins. The Gag-Pol polyprotein contains (within its Pol component) the enzymes viral protease, reverse transcriptase, and integrase. The Gag-Pol polyprotein is produced via a –1 ribosomal frameshift at the end of Gag translation. Because of the lower efficiency of this frameshift, Gag-Pol is synthesized 20-fold less frequently than Gag.5 The frameshift’s mechanism depends upon a slippery heptanucleotide sequence UUUUUUA and a downstream RNA secondary structure called the frameshift stimulatory signal (FSS).6 This FSS controls the efficiency of the frameshift process.
The HIV RNA genome undergoes alternative splicing to produce the rest of the viral proteins. One splicing event produces an RNA that separately encodes the Vpu protein and the Env protein (also called gp160).6–8 A mechanism called ribosome shunting is used to transition from Vpu’s open reading frame to Env’s open reading frame. The Env protein contains the gp41 and gp120 proteins. Env is post-translationally cleaved into gp41 and gp120 by a host furin enzyme in the endoplasmic reticulum.9 It is important to note that Env is also heavily glycosylated post-translationally to help HIV evade the immune system. Several other complex splicing events lead to the production of RNAs encoding Tat, Rev, Nef, Vif, and Vpr.
HIV viral protease cleaves the Gag polyprotein and thus produces structural proteins including the capsid protein CA (also called p24), the matrix protein MA (also called p17), the nucleocapsid protein NC (also called p7), and the p6 peptide.10 The HIV core capsid is shaped like a truncated cone and consists of about 1500 CA monomers. Most of the CA proteins assemble into hexamers, but a few pentamers are present. The pentamers help give the core capsid its conical morphology by providing extra curvature near the top and bottom. Each core capsid contains two copies of the HIV genomic RNA, complexed with NC protein. Reverse transcriptase, integrase, and viral accessory proteins are also held within the core capsid. HIV’s core capsid is packaged into a lipid envelope that bears gp41-gp120 glycoprotein heterodimers. The MA protein forms a layer between the core capsid and the envelope.
Accessory proteins Vpu, Tat, Rev, Nef, Vif, and Vpr facilitate a variety of functions. Vpu induces degradation of CD4 proteins within the endoplasmic reticulum of host CD4+ T cells. It does this by using its cytosolic domain as a molecular adaptor between CD4 and a ubiquitin ligase (which subsequently triggers proteosomal degradation of the CD4).11 The reason that Vpu does this is to prevent HIV superinfection wherein two different types of HIV might infect the same cell and interfere with each other. This is an example of competition between viruses.12 Vpu also enhances release of HIV virions from infected cells by using its cytosolic domain to inhibit a host protein called tetherin (also known as BST-2).11 Without Vpu, tetherin would bind the viral envelope to the cell surface as well to other HIV virus particles, impeding release.
Tat, also called the viral transactivator protein, is necessary for efficient transcriptional elongation of the HIV genome after integration into the host DNA.13 Tat binds the viral transactivation response element (TAR), a structured RNA motif present at the beginning of the HIV transcripts. It then recruits protein positive transcription elongation factor b (P-TEFb). This allows P-TEFb to phosphorylate certain residues in the C-terminal domain of RNA polymerase II, stimulating transcriptional elongation. Tat also recruits several of the host cell’s histone acetyltransferases to the viral 5’-LTR so as to open the chromatin around the U3 promoter and related parts of the integrated HIV genome.3,4 Finally, Tat is secreted from infected cells14 and acts as an autocrine and paracrine signaling molecule.4 It inhibits antigen-specific lymphocyte proliferation, stimulates expression of certain cytokines and cytokine receptors, modulates the activities of various host cell types, causes neurotoxicity in the brain, and more.
Rev facilitates nuclear export of the unspliced and singly spliced HIV RNAs by binding to a sequence located in the Env coding region called the Rev response element (RRE).13 The Rev protein forms a dimer upon binding to the RRE and acts as an adaptor, binding a host nuclear export factor called CRM1. Rev is also known to form higher-order oligomers via cooperative multimerization of the RNA-bound dimers.
Nef is a myristoylated protein that downregulates certain host T cell proteins and thereby increases production of virus. Nef is localized to the cytosol and the plasma membrane. It specifically inhibits CD4, Lck, CTLA-4, and Bad.15 Downregulating CD4 contributes to the prevention of superinfection that also occurs with Vpu’s inhibition of CD4. Nef induces endocytosis of plasma membrane Lck protein and traffics it to recycling endosomes and the trans-Golgi network. At these intracellular compartments, Lck signals for Ras and Erk activation, which triggers IL-2 production. IL-2 causes T cells to grow and proliferate, leading to more T cells that HIV can infect and leading to activation of the machinery HIV needs to replicate itself within infected T cells. Nef triggers lysosomal degradation of CTLA-4. This is because CTLA-4 can serve as an off-switch for T cells, which would lead to inhibition of HIV replication if active. Nef inactivates the Bad protein via phosphorylation. Bad participates in apoptotic cascades, so Nef prevents apoptosis of the infected host cell in this way.
Vif forms a complex with the host antiviral proteins APOBEC3F and APOBEC3G and induces their ubiquitination and subsequent degradation by the proteosome.16 It also may inhibit these proteins through other mechanisms. APOBEC3F and APOBEC3G are cytidine deaminases that hypermutate the negative-sense strand of HIV cDNA, leading to weak or nonviable viruses.17 These proteins also interfere with reverse transcription by blocking tRNALys3 from binding to the HIV RNA 5’UTR (tRNALys3 usually acts as a primer to initiate reverse transcription of the HIV genome).18
Vpr facilitates nuclear import of the HIV pre-integration complex.19 The pre-integration complex consists of viral cDNA and associated proteins (uncoating and reverse transcription have already occurred at this stage). Vpr binds the pre-integration complex and recruits host importins to enable nuclear import. It may further enhance nuclear import through interactions with some of the nuclear pore proteins. In addition to nuclear import, Vpr has several more functions: it acts as a coactivator (along with other proteins) of the HIV 5’UTR’s U3 promoter, might influence NF-κB regulation, may modulate apoptotic pathways, and arrests the cell cycle at the G2 stage.
CD4+ T cells represent the primary targets of HIV, though the virus is also capable of infecting other cell types such as dendritic cells.20 HIV infects CD4+ T cells through binding its gp120 glycoprotein to the CD4 receptor and the CCR5 coreceptor or the CXCR4 coreceptor.10 This triggers fusion of the viral envelope with the plasma membrane and allows the core capsid to enter the cytosol.
HIV’s core capsid is transported by motor proteins along microtubules to dock at nuclear pores. The nuclear pore complex has flexible cytosolic filaments composed primarily of the Nup358 protein, which interacts with the core capsid.21 These interactions guide the narrow end of the core capsid into the nuclear pore’s central channel. Next, the core capsid interacts with the central channel’s unstructured phenylalanine-glycine (FG) repeats that exist in a hydrogel-like liquid phase. As the core capsid translocates through the central pore, it binds the Nup153 protein, a component of the nuclear pore complex’s basket. Finally, many copies of the nucleoplasmic CPSF6 protein coat the core capsid and escort it towards its genomic site of integration. It is thought that the reverse transcription process begins inside of the core capsid at this point, leading to cDNA synthesis.21,22 Buildup of newly made cDNA within the core capsid likely results in pressure that helps rupture the capsid structure, facilitating uncoating.
Tetrameric HIV integrase binds both of the viral LTRs and facilitates integration of the cDNA into the host genome.23 Though integration sites vary widely, they are not entirely random. Host chromatin structure and other factors influence where the viral cDNA integrates.24 Transcription of HIV RNAs can then proceed from the U3 promoter with the aid of the Tat protein and host factors. As described earlier, a series of RNA splicing events produce the various RNAs necessary to synthesize all of the different HIV proteins and polyproteins.
Env protein is trafficked to the cell membrane through the secretory pathway. It is cleaved by a host furin enzyme into gp41 and gp160 components during its time in the endoplasmic reticulum.9 Gag and Gag-Pol polyproteins are expressed cytosolically. Since Gag is post-translationally modified by amino-terminal myristoylation, it anchors to the cell membrane by inserting its myristate tail into the lipid bilayer.25 Gag and a smaller number of Gag-Pol accumulate on the inner membrane surface and incorporate gp41-gp160 complexes. NC domains in the Gag proteins bind and help package the two copies of HIV genomic RNA. The p6 region of the Gag protein (located at the C-terminal end) then recruits host ESCRT-I and ALIX proteins, which subsequently sequester host ESCRT-III and VPS4 complexes to drive budding and membrane scission, releasing virus into the extracellular space. After this, the HIV viral protease (from within the Gag-Pol polyprotein) cleaves the Gag and Gag-Pol polyproteins into their constituent proteins, facilitating maturation of the released HIV particles.
Genome and Structure:
The SARS-CoV-2 genome consists of about 30 kb of linear positive-sense ssRNA. There is a m7G-cap (specifically m7GpppA1) at the 5’ end of the genome and a 30-60 nucleotide poly-A tail at the 3’ end of the genome. These protective structures minimize exonuclease degradation.26 The genome also has a 5’ UTR and a 3’ UTR which contain sequences that aid in transcriptional regulation and in packaging. The SARS-CoV-2 genome directly translates two partially overlapping polyproteins, ORF1a and ORF1b. There is a –1 ribosomal frameshift in ORF1b relative to ORF1a. Within the polyproteins, two self-activating proteases (Papain-like protease PLpro and 3-chymotrypsin-like protease 3CLpro) perform cleavage events that lead to the generation of the virus’s 16 non-structural proteins (nsps). It should be noted that the 3CLpro is also known as the main protease or Mpro. The coronavirus also produces 4 structural proteins, but these are not translated until after the synthesis of corresponding subgenomic RNAs via the viral replication complex. To create these subgenomic RNAs, negative-sense RNA must first be made and then undergo conversion back to positive-sense RNA for translation. Genes encoding the structural proteins are located downstream of the ORF1b section.
SARS-CoV-2’s four structural proteins include the N, E, M, and S proteins. Many copies of the N (nucleocapsid) protein bind the RNA genome and organize it into a helical ribonucelocapsid complex. The complex undergoes packaging into the viral envelope during coronavirus budding. Interactions between the N protein and the other structural proteins may facilitate this packaging process. The N protein also inhibits host immune responses by antagonizing viral suppressor RNAi and by blocking the signaling of interferon production pathways.27
The transmembrane E (envelope) protein forms pentamers and plays a key but poorly understood role in the budding of viral envelopes into the endoplasmic reticulum Golgi intermediate compartment (ERGIC).28–30 Despite its importance in budding, mature viral particles do not incorporate very many E proteins into their envelopes. One of the posttranslational modifications of the E protein is palmitoylation. This aids subcellular trafficking and interactions with membranes. E protein pentamers also act as ion channels that alter membrane potential.31,32 This may lead to inflammasome activation, a contributing factor to cytokine storm induction.
The M (membrane) protein is the most abundant protein in the virion and drives global curvature in the ERGIC membrane to facilitate budding.30,33 It forms transmembrane dimers that likely oligomerize to induce this curvature.34 The M protein also has a cytosolic (and later intravirion) globular domain that likely interacts with the other structural proteins. M protein dimers also induce local curvature through preferential interactions with phosphatidylserine and phosphatidylinositol lipids.29,30 M proteins help sequester S proteins into the envelopes of budding viruses.35
The S (spike) protein of SARS-CoV-2 has been heavily studied due to its central roles in the infectivity and immunogenicity of the coronavirus. It forms a homotrimer that protrudes from the viral envelope and is heavily glycosylated. It binds the host’s ACE2 receptor (angiotensin-converting enzyme 2 receptor) and undergoes conformational changes to promote viral fusion.36 The S protein undergoes cleavage into S1 and S2 subunits by the host’s furin protease during viral maturation.37,38 This enhances SARS-CoV-2 entry into lung cells and may partially explain the virus’s high degree of transmissibility. The S1 fragment contains the receptor binding domain (RBD) and associated machinery while the S2 fragment facilitates fusion. Prior to cellular infection, most S proteins exist in a closed prefusion conformation where the RBDs of each monomer are hidden most of the time.39 After the S protein binds ACE2 during transient exposure of one of its RBDs, the other two RBDs quickly bind as well. This binding triggers a conformational change in the S protein that loosens the structure, unleashing the S2 fusion component and exposing another proteolytic cleavage site called S2’. Host transmembrane proteases such as TMPRSS2 cut at S2’, causing the full activation of the S2 fusion subunit and the dramatic elongation of the S protein into the postfusion conformation. This results in the viral envelope fusing with the host membrane and uptake of the coronavirus’s RNA into the cell.
The 16 nsps of SARS-CoV-2 play a variety of roles. For instance, nsp1 shuts down host cell translation by plugging the mRNA entry channel of the ribosome, inhibiting the host cell’s immune responses and maximizing viral production.40,41 Viral proteins still undergo translation because a conserved sequence in the coronavirus RNA helps circumvent the blockage through a poorly understood mechanism. The nsp5 protein is the protease 3CLpro.42 The nsp3 protein contains several subcomponents, including the protease PLpro. The nsp12, nsp7, and nsp8 proteins come together to form the RNA-dependent RNA polymerase (RdRp) that replicates the viral genome.42,43 The nsp2 protein is likely a topoisomerase which functions in RNA replication. The nsp4 and nsp6 proteins as well as certain subcomponents of nsp3 restructure intracellular host membranes into double-membrane vesicles (DMVs) which compartmentalize viral replication.44
Beyond the 4 structural proteins and 16 nsps of SARS-CoV-2, the coronaviral genome also encodes some poorly understood accessory proteins including ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF9b.45 These accessory proteins are non-essential for replication in vitro, but they are thought to be required for the virus’s full degree of virulence in vivo.
As mentioned, SARS-CoV-2 infects cells by first binding a S protein RBD to the ACE2 receptor. This triggers a conformational change that elongates the S protein’s structure and reveals the S2 fusion fragment, facilitating fusion of the virion envelope with the host cell membrane.39 Cleavage of the S’ site by proteases like TMPRSS2 aid this change from the prefusion to postfusion configurations. Alternatively, SARS-CoV-2 can enter the cell by binding to ACE2, undergoing endocytosis, and fusing with the endosome to release its genome (as induced by endosomal cathepsin proteases).45 After release of the SARS-CoV-2 genome into the cytosol, the N protein disassociates and allows translation of ORF1a and ORF1b, producing polyproteins which are cleaved into mature proteins by the PLpro and 3CLpro proteases as discussed earlier.
The RdRp complex synthesizes negative-sense full genomic RNAs as well as negative-sense subgenomic RNAs. In the latter case, discontinuous transcription is employed, a process by which the RdRp jumps over certain sections of the RNA and initiates transcription separately from the rest of the genome.46 The negative-sense RNAs are subsequently converted back into positive-sense full genomic RNAs and positive-sense subgenomic RNAs. The subgenomic RNAs are translated to make structural proteins and some accessory proteins.45
As described earlier, the nsp4, nsp6, and parts of nsp3 proteins remodel host endoplasmic reticulum (ER) to create DMVs.45 These DMVs are the site of the coronaviral genomic replication and serve to shield the viral RNA and RdRp complex from cellular innate immune factors. DMVs cluster together and are continuous with the ER mostly through small tubular connections. After replication, the newly synthesized coronavirus RNAs undergo export into the cytosol through molecular pore complexes that span both membranes of the DMVs.47 These molecular pore complexes are composed of nsp3 domains and possibly other viral and/or host proteins.
Newly replicated SARS-CoV-2 genomic RNAs complex with N proteins to form helical nucleocapsids. To enable packaging, the nucleocapsids interact with M protein cytosolic domains which protrude at the ERGIC.48 M proteins, E proteins, and S proteins are all localized to the ERGIC membrane. The highly abundant M proteins induce curvature of the membrane to facilitate budding. As mentioned, E proteins also play essential roles in budding, but the mechanisms are poorly understood. Once the virions have budded into the ERGIC, they are shuttled through the Golgi via a series of vesicles and eventually secreted out of the cell.
Adeno-associated virus (AAV)
Genome and Structure:
AAV genomes are about 4.7 kb in length and are composed of ssDNA. Inverted terminal repeats (ITRs) form hairpin structures at ends of the genome. These ITR structures are important for AAV genomic packaging and replication. Rep genes (encoded via overlapping reading frames) include Rep78, Rep68, Rep52, Rep40.49 These proteins facilitate replication of the viral genome. As a Dependoparvovirus, additional helper functions from adenovirus (or certain other viruses) are needed for AAVs to replicate.
AAV capsids are about 25 nm in diameter. Cap genes include VP1, VP2, VP3 and are transcribed from overlapping reading frames.50 The VP3 protein is the smallest capsid protein. The VP2 protein is the same as VP3 except that it includes an N-terminal extension with a nuclear localization sequence. The VP1 protein is the same as VP2 except that it includes a further N-terminal extension encoding a phospholipase A2 (PLA2) that facilitates endosomal escape during infection. In the AAV capsid, VP1, VP2, and VP3 are present at a ratio of roughly 1:1:10. It should be noted that this ratio is actually the average of a distribution, not a fixed number.
Frame-shifted start codons in the Cap gene region transcribe AAP (assembly activating protein) and MAAP (membrane associated accessory protein). These proteins help facilitate packaging and other aspects of the AAV life cycle.
There are a variety of different AAV serotypes (AAV2, AAV6, AAV9, etc.) that selectively infect certain tissue types. AAVs bind to host cell receptors and are internalized by endocytosis. The particular receptors involved can vary depending on the AAV serotype, though some receptors are consistent across many serotypes. Internalization occurs most often via clathrin-coated pits, but some AAVs are internalized by other routes such as macropinocytosis or the CLIC/GEEC tubulovesicular pathway.51
After endocytosis, conformational changes in the AAV capsid lead to exposure of the PLA2 VP1 domain, which facilitates endosomal escape. The AAV is then transported to the nucleus mainly by motor proteins on cytoskeletal highways. It enters via nuclear pores and finishes uncoating its genome.
AAV genomes initiate replication using the ends of their ITR hairpins as primers. This leads to a series of complex steps involving strand displacement and nicking.49 In the end, new copies of the AAV genome are synthesized. The Rep proteins are key players in this process. It is important to realize that AAVs can only replicate in cells which have also been infected by adenovirus or similar helper viruses (this is why they are called “adeno-associated viruses”). Adenoviruses provide helper genes encoding proteins (e.g. E4, E2a, VA) that are vital for the successful completion of the AAV life cycle. After new AAV capsids have assembled from VP1, VP2, and VP3 and once AAV genomes have been replicated, the ssDNA genomes are threaded into the capsids via pores at their five-fold vertices.
AAVs are nonpathogenic, though a large fraction of people possess antibodies against at least some serotypes, so exposure to them is fairly common.
Genome and Structure:
Adenovirus genomes are about 36 kb in size and are composed of linear dsDNA. They possess inverted terminal repeats (ITRs) which help facilitate replication and other functions. These genomes contain a variety of transcriptional units which are expressed at different times during the virus’s life cycle.52 E1A, E1B, E2A, E2B, E3, and E4 transcriptional units are expressed early during cellular infection. Their proteins are involved in DNA replication, transcriptional regulation, and suppression of host immune responses. The L1, L2, L3, L4, and L5 transcriptional units are expressed later in the life cycle. Their products include most of the capsid proteins as well as other proteins involved in packaging and assembly. Each transcriptional unit can produce multiple mRNAs through the host’s alternative splicing machinery.
The capsid of the adenovirus is about 90 nm in diameter and consists of three major proteins (hexon, penton, and fiber proteins) as well as a variety of minor proteins and core proteins. Hexon trimer is the most abundant protein in the capsid, the pentameric pentons occur at the vertices, and trimeric fibers are positioned on top of the pentons.53 The fibers point outwards from the capsid and end in knob domains which bind to cellular receptors. In Ad5, a commonly studied type of adenovirus, the fiber knob primarily binds to the coxsackievirus and adenovirus receptor (CAR). That said, it should be noted that Ad5’s fiber knob can also bind to alternative receptors such as vascular cell adhesion molecule 1 and heparan sulfate proteoglycans.
Minor capsid proteins include pIX, pIIIa, pVI, and pVIII. The pIX protein interlaces between hexons and helps stabilize the capsid. Though pIX is positioned in the crevices between the hexons, it is still exposed to the outside environment. By contrast, the pIIIa, pVI, and pVIII proteins bind to the inside of the capsid and contribute further structural stabilization. When the adenovirus is inside of the acidic endosome during infection, conformational changes in the capsid release the pVI protein, which facilitates endosomal escape through membrane lytic activity.
Adenovirus core proteins include pV, pVII, protein μ (also known as pX), adenovirus proteinase (AVP), pIVa2, and terminal protein (TP).54 The pVII protein has many positively-charged arginine residues and so functions to condense the viral DNA. The pV protein bridges the core with the capsid through interactions with pVII and with pVI. AVP cleaves various adenoviral proteins (pIIIa, TP, pVI, pVII, pVIII, pX) to convert them to their mature forms.55 The pIVa2 and pX proteins interact with the viral DNA and may play roles in packaging or replication. TP binds to the ends of the genome and is essential for localizing the viral DNA in the nucleus and for viral replication.
Adenovirus infects cells by binding its fiber knob to cellular receptors such as CAR (in the case of Ad5). The penton then binds certain αv integrins, positioning the viral capsid for endocytosis.56 When the endosome acidifies, the adenovirus capsid partially disassembles, fibers and pentons fall away, and pVI is released.57 The pVI protein’s membrane lytic activity facilitates endosomal escape. Partially disassembled capsids then undergo dynein-mediated transport along microtubules and dock at the entrance to nuclear pores. The capsids further disassemble and releases DNA through the nuclear pore. This DNA remains complexed with pVII after it enters the nucleus.
Adenoviral transcription is initiated by the E1A protein, inducing expression of early genes.58 This subsequently leads to expression of the E2, E3, and E4 transcriptional units, which help the virus escape immune responses. This cascade leads to expression of the L1, L2, L3, L4, and L5 transcriptional units, which mainly synthesize viral structural proteins and facilitate capsid assembly.
In the nucleus, adenovirus genomes replicate within dense complexes of protein that can be seen as spots via fluorescence microscopy. Replication begins at the ITRs and is primed by TP.59 Several more viral proteins and host proteins also aid the initiation of replication. Nontemplate strands are displaced during replication but may reanneal and act as template strands later. Adenovirus DNA binding protein and adenovirus DNA polymerase play important roles in replication. Once the genome has been replicated, TP undergoes cleavage into its mature form, signaling for packaging of new genomes.
The adenoviral capsid assembly and maturation process occurs in the nucleus.58 Once enough assembled adenoviruses have accumulated, they rupture the nuclear membrane using adenoviral death protein and subsequently lyse the cell, releasing adenoviral particles.
Herpes Simplex Virus 1 (HSV-1)
Genome and Structure:
HSV-1 genomes are about 150 kb in size and are composed of linear dsDNA. These genomes include a unique long (UL) region and a unique short (US) region.60 The UL and US regions are both flanked by their own inverted repeats. The terminal inverted repeats are called TRL and TRS while the internal inverted repeats are called IRL and IRS. HSV-1 contains approximately 80 genes, though the complexity of its genomic organization makes an exact number of genes difficult to obtain. As with many other viruses, HSV-1 genomes encode early, middle, and late genes. The early genes activate and regulate transcription of the middle and late genes. Middle genes facilitate genome replication and late genes mostly encode structural proteins.
The diameter of HSV-1 ranges around 155 nm to 240 nm.61 Its virions include an inner icosahedral capsid (with a 125 nm diameter) surrounded by tegument proteins which are in turn enveloped by a lipid membrane containing glycoproteins.
HSV-1’s icosahedral capsid consists of a variety of proteins. Some of the most important capsid proteins are encoded by the UL19, UL18, UL38, UL6, UL17, and UL25 genes.62 The UL19 gene encodes the major capsid protein VP5, which forms pentamers and hexamers for the capsid. These VP5 pentamers and hexamers are glued together by triplexes consisting of two copies of VP23 (encoded by UL18) and one copy of VP19C (encoded by UL38).63 The UL6 gene encodes the protein that makes up the portal complex, a structure used by HSV-1 to release its DNA during infection. Each HSV-1 capsid has a single portal (composed of 12 copies of the portal protein) located at one of the vertices. UL17 and UL25 encode additional structural proteins that stabilize the capsid by binding on top of the other vertices. These two proteins also serve as a bridge between the capsid core and the tegument proteins.
The tegument of HSV-1 contains dozens of distinct proteins. Some examples include pUL36, pUL37, pUL7, and pUL51 proteins. The major tegument proteins are pUL36 and pUL37. The pUL36 protein binds on top of the UL17-UL25 complexes at the capsid’s vertices.64 The pUL37 protein subsequently associates with pUL36. The pUL51 protein associates with cytoplasmic membranes in infected cells and recruits the pUL7 protein.65 This pUL51-pUL7 interaction is important for HSV-1 assembly. HSV-1 has many more tegument proteins which play various functional roles.
HSV-1’s envelope contains up to 16 unique glycoproteins. Four of these glycoproteins (gB, gD, gH, and gL) are essential for viral entry into cells.66 The gD glycoprotein first binds to one of its cellular receptors (nectin-1, herpesvirus entry mediator or HVEM, or 3-O-sulfated heparan sulfate). This binding event triggers a conformational change in gD that allows it to activate the gH/gL heterodimer. Next, gH/gL activate gB which induces fusion of HSV-1’s envelope with the cell membrane. Though the remaining 12 envelope glycoproteins are poorly understood, it is thought that they also play roles that influence cellular tropism and entry.
After binding to cellular receptors via its glycoproteins, HSV-1 induces fusion of its envelope with the host cell membrane.67 The capsid is trafficked to nuclear pores via microtubules. Since the capsid is too large to pass through a nuclear pore directly, the virus instead ejects its DNA through the pore via the portal complex.68
HSV-1 replicates its genome and assembles its capsids in the nucleus. But the assembled capsids are again too large to exist the nucleus through nuclear pores. To overcome this issue, HSV-1 first buds via the inner nuclear membrane into the perinuclear cleft (the space between nuclear membranes), acquiring a primary envelope.67 This process is driven by a pair of proteins (pUL34 and pUL31) which together form the nuclear egress complex. Next, the primary envelope fuses with the outer nuclear membrane, releasing the assembled capsids into the cytosol.
To acquire its final envelope, the HSV-1 capsid likely buds into the trans-Golgi network or into certain tubular vesicular organelles.69 These membrane sources contain the envelope proteins of the virus as produced by transcription and various secretory pathways. One player is the pUL51 tegument protein that starts associated with the membrane into which the virus buds. The interaction between pUL51 and pUL7 helps facilitate recruitment of the capsid to the membrane. (Capsid envelopment is also coupled in many other ways to formation of the outer tegument). The enveloped virion eventually undergoes trafficking through the secretory system and eventually is packaged into exosomes that fuse with the cell membrane and release completed virions into the extracellular environment.
In humans, HSV-1 infects the epithelial cells first and produces viral particles.70 It subsequently enters the termini of sensory neurons, undergoes retrograde transport into the brain, and remains in the central nervous system in a dormant state. During periods of stress in the host, the virus is reactivated and undergoes anterograde transport to infect epithelial cells once again.
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