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First steps in the development of small-scale 3D printed hydrogel bioreactors for protein production in space travel
By Maya Mysore, Laura Ballou, Anna Rita Moukarzel, Alex Cherry, David Duronslet, Lisette Werba, Nathan Tran, Hannah Mosheim, Stephen Curry, Simon Coelho
Advisors: Kantharakorn Macharoen, Matthew McNulty, Andrew Yao, and Dr. McDonald, Dr. Nandi, and Dr. Facciotti
Author’s Note: My name is Maya Mysore, and I am a team lead on the BioInnovation Group’s Plant Bioprinter project. The BioInnovation Group is a student organization that creates research and leadership opportunities for undergraduates. The Bioprinter project is one of these opportunities.
I joined the BioInnovation group (BIG) in the winter quarter of 2019, as a freshman looking for ways to get involved on campus. I knew I liked research; I had been working in another lab. However, I was looking to explore different aspects of research. I heard about BIG through some friends in my major and went to an information session. There, I tried to join the more tech-based microfluidics project; however, my previous lab experience with cell culture convinced the lead for the Bioprinter project to get me involved in their work. I spent the next couple quarters investigating how to trap viruses in hydrogel. In Fall 2019, I was offered the role of lead. I was shocked, surprised, and a little out of my depth– after all, I had practically joined the project by accident! But I took on the role, excited about the leadership opportunity and the freedom. Now over a year into being project lead, I am planning to transition into the organization’s leadership. However, as a swan song to my time in charge, I wanted to compile all the hard work those involved with the project have accomplished. This paper is a celebration of the work of tens of student researchers over a period of several years. Hopefully, this paper will be the first of many for the Bioprinter project and the BioInnovation Group.
Abstract
As human space exploration expands to include potential settlement on the Moon and Mars, the ability to build shelter, manufacture food, produce medicine, and create other necessities in space will become increasingly important. Currently, the high cost and size constraints of sending payloads into space challenges us to think beyond the traditional manufacturing and agricultural tool-kit. Engineers have proposed that additive manufacturing, particularly 3D printing, is a solution to lower the payload costs and to enable the manufacturing of a variety of products in situ. This study focuses on 3D printing engineered biological cells for the production of biologics (e.g. pharmaceuticals that are living or derived from a biological source). We describe in-progress work to design, build, and test a small and affordable 3D bioprinter capable of printing 3D structured hydrogels that can carry living cells. We provide a general overview of the project, our progress in converting a low-cost and compact 3D printer from printing plastics to printing hydrogels, and preliminary work testing the compatibility of bioink formulations with genetically engineered rice cells that produce and secrete the enzyme butyrylcholinesterase.
Background
As humans continue to explore space and potentially settle in distant locations such as the Moon, Mars, and beyond, it will become increasingly necessary to build shelter, create food, and develop medicine while in space. However, the major costs (roughly $20,000/kg) and size constraints of sending payloads into space create challenges for such long-duration space travel beyond low Earth orbit [1-4]. Challenges include the manufacturing of food, shelter, and even medicine. 3D printing has been proposed as a cost-effective method for addressing some of these challenges, as it might allow the opportunity to ship only the printer to remote sites and to source the majority of the printing materials from the settlement location [5].
Biological systems may also play a large role in this approach. Microorganisms have been envisioned to help construct habitats through biocementation, a process that uses microorganisms to solidify inorganic matter into 3D structures [6-8]. Plants and microbes together are proposed as possible tools for the creation of sustainable ecosystems that recycle and detoxify waste and produce food [9-11]. A purported advantage of biological systems is that they can self-replicate, as each organism carries the full set of genetic instructions to create copies of itself. This means that biological systems could be delivered as light-weight “seeds”, i.e. self-replicating units that can be shipped in small and light quantities and grown to larger quantities upon permanent settlement at remote bases.
We and others envision that the 3D printing of engineered living systems (e.g bioprinting) may prove useful for the manufacturing of biologicals; this includes pharmaceuticals of or derived from a biological source [12]. In this context, the engineered living system serves as an on-demand expandable factory for the production of the biological while the 3D printer serves to produce custom-made culturing and purification hardware that can be produced in the geometries required for specific cells and production sizes. We were interested in exploring this concept and better understanding the challenges associated with the proposed process of drug production through bioprinting. In order to do this, we needed a bioprinter. Depending on their feature sets, commercial bioprinters can cost anywhere between $10,000 and $200,000, which was well outside our budget. Therefore, as a first step, we sought to design, build, and test a low-cost and compact bioprinter that we could later customize and use to explore novel design ideas.
FExisting modalities of bioprinting were considered and four main existing modalities of 3D bioprinting were considered: inkjet, pressure-assisted, laser-assisted, and stereolithography. For a detailed review on this subject, see Li et. al [13]. The major factors that were considered in the selection of a printer were types of usable bioinks, potential for good cell viability, cost, and complexity of the system (e.g. ease with which it can be modified). Inkjet-based bioprinting uses computer controls to drop small drops of bioink onto a surface. This type of printing maintains high short-tem cell viability and is widely available at low cost. However, it is limited in printing materials and creates high thermal and mechanical stress on cells which risks damage to cells and may affect long-term viability. Pressure-assisted bioprinters extrude bioink continuously onto a surface. While the extrusion process is slower and can lower cell viabilities immediately after printing (ranging from 40-80%, compared to 90% for inkjet printing), it allows use of a greater variety of materials and incorporates cells directly into the bioink. Laser-assisted bioprinters use a laser to irradiate a bioink such that the droplets adhere to the desired surface. This method of bioprinting is very precise and results in the highest cell viability; however, it is the most expensive, time-consuming, and has the highest risk of metal contamination. Finally, stereolithography printing uses illumination of a light-sensitive polymer to solidify 3D shapes. This method is fast, cost-effective, and has high final cell viabilities, but it is primarily limited by the need for a light-sensitive bioink, many of which are not biocompatible.
We chose to build a pressure-assisted bioprinter primarily due to practical factors: (a) the availability of low cost and compact fused deposition modeling (FDM) printers that could be used as chassis, theoretically enabling a “simple” swap of printing nozzles and pumps while taking advantage of the existing build platforms and 3D control systems; (b) the easy access to safe and low cost of compatible bioinks, and (c) the ability to incorporate cells directly into the bioink for prototyping.
This paper describes the progress of our project in developing a functional bioprinter. In addition, we describe the chemical assays used to evaluate engineered rice cell viability within hydrogels and these cells’ cell’s ability in gels to produce the pharmacologically-relevant enzyme Butrylcholinesterase (BChE), which is a complex human serine hydrolase enzyme that provides protection against organophosphorus poisoning from toxic agents such as sarin.
Figure 1. This diagram demonstrates the model methodology for seeding the cells into the hydrogel, printing out the cell-gel complex, and extracting the protein of interest from this system.
Methods
Printer selection, modification and testing
Selection of chassis
We sought to find a low-cost and compact FDM printer system that could be reasonably modified to extrude bioink rather than plastic filament. We ultimately selected the Monoprice MP Select Mini 3D Printer V2 because of its high availability, low cost ($250), and relative ease of modification. An accurate open source 3D computer-aided design (CAD) model (https://www.thingiverse.com/thing:2681912) of this printer was already available, making it easier to design new features for this specific unit.
Construction of an bioink extruder
To start converting the 3D plastic printer into a bioprinter, the printer’s original extrusion mechanism was replaced with a standard syringe/syringe-pump mechanism typical of bioprinters [14].
Incorporating the syringe-based bioink extruder required the design and construction of the entire extrusion system. An interchangeable mount was designed to hold the 10 mL syringe on the printer access, as seen in Figures 2b and 2c. In Figure 2b, the interchangeable mount design is shown with a trapezoidal connection piece, allowing the mount to swap between holding the 3D printer plastic extruder and the bioprinter syringe extruder system.
Figure 2. a) Inside of the 3D printer after all electrical components and panels were removed b) 3D printed interchangeable mount used to exchange the plastic extruder and the syringe extruder. c) The hydraulic extrusion system as connected to the bioprinter d) The hydraulic extrusion tubing system
The 10 mL syringe was connected to a hydraulic pumping system through a plastic tube. The hydraulic system is controlled using a Nema 17 Bipolar 40 mm Stepper Motor connected to an 8 mm threaded rod, forming a linear actuation mechanism. Connected to the rod is a 60 mL syringe plunger which is pushed through a 60 mL syringe. A liquid is placed in the 60 mL syringe and the bioink is placed in the 10 mL syringe also with a plunger sitting on top of the syringe. When the motor turns on, this liquid is pushed from the 60 mL syringe through the tubing and into the 10 mL syringe. This system pushes the plunger through the 10 mL syringe and extrudes the bioink onto the printing surface.
A T fitting made from 6 mm brass tubes was attached to the middle of the tubing system in order to remove air bubbles from the tube, as shown in Figure 2d.
Integration of hydraulic motors with chassis
To power the motor for the syringe extruder, the electrical components needed to be rebuilt. With this in mind, an Arduino Mega 2560 was connected with the HiLetGo RAMPS 1.4 control panel and the A4988 stepper motor driver boards using the wiring setup diagrammed in Figure 3.
Figure 3. This diagram shows the wiring for the 3D printer using the Arduino.
The Z-axis switch was then repositioned and mounted to the printer chassis directly under the print head, as seen in Figure 2c.
Firmware
For the firmware, Marlin was selected because it is open sourced and easily modified with the Arduino IDE. After the firmware and electronics were set up, a G code file was needed to determine the print pattern. Cura was used to develop the file due to its compatibility with the Monoprice 3D printer. The Cura profile used with the bioprinter tests followed a cylindrical shape with a square-shaped infill grid. With this information established, the Cura profile was exported as G Code. In the printer design, an SD card is required to flash the firmware and upload the G code to the bioprinter. With the firmware and G code loaded onto the SD card, the bioprinter could be set up to run test prints with the bioink. The final cost spent to make the bioprinter came out to $375. Further information on the process of building the bioprinter can be found at https://www.instructables.com/Low-Cost-Bioprinter/.
Hydrogels
Hydrogels are porous water-based polymers that have many valuable uses, especially in fields such as drug delivery and tissue engineering. Here, we use hydrogels for their ability to selectively trap materials on a size basis, as this is what allows us to trap cells and release the protein of interest. Our hydrogel protocol was adapted from Seidel et al., 2017. Briefly, the hydrogel mixture contained agarose (0.2275% w/v), alginate (2.52% w/v), methyl cellulose (3% w/v), and sucrose (3% w/v). Agarose, alginate, and sucrose were mixed into deionized water at room temperature until dissolved. This mixture and the methyl cellulose powder were then autoclaved in separate containers for 20 minutes at 121 C. Upon completion of the autoclave cycle, methyl cellulose was mixed into the gel. The mixture was then left for 12 to 24 hours in a 2-8 C fridge to allow swelling to occur [15]. After this, the gel was ready to be seeded.
Seeding and Crosslinking the Gels
Transgenic rice cells were supplied by the McDonald-Nandi lab. The cells were genetically modified with the addition of a human BChE gene optimized for rice cell compatibility and cloned into the RAmy3D expression system for transformation into A. tumefaciens to allow insertion into rice cells [16]. This allowed the engineered cells to produce the pharmacologically-relevant BChE protein. The provided cell suspensions were mixed thoroughly via pipetting to obtain even distribution of cells. This suspension was then added directly to the hydrogel in a 50% volume split of cell suspension and gel and gently mixed to distribute cells evenly. To crosslink the gels and create solid structures for later use, a 0.1 M calcium chloride solution was prepared. The hydrogel was loaded into a syringe and deposited into weight boats containing enough CaCl2 solution to half-cover the extruded hydrogel. The hydrogel would then cure in the solution for at least 5 minutes or until the shape solidified. Upon completion of curing, the hydrogel could be removed and used for experiments.
Tetrazolium Chloride Viability Assay on Hydrogels
The TTC (2,3,5-triphenyltetrazolium chloride) assay is a method for testing cell viability. TTC is turned red from a colorless solution in the presence of metabolizing cells, allowing for quantification of cell viability. When used with defined standards and run on a spectrometer, it can be used to monitor cell survival over time.
Preparation of the TTC solution involved mixing 0.4% w/v TTC in 0.05 M sodium phosphate buffer, pH 7.5. Once the TTC solution was prepared, the TTC assay was performed.
5-6 mL of 0.05 M sodium phosphate buffer was added to a 15 mL Falcon tube with cured gel to submerge the cured gel entirely. The gel remained in the solution for 15 minutes. Then the Ellman buffer was removed from the tube and 500 μL of TTC were added to the tube with gel while mixing slightly. This tube was stored in a dark area for 24 hours.
If the gel was not cured, roughly 5 mL of gelled cells were first centrifuged in a 15 mL conical tube at 4500 g for 20 minutes. The supernatant was removed and 1 mL of Ellman buffer was added and mixed. The sample was centrifuged again at 4500 g for 15 minutes, the supernatant was removed, and 500 μL of TTC solution were added to the gel-cell mix. This sample was stored for 24 hours in a dark area.
After the 24 hours period ended, the sample-TTC mix was centrifuged at 4500 g for 15min. The supernatant was removed and the gel-cell mix was washed with 1 mL deionized water. The mixture was re-centrifuged at 4500 g for 10 minutes. The supernatant was removed again and 1 mL of 95% ethanol was added to the gel-cell mix. The sample was transferred to a microcentrifuge tube and placed in a 60C water bath for approximately 10 minutes. The sample is then centrifuged at 21.1 g for 15 minutes to recover the final supernatant. The supernatant was then run on a colorimeter or Tecan and the absorbance value was read at 485 nm. Beer’s law was then used to determine concentration from this value.
Seeded Cell-Ellman BChE Concentration Assay
The Ellman assay was used to measure BChE concentration for a sample at a given time point. This assay uses the kinetics of a color changing reaction to quantify the amount of BChE in solution. When in the presence of specific substrates, BChE turns a colorless solution yellow; the peak rate of this reaction can be determined and used to calculate BChE mass in a sample.
After cells were seeded into a hydrogel complex with a disc shape approximately 7 cm in diameter and 1 cm thick, the complex was suspended in 40 mL sucrose-free nutrient broth (NB-S).
The flask was then covered with a cloth filter and placed in the shaking incubator (37C, 5% CO2, 80 rpm). 50 μL media samples were collected from the flask daily over 14 days and the Ellman assay was run directly following collection of each of these samples.
The Ellman assay protocol was based on the Cerasoli lab protocol, which was adapted from Ellman et al., 1961 [17]. To perform the Ellman assay, a 20 mm stock solution of 5, 5’ – dithiobis-(2–nitrobenzoic acid) (DTNB) was prepared. A 75mM stock solution of S-Butyrylthiocholine (BTCh) iodide was also prepared.
Immediately prior to performing the Ellman assay, the Ellman substrate was prepared. 60 μL of DTNB and 30 μL of BTCh were added to the phosphate buffer in the falcon tube. The tube was temporarily stored in ice with light protection.
Then the Ellman assay was performed. In a 96-well plate, 50 μL of sample containing BChE was were diluted into 0.1 M phosphate buffer, pH 7.4, to ensure the generated? outputted slope readings (mOD/min) would fall in the range of 200-1000 when read for 3-5 minutes at 25 C. This dilution was done by estimating the approximate BChE concentration and estimating the mOD/min based on the expected value. 150 μL of Ellman substrate was added to each sample containing well. The optical density of the sample was immediately read at a wavelength of 405 nm for a total of 300 s (5 min) after the measurement was started.
After collecting data from the assay, Beer’s law was used to determine the concentration of product formed. From that value, we could estimate the mass of functional BChE in the total volume of the sample collected [18].
Results
TTC-Gel compatibility
To measure in-gel cell viability, we evaluated the use of the tetrazolium chloride (TTC) assay. This assay measures metabolic activity in live cells by reducing tetrazolium chloride to red formazan through the process of cell metabolism. Effectively, it provides an indication of how well the cells survive over time. Our team modified the assay for use in gels by including extra Ellman buffer and centrifugation steps to provide more opportunity for cells in the gel to be washed.
Figure 4. This figure shows the results of the TTC assay run on the transgenic rice cells in suspension. The leftmost tube is a positive control showing the TTC assay done on cell aggregates in suspension (i.e. without gel) that have been centrifuged into a pellet after the assay was performed. The middle and rightmost tubes are cells suspended in a hydrogel; the TTC assay was performed on this combination of cells in gels. In each tube, the cells have been stained red from the assay, indicating the presence of metabolic activity. These samples can go on to be washed and suspended in ethanol to obtain a viability data value.
To qualitatively assess how different factors like cell distribution and crosslinking might influence the results of the TTC assay, we performed additional variations of the assay. We first visually examined whether cell homogeneity was impacted by the gel. Then, we performed the TTC assay on E. coli cells alone as a positive control. After that, we tested the effects of non-crosslinked and crosslinked gel to ensure neither condition would prevent the use of the assay. E. coli was used for these tests due to our group’s ability to access it more regularly and grow it more easily than the genetically modified rice cells from Dr. McDonald’s lab. All of these tests together allowed us to determine that cell survival could indeed be measured within the gel, allowing us to monitor culture health over time. This will be critical in future use of the model, allowing us to determine ways to improve cell health and protein output by providing a metric for us to test against.
Homogeneous mixing of biological sample
To determine later TTC accuracy, the first key issue to address was homogeneity of cells in a hydrogel. This would determine whether sectioned off samples of cell-gel complexes would be representative of a whole sample. To ensure that the gel mixing protocol yielded a homogeneous suspension of the cells, we first tested our procedure by mixing E. coli expressing a transgenic green fluorescent protein (GFP) and imaged the suspension under UV light. We expect E. coli to distribute homogeneously in the gel similarly to the transgenic rice cells. This mixture was observed (Figure 5a) and confirmed by visual inspection of a homogeneous mix.
Test of TTC assay with bacterial suspension
To ensure that the TTC assay in later tests would be effective with E. coli, we first tested the TTC assay on an E. coli suspension as a positive control for later tests. We ran the modified TTC assay protocol described in Methods, and observed a color change in the solution. The resultant red solution (Figure 5b) matches the literature expectations for the output of this assay on living cells and indicates the assay is effective for E. coli.
Test of TTC assay with bacteria seeded in hydrogel
After confirming the TTC assay was effective with E. coli, it became important to determine how the presence of gel would affect the assay. We suspended the E. coli cells in the hydrogel and ran the modified TTC assay. The results seen in Figure 4c show the suspension turning red, which visually indicates the presence of cell metabolic activity and the effectiveness of the TTC assay.
Test of TTC assay with bacteria seeded in a crosslinked hydrogel
Upon determining the gel did not qualitatively affect the output of the TTC assay, it became necessary to determine whether crosslinking the gel had any effect on the effectiveness of the TTC assay. We reran the same experiment as the non-crosslinking hydrogel experiment, with the only change being the crosslinking process and the different first wash step. We found that the result of the TTC assay appears to be unaffected by the presence of the crosslinked out layer, as the solution turns red in the same way it does for the positive control and the non-crosslinked gel (Figure 5d).
These experiments allowed us to qualitatively determine whether the TTC assay could be an effective measure of cell viability. They also demonstrated that the introduction of a crosslinked hydrogel will not have visible impacts on measuring cell viability.
Figure 5. Qualitative TTC assays were run on E. coli with the pMax plasmid to test homogeneity within the gel and the effectiveness of the TTC assay in different hydrogel conditions. 5a shows the bacteria mixed homogeneously within the hydrogel, which is visible in the fluorescence that is present homogeneously through the sample. 5b shows the ethanol suspension output for a TTC assay run on a pMax E. coli culture, providing a control for later experiments and showing that the TTC assay is effective for E. coli. The left image is the control and the right image is the test condition. The control is run in the same conditions as the test, except the cells are placed in a 60C water bath for ten minutes prior to adding TTC in order to kill them. 5c shows the output prior to ethanol suspension for a TTC assay on E. coli pMax cells that were suspended in an uncured hydrogel. The left tube is the control and the right tube is the experimental condition. The red color visible in the right tube shows that the presence of the hydrogel does not prevent use of the TTC assay. 5d shows the ethanol suspension output for a TTC assay run on E. coli pMax cells that had been suspended in a cured hydrogel. The left image is the control and the right image is the experimental condition. The red color of the suspension indicates the TTC assay remained effective even with the addition of the crosslinked outer layer of the gel. Throughout this figure, variation in intensity of the redness of the samples is related to variations in time spent in suspension of the TTC solution, with redder samples correlating to longer time.
Initial Attempts at Measuring BChE Production
Our second major goal was to determine whether BChE could be collected from our model system (as seen in figure 1). This would allow us to determine if our model system was an effective way to collect our protein of interest for future space travel applications, as well as confirm that our test for BChE quantity would be effective in this system. To test this, our team ran the seeded cell-Ellman assay as described in methods to assess the amount of BChE that was escaping into the media. We first prepared a hydrogel, mixed the transgenic rice cells in, and cured it into a disc shape roughly 7 cm in diameter and 1 cm in height. We then suspended this cured cell-gel complex in NB-S media to stimulate BChE production, and we kept this mix in a spinning incubator to ensure aeration and adequate diffusion of materials in and out of the gel. Media samples were collected over the course of 14 days and were run with the Ellman assay for BChE detection on a spectrometer. The Ellman assay uses the enzyme kinetics of a color-changing reaction between BChE and a substrate to quantify the amount of BChE present in a sample at a given time point.
It is important to note that this test was intended as a trial run of the system in order to ensure that the assay works and that useful data is being collected. In addition, we sought to assess if BChE could escape from the gel at all. Therefore, no negative control was run and only one run of data was collected (shown in the figure below.) As a result, we cannot conclusively state anything about the data. However, the data does show a trend worth noting for future experimentation. The This is that active BChE concentration in the media increased for the first roughly 100 hours, after which the values dropped off. At the time point marked in figure 5, 96 hours, we see the maximal BChE present. If the unusually low value seen at the roughly 120 hour time point is considered erroneous (which we suspect), the data suggests increased production of BChE over the first 4 days of culture followed by a slow decay thereafter with production ending at around day 8. This provides an early quantitative estimate of the time-dependence of BChE production in this model system. This experiment is a first attempt and will be repeated with various parameter variations in the future.
Figure 6. This plot shows the approximate active concentration of BChE released into the media for various time points over 14 days. Each sample was a 50 μL amount of media pulled from the small scale system model. This figure shows a burst in production of active BChE until the 96 hour time point (denoted with a dashed red line), after which the values drop off. The data point at t=120 hours is most likely an outlier resulting from this data being for one set of samples from one test condition.
Preliminary Bioprinter Testing
The process of building and testing the bioprinter was done in parallel with the TTC and Ellman assay testing. Detailed bioprinter testing has not been performed; however, initial testing of the printer showed its ability to print hydrogel into pre-programmed patterns. The grid pattern seen in Figure 7 was printed into a petri dish containing CaCl2 curing solution. The print shows excess hydrogel accumulation near the edges where the printhead briefly paused and reversed direction. In the center of the print, the lines in the grid averaged 1.25mm +/- 0.4 in width. Further testing and refinement is currently in process.
Figure 7. This shows a test print from our modified 3D printer using the hydrogel described in the methods section and cured in standard CaCl2 curing solution. This structure is described as a lattice shape and will be the primary pattern for future prints.
Discussion
In these experiments we determined that the TTC assay was effective in hydrogels, the Ellman assay showed the ability of protein to be detected from solution, and the bioprinter was able to create the desired lattice shape for later use.
Printer Performance
Our experiments to date have demonstrated our ability to convert a low-cost and compact FDM printer into a preliminarily functional bioprinter. The conversion of the original chassis required the modification of the printhead support, the development of a syringe-based hydraulic pump, and the modification of electronic and software control systems. Preliminary prints indicate that the printer can successfully deposit a programmed pattern with feature sizes in the range of 1.5mm. Existing conventional commercial bioprinters can achieve resolutions of 100-200µm, (some even claim filament diameters as low as 3µm), suggesting that we have room to improve the resolution of our system [19]. In addition to improving the resolution of the prints, we want to explore alternate methods for delivering the CaCl2 curing solution during alginate filament deposition to minimize user interaction and allow complete processing inside a biosafety cabinet; this should allow us to increase sterility during printing and print quality.
Cell Viability
Since it is known that pressure-assisted printing may negatively impact cell viability during printing, a key concern was the resulting cell viability of the system. As a result, our general goal for this phase of the project was to test whether a pressure-assisted bioprinter system could maintain cell viability after extrusion. We adapted the TTC assay for this purpose and tested our protocol to determine the effect of bioink and extrusion on cell viability under conditions mimicking those experienced during bioprinting.
Generally, the TTC assay demonstrated the ability of the assay to cellular viability in the crosslinked hydrogel, despite the unknown nature of how crosslinking affects pore size. Despite this success, the TTC assay remains largely qualitative as it is challenging to get quantitative measurements of cell viability when cells are embedded in a gel. This is further complicated by factors like the heterogeneous distribution of cells (or cellular aggregates) in the gel (see figure 4, rightmost sample). If homogeneity is not maintained, we need to design assays that take into account heterogeneous distribution of cells in the gel. In future experiments, we seek to determine whether samples from a large complex of cells in a hydrogel will provide a representative sample.
In later experiments, additional key variables that may potentially affect viability will be tested. These variables include media composition, culture duration, environmental conditions such as temperature, gel architecture, and the additional variables associated with the printer extrusion process (e.g. pressure, needle pore diameter, etc.). Determining how these specific factors affect viability will allow us to modify the printer design to minimize the drop in cell viability upon extrusion.
Protein production
Having confirmed the effectiveness of the TTC assay in the hydrogel, we moved forward to analyzing BChE production and its diffusion into the media. The assay we adopted allowed us to develop a standard method for data collection that can be used to analyze how various factors impact the cells’ ability to produce BChE. Figure 6, for example, shows that we can measure BChE production and diffusion out of the gel, and that under our preliminary experimental conditions, production peaks at 96 hours and then falls over the next 150 hours. While encouraging, this experiment needs to be repeated with many more samples and replicates to obtain a more reliable assessment of measurement error associated with the assay. Despite needing to replicate the experiment, we are confident that this preliminary experiment answered the core question of whether such a large protein – 85 kDa monomers and 4 units in quaternary form, with a total size of 574 monomers [20] – can effectively diffuse out of the hydrogel and avoid denaturation long enough to be collected and purified.
In addition to replication, future experiments should be explored to further improve protein escape from the hydrogel. These tests could increase the mixing speed to use centrifugal force to free proteins from the gel, increase pore size to create more physical space for protein escape, or print the 3-dimensional lattice structure to increase surface area and allow greater escape. Other relevant variables whose impact on BChE production should be tested include media composition and media changing schedules, culture duration, environmental conditions, gel architecture, and growth temperature. In our initial experiments, plant cells were grown in a shaking incubator at 37C to mimic the environment of protein production in mammalian hosts. However, this growth condition may have stressed the plant cells for which growth at 27C is more typical [16, 21]. This may explain the trend shown in figure 6, where die-off occurs after 96 hours.
Finally, in our current studies, the presence of sucrose in gel formulation (which inhibits BChE production) may have adversely impacted the amount of protein produced. While we expected that overlaying a relatively large volume of sucrose-free media would effectively dilute the sucrose to low levels, the presence of sucrose in the initial formulation could have nevertheless impacted the cells’ initial states and therefore protein production. A followup experiment that more stringently controls for the presence of sucrose in the gel than in the studies described above seems warranted.
Conclusion
In this work, we successfully modified an off-the-shelf pressure-assisted 3D printer into a working bioprinter. In addition, we established that BChE producing rice cells are biocompatible with the different bioink gel formulations and that our assays for testing cell viability and protein production are effective when analyzing the cells within the gel. Having shown that we can print gel, assess cell survival, produce BChE, and quantify its abundance, we next seek to optimize both printer function and the measurement assays for cell viability and protein concentration in ways that provide more quantitative data and more refined control over printed structures. Eventually, we expect that such advances will allow us to optimize protein production itself and ultimately develop a bioprinter suitable for protein production during space travel or in other remote locations.
Acknowledgements
Thank you to the Molecular Prototyping and BioInnovation Lab for the lab space, the BioInnovation Group for the administrative, scientific, safety, and monetary support, the McDonald-Nandi lab for materials and mentorship, and all past, present and future members of the Bioprinter team for contributing to these experiments.
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- Lopez JV, Peixoto RS, Rosado AS. 2019. Inevitable future: space colonization beyond Earth with microbes first. FEMS Microbiol Ecology [Internet]. 95(10). doi.org/10.1093/femsec/fiz127
- Bornemann G, Waßer K, Tonat T, Moeller R, Bohmeier M, Hauslage J. 2015. Natural microbial populations in a water-based biowaste management system for space life support. Life Sci Space Res [Internet]. 7:39–52. doi.org/10.1016/j.lssr.2015.09.002
- Bokulich NA, Lewis ZT., Boundy-Mills K, Mills DA. 2016. A new perspective on microbial landscapes within food production. Curr Opin Biotechnol [Internet]. 37:182–189. doi.org/10.1016/j.copbio.2015.12.008
- Varma A, Gemeda HB, McNulty MJ, McDonald KA, Nandi S, Knipe JM. 2021. Bioprinting transgenic plant cells for production of a recombinant biodefense agent. BioRxiv [Internet]. 2021.02.01.429263. doi.org/10.1101/2021.02.01.429263
- Li J, Chen M, Fan X, Zhou H. 2016. Recent advances in bioprinting techniques: approaches, applications and future prospects. J Transl Med [Internet]. 14:271. doi.org/10.1186/s12967-016-1028-0
- Pusch K, Hinton TJ, Feinberg AW. 2018. Large volume syringe pump extruder for desktop 3D printers. HardwareX [Internet]. 3:49–61. doi.org/10.1016/j.ohx.2018.02.001
- Seidel J, Ahlfeld T, Adolph M, Kümmritz S, Steingroewer J Krujatz F, … Lode A. 2017. Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds. Biofabrication [Internet]. 9(4):045011. doi.org/10.1088/1758-5090/aa8854
- Corbin JM, Hashimoto BI, Karuppanan K, Kyser ZR, Wu L, Roberts BA, … Nandi S. 2016. Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures. Front Plant Sci [Internet]. 7:412. doi.org/10.3389/fpls.2016.00412
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Through War and Peace, These Doves Rock
By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21
“The diversity of the breeds is something astonishing,” Charles Darwin wrote in “On the Origin of Species.” He was not referring to his famous Galápagos finches. Instead, Darwin opened his foundational work by commenting on various breeds of the domestic pigeon, all descended from a common ancestor: Columba livia. Widely known as the rock dove, this species has adapted to urban environments throughout human history. Over time, we have kept pigeons for fairs, racing, message carrying in wartime, and even scientific research.
Since joining the B3 Lab at UC Davis in 2020, I have contributed to research on this model organism. The B3 name, short for Birds, Brains, and Banter, represents the lab’s main goals: to study rock doves and how stress affects their reproductive behaviors, and to advance culturally relevant science communication research and training. In April, I presented a project on how single parenting affects the amygdala, often considered the brain’s “emotional center,” at the UC Davis Undergraduate Research, Scholarship, and Creative Activities Conference. This research helps us to understand the impacts of single parenting in humans, and it could lead to insights that mitigate stresses felt by single parents and their children.
These photographs were captured in the B3 aviary via iPhone 7 camera during 2020 and 2021.
Lazarus Dies, Lazarus Lives Again
By Jesse Kireyev, History ‘21
Each of these photos captures a landscape in slow degradation. Berryessa, for all the wintergreen beauty that it holds, has experienced horrifying fires numerous times over the past few years. The natural bridge that dominates the landscape of its namesake park in Santa Cruz now remains alone, at risk of collapsing like its sibling did, forever leaving the shoreline empty of its beauty. This risk only grows as sea levels rise and as human interaction puts it at greater risk. The salt flats of the Dead Sea used to be covered in water — now nature struggles to fill the few remaining pools as the sea rapidly shrinks. Captured in these three horizons are the struggles of nature to sustain itself despite the present beauty. For all the tranquility of the Ansel Adams-esque lines jutting forth from the foreground, a great and slow war is playing itself out in the back, often hidden to the gazing eye of the unaware viewer. The horizons both serve as a reminder of the danger that lurks in our future, as well as the distant (and perhaps unreachable) hope of resurrection in the face of annihilation.
1. Berryessa Foothills, Solano, California.
Storm clouds move over the fields and lush wetlands, both morphing into the mountains hugging Lake Berryessa. Just a few months prior, the mountains had been scorched by the dizzying flames of the LNU Lightning Complex Fire, a fire whose smoke blotted out the sun for weeks in two of the largest metropolitan areas in America. The ebb-and-flow of the surroundings give us a stark reminder of just how fast a place can be destroyed and can flourish once again from the ashes. Canon EOS 5D Mark III. April, 2021.
2. West Cliff, Natural Bridges State Park, Santa Cruz, California.
Pelicans and seagulls huddle together as they hunt for fish and fight the buffeting winds. The remainders of the natural bridges, which once dominated the state beach, still serve as a helpful vantage point for the seabirds. Locals hope that this vantage point can survive, even as climate change puts the bridge at greater risk every year. Canon EOS 630, Kodak Tri-X 400TX 35mm film. June, 2017.
3. Dead Sea Salt Flats, Masada, Israel.
The salt flats are all that is left of the once sea-filled expanse below Masada. A combination of climate change and human changes to the environment are driving the evaporation of the Dead Sea, which at current rates is expected to be gone in the next three decades. Sony a700. December, 2018.
Review of recent progress in development of genetically encoded calcium indicators in imaging neural activity
By Lia Freed-Doerr, Cognitive Science, Neurobiology, Physiology & Behavior ‘22
Author’s Note: In fall quarter, I got into contact with the Tian lab in the Department of Biochemistry and Molecular Medicine in order to learn more about optogenetic techniques and the difficulties of in vivo sensing of neural dynamics and, with the mentorship of a postdoctoral researcher, I have learned more about different high-resolution sensors (or indicators) and expanded my interests to genetically encoded sensors of cellular dynamics. As I began learning about various types of imaging sensors, calcium ion (Ca2+) indicators, in particular, stuck out to me due to their variety and depth of development. As I am unable to take part in in-person projects due to COVID restrictions, to ensure my understanding of the topics I was reading about, I began to write this review.
Abstract
Methods of performing neuroscience research have progressed remarkably in recent years, providing answers to many different types of questions. Genetically encoded indicators (sensors) are of particular interest for use in answering questions about neural circuits, cell specific populations, and single cell dynamics. These indicators modulate their fluorescence in different cellular environments and allow for optical observations. Of the various cellular activities that can be measured by genetically encoded indicators, the dynamics of calcium ions (Ca2+) are of interest due to their fundamental importance in neuronal signaling. In this review, we introduce the basic underlying features of genetically encoded calcium indicators (GECIs) including characteristics of fluorescence, an overview of GECI engineering, and a brief discussion of some common variants of GECIs and their uses.
Introduction
Modern neuroscientists have found many ways to analyze the information-carrying neuronal circuits and dynamics within the brain. The continued development of genetically encoded optical indicators, specifically Ca2+ indicators, is particularly promising for analyses of single neurons or neural circuits. Optical fluorescence imaging allows large populations of neurons to be examined simultaneously and avoids major damage to the cells of interest [1]. In particular, measuring the dynamics of Ca2+ can be useful in inferring spiking activity in neurons, as Ca2+ is involved in neuronal action potentials. In this review, we will introduce the basic workings of genetically encoded sensors beginning with a ground-up introduction of fluorescence measurements and the process of engineering genetically encoded sensors. Several Ca2+ indicators will be briefly discussed in order to examine recent progress and how this can impact future studies of the brain.
Mechanics of Fluorescence Indicators
Fluorescence imaging is a valuable tool for visualizing populations of cells. It is relatively non-invasive; but, in order to use optical tools to study the cortex, some surgical procedures must still be performed. A cranial window might be installed in the animal to shine light through; alternatively, an endoscope or fiber optic cable could be installed at the desired depth within brain tissue [2].
Fluorescent proteins (FPs) internally form a barrel-like structure containing the chromophore (also known as the fluorophore), which is a trio of amino acids responsible for the protein’s fluorescence. The chromophore is autocatalytically formed as a post-translational modification, requiring just atmospheric oxygen. Genetically encoded indicators rely on a change in the chromophore environment within the labeling FP. Fluorescence is observed when light of an appropriate wavelength excites the chromophore’s electrons, which then results in the emission of a lower energy photon as the excited electrons return to a lower energy state. FPs are often connected to sensing domains, which induce the change in the chromophore environment after detecting the event of interest (e.g., Ca2+ binding for GECIs). Any number of cellular activities may induce a conformational change such as changes in pH or the binding of a ligand. Sensors can have one or two FPs with partially overlapping fluorescence spectra. Single FP-based sensors are generally preferred as indicators; the green fluorescent protein (GFP), cloned from the jellyfish Aequorea Victoria, is the most commonly used FP for single FP sensors [3]. In systems with two FPs, a Förster or fluorescence resonance energy transfer (FRET) occurs. FRET involves an energy transfer from the higher energy (more blue-shifted) donor FP to the lower energy (more red-shifted) acceptor FP. Genetically manipulating FP systems by circularly permuting FPs (fusing the original termini of the FP and introducing a new opening closer to the chromophore) can improve their performance in sensors by making the chromophore more accessible to the outside environment and, thus, more susceptible to environmental changes [4]. FPs like GFPs are also typically oligomeric in their natural environment (i.e. multiple copies stick together); but, in order to help prevent breakdown and allow for better combination with sensing domains in indicators, FPs must also be mutated to become monomeric [5].
Figure 1: A Jablonski diagram that visualizes an electron’s excitation to a higher energy level by absorption of a photon and subsequent fluorescence emission with energy decay.
To image fluorescent systems, we can use fluorescence microscopy with one or multiple photons (Fig. 2) [2]. In one-photon systems, the fluorophore absorbs energy from a light source and is excited by a single photon. Some energy is lost non-radiatively (without light) resulting in the emittance of lower energy, visible photons from the fluorophore. One-photon systems are relatively inexpensive and fast but can only penetrate tissue to a shallow depth. In contrast, multi-photon microscopy shows more promise for in vivo imaging because of its reduced out of focus emission, light scattering, and phototoxicity. The combination of the energy of multiple photons is required for excitation in such systems.
Figure 2: A diagram outlining the setup for a standard fluorescence microscopy experiment.
Genetic encoding of sensors
To introduce indicator genes into a system, methods like in utero electroporation or viral vectors can be used [6, 7]. DNA promoters or localization sequences can be used to target specific subtypes of neurons in organisms to produce transgenic animals. Transgenic animal genomes that have been modified by artificial bacterial chromosomes, CRISPR, or effector nucleases, and are particularly useful when longitudinal and intensive sampling is required. Genetic changes can be maintained throughout an animal’s lifespan and lines of transgenic animals can be bred for further testing [2]. A recombinase system administered via viral vector, like the popular Cre/loxP system, can be used to achieve high specificity [6]. In the Cre/loxP system, the loxP sequences are placed at specific target sites of genomic DNA. The Cre-recombinase protein can then target loxP sequences to modify the genetic sequence. Two mouse lines, one carrying the gene of interest flanked by loxP sequences, and the other line expressing Cre-recombinase, can be bred to produce mice expressing the gene of interest. The Cre Driver mouse line expressing Cre-recombinase can be designed to only express the gene under certain conditions. To apply Cre/loxP to genetically encoded indicator systems, a viral vector injects the indicator genes into the brain cells of a Cre Driver mouse. The indicator is only expressed where the Cre-recombinase is active. Expression would continue through one animal’s lifetime; to create a line of mice that express the desired indicator, other methods must be used [6]. Through recombinase methods, the development of transgenic animal lines is an area of active improvement.
There are several advantages to genetically encoding indicators over other methods of imaging. There are a wide variety of neuronal events that can be observed by constructing indicators from proteins that respond to cellular events, including changes in neurotransmitter concentrations, transmembrane voltage, Ca2+ dynamics, and pH [1]. Genetic encoding also allows for selective sampling of cells based on genotype. Selective sampling is not possible with chemical dyes, nor is the viewing of the evolution of neuronal dynamics during learning or development processes [8, 1]. Similar to chemical dyes, genetically encoded indicators allow for the imaging of brain activity in neurons in vitro and in animals [9]. Neurons have the machinery implanted within them to automatically report cellular dynamics of interest.
There are several different broad classes of genetically encoded indicators that are based on the dynamics of the action potential [7]. Genetically encoded voltage indicators (GEVIs) operate based on the membrane depolarization that occurs during action potentials. Other indicators, like pH and neurotransmitter sensors, detect vesicular release. Genetically encoded pH sensors (GEPIs) react to the decrease in acidity as vesicles fuse with the membrane, and genetically encoded transmitter indicators (GETIs) are used to visualize the release of neurotransmitters into the synapse [1]. Genetically encoded calcium indicators (GECIs) operate based on the rise in cytosolic Ca2+ during an action potential; however, they do not directly measure spiking activity. When an action potential occurs, Ca2+ floods into the cell. Ca2+ influx is important because calcium ions are crucial for the release of neurotransmitters from vesicles, which then go on to produce signals in other neurons. More mild calcium ion dynamics are always present in neurons, even in a resting state. Among these various classes, GECIs have been perhaps some of the most developed of these indicators and, thus, some of the most promising.
Engineering genetically-encoded calcium indicators
Performance Criteria
As GECIs are engineered, many performance criteria must be considered. Tradeoffs often occur between the various important qualities of an indicator’s performance [10]. As we optimize the indicator to produce a desired result in one criterion, another criterion often decreases in quality. Thus, development of sensors optimized for specific applications is continuous. Some of these criteria are affinity, sensitivity, kinetics, localization, and photophysical characteristics.
Affinity, represented as the dissociation constant Kd, describes what percentage of the indicator is unbound given a particular concentration of ligand.
Specificity refers to the indicator system’s ability to respond only to the target of interest, as opposed to perhaps similar molecules.
Sensitivity is usually represented by ΔF/F0, the fractional fluorescence change, which is the fluorescence signal change over a change in concentration of the target molecule. It can also be represented by signal-to-noise ratio (SNR), the relative difference between the signal of interest and background noise.
Kinetics is the rate of change in fluorescence intensity of the indicator in response to the change in ligand concentration. There tends to be a tradeoff between affinity and kinetics [8].
Photophysical qualities like brightness, photostability, and photoswitching behaviors are also important considerations. In general, brighter or more intensely emitting indicators are desired. Photostability is inversely proportional to the rate of photobleaching (the damaging of the FP so that it becomes unable to fluoresce). Additionally, some indicators have broader ranges of excitation than others, or may change their intensity or sensitivity in different light conditions, which would limit usage.
GECIs are some of the most widely used genetically encoded indicators in vivo because of their relatively high SNR and improved properties like brightness, photostability, and dynamic range [2]. However, there are still numerous obstacles to be faced in designing GECIS, and only certain variants have faced success in vivo.
Engineering GECIs
Genetically encoded indicators generally are composed of an analyte-binding (sensing domain) and a fluorescent protein (reporting domain), though there are additional peptide complexes that assist in changing the conformation of the system [2]. Upon the occurrence of a sensing event, the sensing domain undergoes a conformational change which, in turn, induces a conformational change in the FP, resulting in fluorescent activity. Engineers of GECIs use two different strategies for constructing reporting domains: FRET-based indicators and single FP-based indicators [7]. When Ca2+ binds to FRET-based indicators, the spatial relationship between the donor and acceptor FPs changes so that there is a transfer of energy from the donor FP to the acceptor FP [2]. One family of indicators, Cameleon, has had some success. In this family, the sensing and peptide complex is located between two FPs with overlapping spectra. FRET-based indicators’ SNR tends to be lower, meaning it is harder to isolate the activity of a neuron from background noise. Because of these drawbacks, we mostly examine the engineering of the more commonly used single FP-based GECIs.
There are two popular designs among developers of single FP GECIs [8]. One is based on one of the earliest lines of calcium indicators, GCaMP. GCaMP consists of a circularly permuted green fluorescent protein (cpGFP) inserted between the Ca2+-binding protein, calmodulin (CaM), and another peptide called RS20, which binds CaM upon Ca2+ binding. When CaM binds Ca2+, a conformational change is induced in the cpGFP and the sensor fluoresces [10, 11]. Another recent design, the NTnC family of indicators, inserts a calcium-binding domain into a split FP [8]. Unlike GCaMP-type indicators, NTnC indicators display an inverted fluorescence response upon calcium binding (i.e., fluorescence decreases upon Ca2+ binding). They are less optimized than GCaMP variants, but it is hypothesized that their lesser Ca2+ binding capacity would interfere less with normal calcium dynamics.
Figure 3: A basic representation of the GCaMP structure.
There have been efforts to expand the color variants of GECIs. In particular, there has been much effort to develop red-fluorescing GECIs because longer red wavelengths reduce phototoxicity and have better tissue penetration [12]. However, there have been many obstacles to producing red-fluorescing GECIs. Unlike GFP, inserting calcium binding domains into red fluorescent proteins (RFPs) disrupts folding and chromophore maturation [8]. A more popular design choice is to replace the GFP in a GCaMP-style indicator with an RFP and optimize the sensor for a new FP [1].
GECIs are improved iteratively through directed evolution and linker optimization between the cpFP and the sensing domains. Site-directed mutagenesis can be used to mutate specific locations to produce novel variants. In the development of one variant of GCaMP, mutations were specifically introduced in the calcium-binding domain-cpGFP linker in a GCaMP5 scaffold to increase sensitivity [11]. Using directed evolution, mutations are randomly introduced. Then, upon testing for desired effects, the variants that produced the best results may be preserved and propagated. This process may repeat many times, producing increasingly successful indicators as the best-performing mutations survive across generations.
Challenges
Genetically encoding indicators, as a rule, comes with challenges. If we choose to use viral infection as our genetic encoding scheme, consideration must be taken to the many viral serotypes, which have varying levels of efficiency and can be toxic. Furthermore, in utero electroporation can be unpredictable, and transgenic animals may not express indicators at sufficiently high levels to be useful [1].
Sensors may affect the natural dynamics of their measured systems, affecting accuracy of results. GECIs, particularly GCaMP-based designs, may interfere with regular Ca2+ dynamics and gene expression [8]. This interference is likely due to interactions between the calcium-binding sensing domain with native proteins and the lack of availability of calcium once bound to the indicators. There have been efforts to improve and modify the calcium-binding domain so that it can bind fewer Ca2+ or otherwise improve affinity so that the indicator operates at lower concentrations of calcium.
There is also difficulty in using these indicators in vivo [2]. Especially in the mammalian brain, the SNR is highly decreased due to the amount of background noise. This reduced SNR is putting aside the level of breakdown that naturally happens in vivo vs. conditioned, cultured environments. Although many indicators have improved structural integrity in vivo, there are many that still cannot be used in living organisms.
Progress in GFP-based GECIs
There has been much development in the GCaMP series as variants are continuously improved by site-directed mutagenesis and computational design efforts [2]. The jGCaMP7 series, built from the GCaMP-6 series, provides a good example of optimization of indicators for different purposes: jGCaMP7f is optimized for fast kinetics, jGCaMP7s is optimized for high sensitivity (though it has slower kinetics), and jGCaMP7b is optimized to have a brighter baseline fluorescence [11]. All of these indicators are based on the same base scaffold but differ drastically in performance because of just a few mutations in the CaM-binding peptide, the GFP, the CaM domain, or the linkers between domains.
Progress in RFP-based GECIs
RFP-based GECIs have important advantages over GFP-based ones. Beyond the importance of color variety in tracking distinct populations of cells at once, red GECIs are also promising for reducing phototoxicity and allowing deeper imaging [12]. There are many promising RFP-based GECIs being developed, though they are generally dimmer than GFP-based indicators and may display photobleaching behaviors under blue light [1]. In particular, there are R-GECO1 variants like jRGECO1a, the RCaMP series, and, perhaps most promising, K-GECO1 [12]. There are three widely used RFPs from which red GECIs are developed; each red indicator family was generated from different RFPs. K-GECO1 has shown particular promise as it works at a distinct spectral range, allowing researchers to simultaneously work with other indicators in multicolor imaging experiments, and it also shows minimal fluorescent noise [9].
Designs of red GECIs often expand on the GCaMP design–for example, K-GECO1 follows a similar design of sandwiching the circularly permuted FP between the Ca2+-sensing domain, CaM, and a CaM-binding peptide [12]. Switching the GFP in GCaMP with an RFP comes with engineering challenges of linker optimization and preventing the breakdown of the sensor. The increased penetrative depth of red GECIs has been used to image subcortical areas like the hippocampus or medial prefrontal cortex relatively noninvasively, demonstrating the applicability of GECIs in neuroscience research [13].
There are other FP-based GECIs in development, but of particular interest is the development of near-infrared GECIs, whose spectral distinction from other indicators would help prevent photoswitching when used with optogenetic tools [8, 14].
Uses and Applications
The applications of GECIs are varied and powerful. The use of genetically encoded indicators allows for the analysis of cells of a specific type or subpopulation as they select for specific genetic qualities. The first transgenic mouse line expressing GCaMP2 in the cerebellar cortex was generated in 2007 and has allowed for characterization of certain synapses [6]. GECIs have been used to provide single-cell resolution to the decades-long study of various topographic maps in the brain and to track the communication of neural circuits [2]. In rats, GECIs have been used to monitor neural population behavior during motor learning tasks and observe the response of cells to sensory deprivation in the primary visual cortex after retinal lesion. They allow examination of ensemble and single cell-scale neural events at more and more temporally precise levels. Broadly, and perhaps more importantly, they are often used in conjunction with optogenetic and other experimental methods that allow for the inference of causation. In using these indicators, the stimulation techniques used in optogenetic experiments can also involve precise tracking of calcium or other dynamics in cells of interest [8]. These experimental approaches have caused excitement as they allow for the examination of behaviors of cells or whole organisms upon physical stimulation of even just single cells. The continued expansion of these approaches is promising.
Conclusion
Many researchers are devoted to developing new and distinct calcium indicators based on existing indicator series. With more GECIs than ever available to neuroscientists, there is some challenge in choosing which is best suited to the exploration of a particular question. With the continuing development of mouse lines and methods of genetically encoding more potent indicators with high temporal resolution, GECIs will continue to be an increasingly important tool within the neuroscientist’s toolkit that allows for population or single-cell imaging with greater resolution than ever before.
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COVID-19 Cover Art Gallery
This year, for the first time, The Aggie Transcript accepted submissions for our journal’s cover from the wider undergraduate community at UC Davis. To celebrate the release of our fifth annual print edition, we present three of the submissions that we received in this art gallery. The winning submission appears first, followed by our honorable mentions. We sincerely thank the authors and artists who submitted to our journal this year for sharing their work with us.
Unprecedented: The Science of COVID-19
By Mario Rodriguez, Wildlife, Fish & Conservation Biology, Design ‘22
This work was created with the intention of highlighting those in the medical profession and the timeline of the COVID-19 pandemic, from bottom to top: the death of loved ones, the medication and hospitalization of patients, and then the development of vaccines to combat the virus. The digital artwork was created on an iPad in the digital painting app Procreate.
Working Together to Catch Covid
By Daria Beniakoff, Biochemistry & Molecular Biology ‘21
Last year, the COVID-19 pandemic affected everyone. The hands of so many people from every direction were put to work figuring out what the virus was and how to mitigate and fight its effects, from the doctors treating patients to the scientists trying to develop treatments and vaccines to the everyday people who had to work around the circumstances. I wanted this digital medium piece to reflect the collaborative effort to contain and stop the pandemic.
Light of Hope
By Bianca Law, Design ‘23
COVID-19 survivors can retrain their smell to enjoy food and wine again
By Daniel Erenstein, Neurobiology, Physiology & Behavior ‘21
Author’s Note: Last spring, I enrolled in the inaugural offering of the University Writing Program’s wine writing course. Our instructor, Dr. Alison Bright, encouraged us to report on topics of personal interest through our news stories on the wine industry, viticulture, enology, and more. In this article, which was prepared for an audience of general science enthusiasts, I examine how biologists are making sense of a puzzling COVID-19 symptom — anosmia, or loss of smell — and what COVID-19 patients with this condition can do to overcome it. Eighteen months into this pandemic, scientists continue to study cases of COVID-19-related anosmia with dreams of a treatment on the horizon. I hope that readers feel inspired by this article to follow this in-progress scientific story. I extend my appreciation to Dr. Bright, who throughout the quarter shared approaches to rhetorical awareness that elevated my grasp of effective writing.
Image caption: Anton Ego, the “Grim Eater” from PIXAR’s Ratatouille, is reminded of his childhood by Remy’s rendition of ratatouille, a Provençal dish of stewed vegetables.
With a single bite of Remy’s latest culinary creation, the eyes of Anton Ego, a notoriously harsh food critic, dilate, and Ratatouille’s viewers are transported back in time with Monsieur Ego. The meal — a simple yet elegant serving of ratatouille, accompanied by a glass of 1947 Château Cheval Blanc — has triggered a flashback to one singular moment, a home-cooked meal during his childhood. The universal charm of this enduring scene resonates; in Ego’s eyes, many recognize how our senses of smell and taste can impact a culinary experience.
Imagine how a real-life version of this scene might change for the millions of COVID-19 patients who have lost their sense of smell [1]. Anosmia, the phenomenon of smell loss, has become one of the more perplexing COVID-19 symptoms since first observed in patients during the earliest months of the pandemic [2].
What happens when we lose our sense of smell? During the pandemic, scientists have studied smell loss, which affects more than 85 percent of COVID-19 patients according to research published this year in the Journal of Internal Medicine [3]. In fact, anosmia is so common in COVID-19 patients that physicians were offered guidance for testing olfactory function as an indicator of infection last year [4].
To simplify studies of these complicated senses, taste and smell are often examined independently of one another, even though these senses are usually experienced simultaneously.
“Smell is just — it’s so crucial to taste, which means it’s really crucial to everything that I do,” said Tejal Rao, a New York Times food critic, in a March episode of The Daily [5]. “And it’s really difficult to cook without a sense of smell if you’re not used to it. You don’t know what’s going on. It’s almost like wearing a blindfold.”
Rao, who lost her sense of smell in mid-January after contracting COVID-19, began to search for answers to this mystery from scientists. Rao’s journey started with TikTok “miracle cures” and other aromatherapies — unfortunately, they were too good to be true — but she eventually discovered the work of Dr. Pamela Dalton, a scientist at the Monell Chemical Senses Center in Philadelphia [6]. At the center, Dalton studies the emotions that are triggered by our sense of smell [7].
During simple colds or viral infections, smell is normally affected when the molecules in food and other aromas are physically blocked off from chemoreceptors in our nose by congestion. Scientists have also cited Alzheimer’s and Parkinson’s diseases, head trauma, and chemotherapy as triggers for anosmia [8]. But a separate phenomenon was occurring in the case of COVID-19.
“COVID is different in that way, because most people who lost their sense of smell did so without having any nasal congestion whatsoever,” Dalton told Rao during an interview.
One study published in October of last year by Dr. Nicolas Meunier, a French neuroscientist, aimed to investigate how the SARS-CoV-2 virus, which causes COVID-19, may disrupt sustentacular cells [9]. These structural cells express the ACE2 receptor, which the virus hijacks to gain entry into our cells, at higher levels [10]. Sustentacular cells support the specialized neurons that transmit signals from the nose to the brain.
When Meunier and his team at Paris-Saclay University in France infected hamsters with the virus, tiny hair-like projections known as cilia on the surfaces of olfactory neurons began to peel back from sustentacular cells. This disruption is a possible explanation for the difficulties with smell that COVID-19 patients experience.
If it is true that damage to sustentacular cells causes anosmia, loss of smell is not an irreversible brain condition. In this case, the poor connection between incoming odors and brain networks that typically process these stimuli is at fault, not direct damage to the brain itself. The sudden onset of smell loss in COVID-19 patients supports this thinking.
“It was just like a light bulb got turned off or a switch got flicked to off,” Dalton said. “And one moment they could smell. And the next moment, nothing smelled.”
But because olfactory support cells regularly regenerate, this loss of smell is only temporary, which allows for retraining of our senses. Two months of smell training, also known as olfactory training, allowed Rao to regain her sense of smell.
Olfactory training gradually exposes patients to particularly strong smells. Spices such as cinnamon or cumin, for example, were perfect for Rao’s first smell training session [5], and AbScent, a British charity offering support to patients with anosmia, sells kits with rose, lemon, and eucalyptus [8]. Scientists have found that recurring exposure to these strong scents gives the brain time to recalibrate its networks, a feature known as neuroplasticity [11].
But “you don’t just go from hurt to healed overnight,” Rao said. “It’s more like adjusting and learning how to live in a new space. That’s really just the beginning.”
Our chemical senses have the power to satisfy, to inspire, even to cause our memory to reveal itself, as 20th-century French author Marcel Proust observed in his seven-volume novel À la recherche du temps perdu, or In Search of Lost Time. Researchers have even speculated that our sense of smell can facilitate learning in other sensory domains, including vision [12].
While scientists further investigate how coronavirus causes loss of smell, olfactory training can provide an avenue in the meantime for COVID-19 patients to recover this crucial sense. Indeed, many patients are “in search of lost time,” and smell training can help them to once again experience food and wine in its sensory entirety.
References:
- Allen J, Almukhtar S, Aufrichtig A, Barnard A, Bloch M, Cahalan S, Cai W, Calderone J, Collins K, Conlen M, et al. 2021. Coronavirus in the U.S.: Latest Map and Case Count. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/interactive/2021/us/covid-cases.html.
- Symptoms of COVID-19. 2021. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Viral Diseases; [accessed 28 July 2021]. https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html.
- Lechien JR, Chiesa-Estomba CM, Beckers E, Mustin V, Ducarme M, Journe F, Marchant A, Jouffe L, Barillari MR, Cammaroto G, et al. 2021. Prevalence and 6-month recovery of olfactory dysfunction: a multicentre study of 1363 COVID-19 patients. J Intern Med. 290(2):451–461. https://doi.org/10.1111/joim.13209.
- Whitcroft KL, Hummel T. 2020. Olfactory Dysfunction in COVID-19: Diagnosis and Management. JAMA. 323(24):2512–2514. https://doi.org/10.1001/jama.2020.8391.
- Antolini T, Dorr W, Powell D, Schreppel C. 2021. A Food Critic Loses Her Sense of Smell. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/03/23/podcasts/the-daily/coronavirus-smell-food.html.
- Rao T. 2021. Will Fish Sauce and Charred Oranges Return the World Covid Took From Me? New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/03/02/dining/covid-loss-of-smell.html.
- What COVID 19 is teaching us about the importance of smell, with Pamela Dalton, PhD. 17 Mar 2021, 33:31 minutes. American Psychological Association; [accessed 28 July 2021]. https://youtu.be/0pG_U13XDog.
- Schoch D. 2021. Distorted, Bizarre Food Smells Haunt Covid Survivors. New York (NY): New York Times; [accessed 28 July 2021]. https://www.nytimes.com/2021/06/15/health/covid-smells-food.html
- Bryche B, St Albin A, Murri S, Lacôte S, Pulido C, Ar Gouilh M, Lesellier S, Servat A, Wasniewski M, Picard-Meyer E, et al. 2020. Massive transient damage of the olfactory epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian hamsters. Brain Behav Immun. 89(2):579–586. https://doi.org/10.1016/j.bbi.2020.06.032.
- Brann DH, Tsukahara T, Weinreb C, Lipovsek M, Van den Berge K, Gong B, Chance R, Macaulay IC, Chou HJ, Fletcher RB, et al. 2020. Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia. Sci Adv. 6(31): eabc5801.
- Kollndorfer K, Kowalczyk K, Hoche E, Mueller CA, Pollak M, Trattnig S, Schöpf V. 2014. Recovery of Olfactory Function Induces Neuroplasticity Effects in Patients with Smell Loss. Neural Plast. 1–7. https://doi.org/10.1155/2014/140419.
- Olofsson JK, Ekström I, Lindström J, Syrjänen E, Stigsdotter-Neely A, Nyberg L, Jonsson S, Larsson M. 2020. Smell-Based Memory Training: Evidence of Olfactory Learning and Transfer to the Visual Domain. Chem Senses. 45(7):593–600. https://doi.org/10.1093/chemse/bjaa049.
Surviving COVID-19: Variables of Immune Response
By La Rissa Vasquez, Neurobiology, Physiology & Behavior ‘23
Author’s Note: In this paper, I analyze autopsy reports conducted on deceased COVID-19 patients and supply a breakdown of the body’s immune response. The purpose of this paper is to provide a more generalized synopsis of how the body is affected by the virus from the onset of infection to the escalating factors that contribute to cause of death. COVID-19 and SARS-CoV-2 are referenced countless times throughout this paper, but they should not be used interchangeably. The name of the pathogenic virus is “Severe Acute Respiratory Syndrome Coronavirus 2” (SARS-CoV-2), and the name of the illness is called COVID-19 and is the common usage in forms of discussion. This paper only scratches the surface of the virus’s complexity and its effects upon the body and societies around the world.
Introduction
On December 31, 2019, the first case of the novel coronavirus was reported in Wuhan, China [1]. The first case of the virus reported in the United States was on January 22, 2020 [2]. Within 22 days, the Coronavirus had traveled across the Pacific to wreak havoc upon countries woefully unprepared. Within a year, COVID-19 has managed to bring some of the most powerful countries in the world to heel. Economies and healthcare systems across the world continue to be devastated by an adversary only 60 to 140 nanometers in diameter [3]. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) formally identified the virus as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). On March 11, 2020, the World Health Organization classified COVID-19 as a worldwide pandemic and global health crisis [4]. As of May 2021, the CDC has confirmed that the U.S. has over 32 million cases. Healthcare systems across the nation and around the world are overwhelmed by the number of infected patients. Many of them perish due to either a lack of resources or accurate and efficient testing.
SARS-CoV-2 Viral Pathogenesis
Humans have two levels of immunity. Innate immunity is the body’s first line of contact and defense against invading pathogens. Adaptive immunity learns and remembers how to effectively target and eliminate these pathogens.
Innate Immunity
Our innate immune system is composed of barrier tissues and cells specialized for defense against pathogens [5]. Barrier tissues are the first line of defense, and inside barrier tissues reside sentinel cells, which are capable of consistently recognizing repeated exposure to pathogen associated molecular patterns (PAMPs). The sentinel cells release proinflammatory mediators like cytokines, chemokines, or histamines and circulate within the blood vessels inviting more immune cells from the surrounding tissue into the bloodstream [5]. Cells such as neutrophils or monocytes differentiate into macrophages and migrate from the bloodstream and phagocytose (eat) the pathogens. Neutrophils will undergo programmed cell death, referred to as apoptosis. Macrophages will continue to phagocytose the rest of the pathogens and restore homeostasis by consuming the dead neutrophils [5].
Infection occurs when these viral pathogens in respiratory droplets from a sneeze or a cough enter a person’s mouth, nose, or eyes and attach to the ACE-2 receptors in the nose, throat, and especially the lungs. Like any virus, SARS-CoV-2 cannot replicate on its own and instead hijacks the body’s own cellular machinery. The virus inserts its own genetic information into the host cell to produce more copies of itself until the cell bursts and dies, spreading more of the virus around the body to infect more cells [6]. Infection of the host cell consists of the following five steps: attachment, penetration, biosynthesis, maturation, and release. Once a virus binds to host receptors (attachment), it enters host cells via endocytosis or membrane fusion (penetration). Once the viral contents are released inside the host cells, viral RNA are transported by protein molecules in the host cell’s cytoplasm and travel into the nucleus for replication via the nuclear pore complex (NPC). Viral mRNA then makes viral proteins (biosynthesis). Lastly, novel viral particles are made (maturation) and released [7]. This innate immune response is not as effective against SARS-CoV-2 due to the strength of the various proteins displayed in Figure 1, an ultrastructural morphology rendering, provided by the Centers for Disease Control and Prevention (CDC) Image Library on February 10 [8].
Figure 1
The SARS-CoV-2 virus contains “M (membrane), S (spike), E (envelope), and N (nucleocapsid)” proteins, which envelop the virion and act as a defensive shield [9]. The S or Spike viral surface protein, which consists of two subunits, S1 and S2, binds to the angiotensin converting enzyme 2 (ACE2) receptors of the host cells [7]. The primary role of ACE2 is the breakdown of the angiotensin II (ANG II) protein into molecules that neutralize its harmful effects. ANG II is responsible for increased inflammation and death of alveolar cells in the lungs, which reduces oxygen uptake. When the S (spike) protein of SARS-CoV-2 binds to the ACE2 receptors, they inhibit ACE2 from doing its job of regulating ANG II, allowing ANG II to freely damage tissue in the lungs. These ACE2 receptors are naturally present on the surface of the lung’s epithelial cells and other organs throughout the body, but the virus’ S protein uses these receptors to penetrate the cell membrane and replicate inside host cells. The N (nucleocapsid) protein is another viral surface protein of SARS-CoV-2, which inhibits interferons (IFN1 and IFN-β) responsible for cytokine production [10]. But if the signals for regulating proinflammatory response are disrupted by the pathogen’s surface proteins, the innate immune response becomes hyperactive and self-destructive. A malfunctioning innate immune response also compromises an adequate adaptive immune response [9].
Adaptive Immunity
Adaptive immunity consists of B-cell and T-cell responses. B-cells produce antibodies to trigger an immune response, while T-cells actively target and eliminate infected cells.
B-Cell Response
The innate immune response is not particularly equipped to combat pathogens that are especially complex and vicious because the innate immune response is non-specific and will attack anything it identifies as an invader. The adaptive immune response can target pathogens more precisely and powerfully by using proteins called antibodies, which are produced by B-cell lymphocytes that bind to antigens on the surface of pathogens [5]. Adaptive immunity can more efficiently handle foreign pathogens, like a virus, because antibodies can see through the debris of proteins and dead cells left by the cytokine storm. Antibodies uniquely bind to antigens, acting as a beacon for the adaptive immune response to converge on the invading pathogen [5]. More importantly, adaptive immunity has memory and learns how to become more effective by retaining its response to pathogens so that it can be even quicker at eliminating them after repeated exposure [5]. Widespread pandemics like COVID-19 occur because of a lack of protective antibodies in populations that have never been exposed to or vaccinated against the specificity of SARS-CoV-2 [5]. Figure 2 depicts the four ways in which antibodies attack pathogens: neutralization, complement fixation, opsonization, and antibody dependent cellular cytotoxicity.
Figure 2
Figure 2 – “Immunopathogenesis of Coronavirus Disease 2019 (COVID-19)” [3].
Neutralization is the process by which antibodies immediately bind to the surface antigens of a pathogen and block their S protein from attaching to the receptors of healthy cells, thereby neutralizing the virus’ ability to attach and insert its genetic information. Complement fixation occurs when antibodies are responsible for inviting complement proteins to bind to the antigens of the pathogen. This process coats the pathogen in attack proteins that can either initiate the complement cascade leading to cell lysis, the breakdown of the cell, or it can induce the third stage, opsonization. During opsonization, proteins called opsonins bind to the invading pathogen, acting as markers for phagocytotic cells like macrophages to identify and consume the pathogen. Lastly, antibody dependent cellular cytotoxicity (ADCC) is the process by which antibodies recognize the antigen of a pathogen and signal for natural-killer cells (NK cells) to release cytotoxic molecules which kill off the virally infected cell [5].
T-Cell Response
T-cell lymphocytes are produced by the bone marrow and mature in the thymus. They form the basis of cellular immunity because they directly attack foreign pathogens. Consequently, they are more effective than innate immune or B-cell responses at targeting intracellular pathogens like viruses [5]. Antibodies can get distracted by viral particles and proteins, so they rely on the blind T-cell lymphocytes to ignore the surrounding virus particles and eliminate the infected host cell at the source. As naive T-cells circulate the lymph nodes and spleen, they express T-cell receptors (TCR) that recognize cell surface peptides (antigens) attached to major histocompatibility complex (MHC) molecules on the surface of a specific pathogen. These surface MHC proteins tell the T-cells where to attack [5]. The dendritic cells work to activate the adaptive immune response by ingesting viral proteins and turning them into cell surface peptides that bind to MHC molecules, forming peptide-MHC complexes. The TCR of naive T-cells recognize the peptide-MHC complexes and activate the T-cell. For T-cells to become active, they also need to bind to proteins from the dendritic cell via co-simulation. They then undergo clonal expansion and differentiate into effector T-cells [5]. Effector T-cells are also referred to as cytotoxic T lymphocytes (CTLs). They travel through the body to hunt down peptide-MHC presenting pathogens and kill the infected cells by releasing cytotoxic molecules [5].
The adaptive immune response is stimulated by the recognition of pathogen-associated molecular patterns (PAMPs). Within 1-2 weeks after infection, the B-cells produce antibodies while T-cells simultaneously increase proinflammatory cytotoxic molecules in a forceful attempt to eliminate the virus [7]. The uptick in Interleukin cytokines abbreviated as IL-1, IL-6, IL-8, and so on, flood the body with proinflammatory substances, which “chronically increase the stimulation of T-cells, resulting in a cytokine storm and T-cell exhaustion” [9]. T-cell exhaustion not only means that the virus is overwhelming the body’s antibodies but also draining the strength of the T-cell’s ability to eliminate the virus at the source of infected host cells. SARS-CoV-2 is a “high-grade chronic viral infection because it decreases the responsiveness of T-cells leading to a decreased effector function and lower proliferative capacity” [9]. T-cell exhaustion is also linked to an increase in inhibitory receptors that can initiate apoptosis in T-cells. This results in the destruction of T-cells and their co-receptors, further suppressing the T-cells, as well as B-cells and NK cells, all of which are white blood cells (lymphocytes). Thus, explaining the general lymphopenia (the lack of lymphocytes) observed in severe COVID-19 cases and the increased number of cytokines [9]. Viral entry and attachment to ACE2 receptors trigger a vicious cycle of both innate and adaptive immune responses, mounting an intense attack by secreting proinflammatory substances that invite more lymphocytes to try and kill the virus. This releases more cytokines and chemokines [11]. The downregulation of the ACE2 enzyme results in a cascade of chemical reactions that lead to further inflammation and destruction of cells, weakening and damaging the body’s own immune response.
pathologies of a pandemic:
COVID-19 Autopsies
Once the SARS-CoV-2 attaches to alveolar type II cells, it propagates within the cells. Most viral particles cause apoptosis, releasing more self-replicating pulmonary toxins. Figure 3 displays normal ACE2 receptors located in the type II pneumocytes. Healthy alveoli are unobstructed to allow efficient diffusion of oxygen and carbon dioxide with red blood cells.
Figure 3
Figure 3 – “Type I pneumocytes are very thin in order to mediate gas exchange with the bloodstream (via diffusion). Type II pneumocytes secrete a pulmonary surfactant in order to reduce the surface tension within the alveoli” [12].
In contrast to Figure 3, Figure 4 shows the histopathology of alveolar damage (A) and inflammation (B) of the epithelial cells. As the epithelial cells detach from the alveolar wall the alveoli structures collapse and no longer inflate making it hard for patients with severe cases of COVID to breathe [13]. This results in diffuse alveolar damage with fibrin rich hyaline membranes and hemorrhages in the lungs [13]. The histopathology also detected multinucleated cells that lead to pulmonary fibrosis (scarring in the lungs). Infected cells are “abnormally large and often polynucleated cells showing a large cytoplasm with intense staining for the COVID-19 RNA probe” [13]. The viral Spike protein is also largely detected in the histopathology of COVID cases (C). The nuclei of Spike-positive cells appear an intense red stain and have abnormally enlarged cytoplasts (panel h) [13].
Figure 4
Figure 4 – “Histopathological evidence of alveolar damage, inflammation and SARS-CoV-2 infection in COVID-19 lungs” [13].
The cellular destruction detected in the histopathology is macroscopically reflected in the physical damage of lung tissue displayed in Figure 5.
Figure 5
Figure 5 – “Macroscopic appearance of COVID lungs” [13].
In all pathological examinations of patients that died of COVID, their lungs sustained macroscopic damage [13]. Severe cases of COVID reveal congested and firm lungs (A) with “hemorrhagic areas and loss of air spaces (a’, c’)” [13]. As the virus ravages the body, some patients rapidly deteriorate and develop severe inflammation and clotting in the lungs (B) which shows “the thrombosis of large pulmonary vessels, often with multiple thrombi and in one case determining an extensive infarction in the right lobe (Fig. 5B panels a and b)” [13]. The lung’s architecture crumbles as cells lose their integrity and continue to die, thus resulting in the development of Acute Respiratory Distress (ARDS). ARDS develops in about 5% of COVID-19 patients, and of all the infected, the mortality rate remains around 1 to 2% [14]. Autopsies are beginning to reveal that rather than a singular cause of death, many factors seem to be responsible for higher mortality rates in patients that develop critical cases of COVID-19.
The fallout from the hyperactive immune response disrupts regular oxygen diffusion from the alveoli into the capillaries and consequently to the rest of the body. This commonly leaves fluid and dead cells, resulting in pneumonia, a condition in which patients experience symptoms such as coughing, fever, and rapid or shallow breathing [14]. If oxygen levels in the blood continue to drop, patients rely on breathing assistance by a ventilator to forcefully push oxygen into damaged lungs “riddled with white opacities where black space—air—should be” [14]. The presence of opacities in the lungs indicate the development of pneumonia into ARDS, which was found in the autopsy of a 77-year-old man with a history of comorbidities, including hypertension and the removal of his spleen (splenectomy) [15]. The decedent exhibited chills and an intermittent fever but no cough for 6 days. On March 20, 2020, emergency medical services responded to a call, stating that the deceased was experiencing weakness, fever, and shortness of breath. In route to the hospital, the decedent went into cardiac arrest and died shortly after reaching the hospital [15]. A postmortem nasopharyngeal swab was administered and came back positive for SARS-CoV-2.
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Figure 6 |
Figure 7 |
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| Figure 6 – Normal chest X-Ray of healthy lungs [16]. | Figure 7 – “Lesion segmentation results of three COVID-19 cases displayed using the software post-processing platform” [17]. |
Figure 7 shows opacities in the CT “of typical COVID-19 infection cases at three different infection stages: the early stage, progressive stage, and severe stage” [17]. Figure 7 highlights these opacities in red, which appear to intensify and cover more of the lung CT as the virus increases in severity (a-c). Patient 4 (c) exhibits what medical examiners refer to as a “complete whiteout” of the lungs. Indicating reduced air flow, whereas the normal scan of healthy lungs (Figure 6) has a black background, representing the transparency of free and unrestricted airflow.
The postmortem radiography of the deceased 77-year-old man showed “Diffuse, dense bilateral airspace consolidations (complete “whiteout”)” [15]. In most cases of severe COVID-19 “the greatest severity of CT findings is visible around day 10 after the symptom onset. Acute respiratory distress syndrome is the most common indication for transferring patients with COVID-19 to the ICU” [18].
ARDS in connection to SARS-CoV-2 was first documented in Wuhan, Hubei, China in December 2019 with over 90,000 deaths associated with organ dysfunction, particularly progressive respiratory failure and the formation of blood clots resulting in the highest mortality rates [19]. Another autopsy from Hamburg, Germany conducted on the first 12 documented consecutive cases of COVID-19 related deaths revealed that there was not only profuse alveolar damage in 8 out of the 12 patients but also a high rate of clotting resulting in death. 75% of patients that died were males within an age range of 52 to 87 years and 7 out of 12 patients autopsied (58%) presented venous thromboembolism, as displayed in Figure 7. A pulmonary embolism was the direct cause of death in 4 of the deceased [20].
Figure 8
Figure 8 – “Macroscopic autopsy findings: A. Patchy aspect of the lung surface (case 1). B. Cutting surface of the lung in case 4. C. Pulmonary embolism (case 3). D. Deep venous thrombosis (case 5)” [20].
The formation of clots results in pulmonary vasoconstriction, or the constriction of arteries and halting of blood delivery to the arteries and capillaries in the lungs. Blood cannot travel to the lungs, so oxygen levels drop. As a result, a cytokine storm from our hyperactive immune system occurs, destroying the alveolus and the endothelium and causing clots to form. Smaller clots come together and form a fatal giant blood clot, or the clots can break apart and travel to other parts of the body, causing a blockage and inadequate blood supply to organs or other parts of the body [19]. If the blood supply to fingers, toes, and other extremities is cut off by a clot, it is referred to as ischemia and often results in the amputation of digits and appendages once the flesh begins to die [19].
When SARS-CoV-2 enters the alveolar cells in the lungs via the ACE2 receptors, it can directly attack organs and indirectly cause damage to other organs by triggering a hyperactive immune response (cytokine storm). When the viral particles trigger a cytokine storm, they cause further inflammation of the lungs resulting in plummeting oxygen levels and the formation of blood clots in the arteries (arterial thrombosis).
Conclusion
SARS-CoV-2 is a multi-organ scourge, but it primarily attacks the lung by first attaching its spike protein to the host cell’s ACE2 receptors. This prevents the lungs from regulating their function because it inhibits ANG II protein breakdown, causing increased alveolar damage and inflammation of the lungs. The virion proteins create proinflammatory responses in the innate immune response and compromise an effective adaptive immune response. As the virus progresses the number of neutrophils from the innate immune response increase while the number of helpful lymphocytes (T-cells and B-cells) decrease. The ACE2 receptors overstimulate the innate and adaptive immune response to produce more proinflammatory molecules to eliminate the virus, thus causing more destruction to the body and its immune response. Autopsies of COVID-19 victims show ongoing cellular death and collapse of the respiratory system caused by inflammation and alveolar damage that eventually develop into ARDS. Extreme inflammation induced by the immune response causes difficulties in breathing and clotting in the lungs. Radiography of progressive stages of COVID identify opacities in lung CTs indicating obstructed airways and alveolar deterioration. Postmortem examinations reveal gross destruction of the lung tissue, such as pulmonary artery thrombosis, vasoconstriction, lung infarction, or pulmonary embolism. Progressive organ and respiratory failure and abnormal clotting are all contributing factors to the cause of death in the most severe cases of COVID-19.
SARS-CoV-2 efficiently exploits weaknesses not only within our innate and adaptive immune systems across sex, age, race, and ethnicity, but it also exploits weaknesses within our societies. The etymological origins of Pandemic are rooted in pandēmos , which is Greek for ‘all’ (pan)+ ‘people’ (demos). When simplified, pandemic literally means “all people” but the priorities of leadership across the world reveal that not all people suffer the burden of this pandemic equally. Regarding the United States’ approach to the pandemic, this quote from the Atlantic’s article “Why Some People Get Sicker Than Others” is sufficient; “the damage of disease and a global pandemic is not a mystery. Often, it’s a matter of what societies choose to tolerate. America has empty hotels while people sleep in parking lots. Food is destroyed every day while people go hungry. Americans are forced to endure the physiological stresses of financial catastrophe while corporations are bailed out. With the coronavirus, we do not have vulnerable populations so much as we have vulnerabilities as a population. Our immune system is not strong” [21].
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The Relationship Between Itch and Pain in Itch Pathways
By Nathifa Nasim, Neurobiology, Physiology & Behavior ‘22
Author’s Note: Itch is not a stranger to any of us, but growing up with eczema, I have always been hyper aware of it. As far back as I can remember, burning hot showers and painful levels of scratching temporarily alleviated the maddening sensation of itch without my understanding of how pain was linked to itch. Once I joined the Carstens Lab studying the relationship between itch and pain, these memories were rekindled, and I became interested in not only understanding itch, which we know so little of, but how these two sensations interact. This paper was also written for my UWP 104E class.
Introduction
Itch is an everyday sensation that nearly all people have experienced. Its origins lie in its role as a defense mechanism — when faced with irritant stimuli, the scratching urge produced by itch can remove potentially harmful substances [1]. However, despite its evolutionary advantages, itch is often a source of discomfort and for many can dramatically impact their quality of life. Itch not only includes acute itch such as mosquito bites, but debilitating chronic itch can stem from different diseases such as cancer, HIV/AIDS, liver/kidney failure, atopic dermatitis, and other skin disorders [2]. However, despite the widespread impacts of itch, much of its mechanisms and pathways still remain elusive.
Itch is a somatosensory sensation, relying on the nervous system for detection and perception, therefore similar to other somatosensory sensations such as heat, touch, vibration, and most importantly, pain. Pain and itch have an antagonistic relationship, meaning each sensation has an opposing effect on the other. This is evident in “painful” scratching which relieves the feeling of itch, and that morphine administration reduces pain while increasing itch [3]. The intersection between the two sensations translates to potential treatment as well: chronic itch, for example, can be treated by medications similar to chronic pain [2]. Researching the interplay between itch and pain can help illuminate the pathophysiology of itch and how it is perceived as a sensation different from pain, and consequently lead to a better understanding of treating itch.
Currently, there are numerous models and theories proposed to explain this overlap, however, there is no consensus amongst itch researchers on which model(s) may best explain the relationship between pain and itch. This review will be an overview of the various models of itch transduction and perception and how they have evolved with the accumulation of new research. It will also cover the basic mechanisms of itch at the level of the periphery and spinal cord and how it interacts with pain.
Overview of Itch Mechanisms
Itch Activation at the Level of the Epidermis
Pruriceptors are neurons capable of detecting itch; these can be activated by either mechanical stimuli, a scratchy fabric for instance, or chemical stimuli, such as poison ivy. For simplicity, and as the chemical pathway is currently better understood, the paper will focus on chemical itch from here onwards. Similar to other somatosensory neurons, the cell bodies of the primary pruriceptive neurons reside in the dorsal root ganglion (DRG), close to the spinal cord, with axons stretching to both the periphery and the spinal cord [4, 5]. Unlike other sensory modalities, itch is specific to the outermost epidermis only, as opposed to pain, which can be felt in the muscle and bone. The pruriceptors’ branched sensory nerve endings which terminate in the epidermis are studded with membrane receptors activated by various “itchy” mediators [4]. The receptors differ in the mediators they respond to but can be broadly grouped into histamine receptors, serotonin receptors, G Protein-coupled receptors (GPCRs), toll-like receptors (TLRs), or cytokine and chemokine receptors [4,5].
Once acute itch is triggered by an irritant, keratinocytes, mast cells, and immune cells release chemical mediators which trigger vasodilation, inflammation, and the arrival of more immune cells to clear the irritant. The chemical mediators can include histamine, serotonin, proteases, cytokines, and chemokines, each of which is associated with a certain receptor [4]. The activation of itch from internal factors in disease differs from acute itch in that it is instead dependent on unknown mediators in the bloodstream from drugs or diseased organs [4]. Despite the origin of the chemical mediators, however, once released they bind to the receptors on the free nerve endings and activate them. The receptors then depend on various ion channels to depolarize the pruriceptor neuron which conveys the sensory information to the spinal cord via its axon [4, 5].
Itch Transmission to the Spinal Cord
The pruriceptive DRG neurons’ axon also ends in the spinal dorsal horn. These then synapse onto interneurons in the spinal cord which connect to projection neurons that carry the sensory information to the brain. The interneurons are important for transmission as well as modulation of itch via excitatory and inhibitory synapses [4, 5, 6]. Electrophysiological responses to itch stimuli in primates have identified the projection neurons as belonging to the spinothalamic tract, which carries axons to the thalamus. This tract also conveys pain and temperature and is consequently an area of itch interaction with other modalities [5, 7]. In addition to interneurons, descending modulation in the spinal cord can also regulate itch. After applying a cervical cold block to mice – activity of the upper cervical spinal cord level was essentially stopped – mice were unable to relieve itch and decrease neuronal firing when the lumbar spinal cord neurons below were stimulated by an itchy substance. This suggests that there is some level of descending modulation that was disrupted when the upper spinal cord was damaged [8].
Areas of Itch and Pain Interaction
Having briefly discussed the pathways for itch perception, it is important to note how often it converges with that of pain. Firstly, pruriceptors are in fact pruriceptive nociceptive neurons, meaning they are a subset of nociceptors, or pain-sensitive neurons. Although there are many non-pruriceptive nociceptors (neurons sensitive to pain but not itch), studies have pointed towards most pruriceptors being stimulated by pain as well as itch [1, 2]. One method of explaining this convergence is the expression of TRPV1 ion channels in pruriceptors. Although these are important for detecting itch, it is also expressed in nociceptors, and is stimulated by the classic pain stimulus found in peppers, capsaicin [4].
The relationship between itch and pain continues to the spinal cord. As mentioned, the spinothalamic tract (STT) is of special interest in understanding the distinction between itch and pain, as both sensations traverse the same pathway. Transecting the anterolateral funiculus where the tract ascends has eliminated sensitivity to itch, pain, and temperature, establishing the common usage of the tract by these sensations [6]. Electrophysiological recordings of primate STT neurons when given different types of sensory stimuli also revealed that two thirds of the nociceptors were sensitive to itch stimuli as well as pain, again highlighting the apparent overlap between itch and pain in the spinal cord [9].
The relationship between itch and pain is best understood as antagonistic. Recordings of STT pruriceptive neurons showed that after being stimulated by histamine (itch/pruritic stimuli) the neuronal firing decreased when the skin was scratched. However, the same neuron increased firing after scratching in response to capsaicin. Although being activated by both pain and itch stimuli, the difference in response to scratching suggests an antagonistic relationship between pruriceptive and nociceptive neurons via inhibitory interneurons [6].
The intersection between pain and itch raises the question of the brain’s perception of pain and itch as distinct in the presence of much overlap. There are numerous theories and models attempting to explain the nature of this relationship, which will be overviewed in the following sections.
Classical Models of Itch and Pain Discrimination
Intensity Model
Given observations on the overlaps between itch and pain, itch was first theorized to be a subset of pain in the intensity model. This postulates that polymodal neurons (sensitive to many modalities) differentiate between itch and pain through patterns of firing or “intensity” due to weak or strong stimulation [1, 4, 6, 10]. The model was tested by delivering electrical pulses to the skin that varied in frequency. Although the results seemingly disproved the theory as it only increased the intensity of itch felt, rather than transforming it to pain, the theory has not yet been discounted [6]. Itch stimuli has been shown to trigger lower firing rates compared to painful stimuli in both peripheral and STT neurons, suggesting that firing rates do have some role in itch perception [6, 9]. Furthermore, both itch and pain stimuli give rise to “bursting” patterns of action potentials, and the interburst interval is shorter in response to capsaicin/pain. This suggests some level of temporal coding, when information is coded based on the timing of action potentials or intervals between them. This aligns with the intensity model as a polymodal neuron could code for itch and pain depending on the rate of action potentials or their intervals [9].
The intensity model’s basic principle lies in neurons activated by both pain and itch, and seemingly aligned with the previously mentioned research identifying pruriceptors as a subset of nociceptors and activated by both pain and itch. However, the discovery of itch-specific neurons further complicated the validity of the model, lending support to the labeled line model instead.
Labeled Line or Specificity Theory
Labeled line refers to the idea that there exists a specific, separate “line” or neural pathway devoted to the sensation of itch – the opposite of the intensity model’s polymodal neurons. As early as the 1800s, researchers discovered there are specific spots on the skin which are activated by different sensory modalities: coolness, heat, pain, etc., giving rise to the labeled line theory. Recent electrophysiological studies have supported this for different sensations through establishing the presence of sensory fibers and spinal relay neurons tuned to only one sensory modality [11].
The labeled line theory’s validity for itch was confirmed by the presence of itch specific neurons. GRPR3+ neurons in the spinal cord were identified that differed from STT neurons in that they carried purely itch information [3, 12]. This was evident as when these neurons were treated with a toxin, not only was there loss of itch behavior (scratching), there was no change in pain behavior (wiping) [12]. The discovery of these itch specific neurons was emphasized by the consequent discovery of MrgprA3+ neurons in the dorsal horn which were itch specific as well; their deletion also resulted in loss of itch behavior only [7]. Furthermore, the neurons gave rise to purely itch behavior regardless of the nature of the stimulus – precisely as predicted by the labeled line [7]. These discoveries gave significant support to the labeled line theory, yet the presence of itch neurons activated by pain remained a dilemma.
Modified Models of Itch and Pain
The theories of intensity and labeled line represent the two ends of the spectrum in understanding pain signalling – the first depends on polymodal neurons, and the latter on itch specific neurons. The discovery of neurons that fall under both complicate their validity, and suggest that an accurate model should include neurons sensitive to both itch and pain while capable of differentiating between the two [1].
Spatial Contrast Model
Spatial models expand on the intensity model, and do not require itch and pain specific neurons. It proposes that itch is felt in “spatial contrast,” or when a small population of nociceptors are activated, and pain is felt when a larger population is activated due to a stronger stimulus [6,10]. In a study, it was observed that a spicule (small pointed end) of both histamine (itch stimuli) and capsaicin resulted in itch sensation, yet an injection of only capsaicin resulted in pain activation [13]. This could be explained by the spatial contrast theory in that the spicule activated a small number of nociceptors, resulting in itch, whereas the more widespread injection stimulated a larger number of nociceptors, resulting in pain.
According to the model, a small number of even non-pruriceptive nociceptors activated should result in itch, eliminating the need for a labeled line. However, there remains an obstacle in this model as well – there was no decrease of itch sensation relative to pain when the area of exposure to stimuli increased, although the model predicts this should in theory activate a greater number of receptors [6, 13].
Selectivity Theory and Population Coding
The population coding theory – also known as the selectivity theory – modifies the labeled line, proposing that although there are specific sensory labeled lines, the antagonistic interaction between them shapes perception of itch. It takes into account the overlap between nociceptors and pruriceptors as well as pain’s inhibition of itch, proposing that pruriceptors are a smaller subset of nociceptors and are linked to them by inhibitory interneurons [1,11]. Theoretically, activation of the larger nociceptive population – including pruriceptors – is felt as pain, as the activation of the pain neurons “masks” the sensation of itch. Yet if only the smaller itch specific subset is activated, this is felt as purely itch, as there is no activation, and consequently no inhibition from the nociceptive neurons [1, 11, 14].
There have been numerous studies that appear to support this hypothesis. In one, the vesicular glutamate transporter VGLUT2 was deleted from DRG nociceptive neurons, affecting their ability to signal. This resulted in spontaneous itch in mice along with a decrease in pain behavior and, importantly, itch behavior resulting from capsaicin injections [14]. These results were paralleled in another study where blocking pruriceptors had no effect on pain, yet deleting TRPV1 in a group of nociceptive neurons led to capsaicin to be perceived as itch [15]. These two studies suggest groups of nociceptors are involved in inhibiting and masking itch, as deleting their receptors results in itch signaling instead, supporting the population coding theory.
It is also necessary to identify an inhibitory neuron to explain the antagonistic relationship between the nociceptors and pruriceptors; Bhlhb5 neurons are one such interneuron. When Bhlhb5 was knocked out in mice, there was increased itch behavior that ultimately resulted in lesions from itching and licking [16]. This suggests that the interneuron, and perhaps other interneurons as well, are responsible for inhibiting and regulating itch, further bolstering the support for the population coding model.
Gate Control and Leaky Gate Model
The gate control theory hypothesizes that nociceptive transmission neurons in the spinal cord receive input from both nociceptive primary neurons, and Aβ fibers: primary neurons attuned to non-nociceptive stimuli such as touch. These Aβ fibers in turn inhibit the nociceptive neurons via interneurons, effectively creating a “gate” that can halt transmission of pain or itch [10]. The previously mentioned Bhlhb+ interneurons support this gate control model as well [10, 16].
This model was recently further refined into the “leaky gate” theory. This builds on the intensity theory and modifies the gate control theory by substituting Grp+ neurons in the role of Aβ fibers. Grp+ spinal cord neurons receive strong input from pain sensory neurons and weak input from itch specific neurons, coding for itch in an intensity dependent manner and inhibiting pain. This model is different from gate control in that it lets weak pain signals “leak” through while suppressing strong pain signals to prevent an overwhelming pain sensation. When strongly activated by pain, these interneurons inhibit pain, whereas due to weak activation from itch, it does not inhibit pain [10]. This model is able to explain the phenomenon that itch is often accompanied with a prickly, burning pain: it proposes that itch is not strong enough to inhibit pain sensation, resulting in a weak pain sensation accompanying itch [10].
Conclusion
A few of the major theories of itch perception have been discussed in an attempt to illuminate how itch is attenuated by the presence of pain in an inverse relationship. The intensity theory and the labeled line theory are both supported by the presence of polymodal neurons and itch specific neurons, respectively. However, given their opposing views, the accuracy of both theories is undermined by support for the other; this indicates the need for a model that is able to reconcile itch specificity with neurons attuned to both itch and pain.
The following models attempted to ease the apparent discord between the two previous models. The spatial model expands on the intensity model while providing a possible mechanism by which pain and itch could be felt from the same population of neurons. On the other hand, the population coding model expands on itch specific neurons of the labeled line while accommodating the inverse relationship between itch and pain. Lastly, the leaky gate model combines aspects of both intensity and selectivity theories.
These theories attempt to explain itch and pain crosstalk; the importance of understanding this relationship is seen in both acute and chronic itch pathophysiology in cases of crosstalk dysfunction. The previously discussed Bhlhb+ neurons are a prime example of the consequences of impaired itch and pain interaction [2, 16]. Research has shown that knocking out these interneurons – thereby severing the connection between itch and pain – results in chronic itch-like behavior such as lesions from scratching [16]. This suggests that chronic itch may result from uninhibited, unregulated itch when pain is no longer permitted to suppress itch [2, 16].
This example highlights the importance of the application of the interaction between pain and itch. Not only does understanding the intersection between the two sensations provide a better understanding of itch mechanisms, the very intersection itself has an important role in itch pathophysiology, of which there is much that is still unknown. With the advent of new discoveries of new aspects of the itch pathway, these current models will continue to develop.
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Transgender Health: Barriers to Healthcare and Physiological Differences
By Ana Nazmi Glosson, Neurobiology, Physiology & Behavior ‘21
Author’s Note: I initially wrote this literature review for UWP 104F in Winter 2020. I chose to focus on a topic that was, and is, very dear to me. I believe that readers would benefit from an overview of transgender specific health, as it is a subsection of science that is often unknown or overlooked. I wrote this while personally researching TGD healthcare and the availability of transitional therapies, and realizing firsthand the barriers to access, and lack of available information.
ABSTRACT
Transgender and gender diverse (TGD) individuals are people whose gender identity does not match the biological sex they were assigned at birth. Transgender is an umbrella term for many gender identities, and individuals may identify as male, female, or outside the gender binary. This population faces more barriers to healthcare access than cisgender individuals, or people whose gender identity does match their sex assigned at birth. Lack of access to knowledgeable healthcare providers, as well as provider bias, creates an environment of hostility for a TGD patient. Transgender people have unique health needs that healthcare professionals must be educated on in order to properly serve this community. Emerging literature is beginning to identify health concerns among transgender people who have undergone hormone replacement therapy (HRT) that may require specialized treatment and attention. This review attempts to answer the following question: What does current research tell us about barriers and educational gaps in healthcare of transgender individuals, and what physiological differences in this population, compared to cisgender individuals, make this research important? Further studies are essential to properly providing healthcare to this population.
Key Concepts: Transgender and gender diverse, hormone replacement therapy, culturally competent healthcare.
INTRODUCTION
Historically, TGD individuals have faced many barriers to healthcare access—and many of these barriers still exist [1-9]. This paper aims to review the specifics of these barriers and educational gaps. Current research suggests that a lack of education from physicians and provider biases against transgender people are primary reasons why transgender individuals—especially TGD youth— struggle to access safe and culturally component heathcare [3,5-7]. Transgender people are less likely to seek healthcare, and if they do seek it, they are less likely to receive proper, unbiased access with educated professionals [1,3]. This review also presents literature on unique physiological differences between transgender and cisgender individuals in order to properly express why clinical research is needed to increase baseline education [10-16]. The critical health differences between the TGD population and other groups means a team of doctors and specialists—primary care physician, gender specialist, surgeon, and endocrinologist—must collaborate to provide culturally competent care. TGD individuals may choose to medically transition and undergo gender-affirming therapies such as gender-affirming surgery (GAS) or hormone replacement therapy (HRT). Given the nature of this topic, it is important to note that much of the research in this review is from ground-breaking preliminary studies that have not yet been repeated with larger sample sizes beyond initial investigation.
DISCUSSION
Healthcare Access Among TGD Individuals
TGD individuals of all ages face challenges to healthcare on both a personal and institutional level. Increasing numbers of TGD people, including older adults, are openly living with their gender identity, meaning this is a critical area of research. TGD adults frequently struggle with insurance access; they are less likely to have insurance access compared to non-TGD LGBTQ+ individuals, and those that do have access are more likely to face healthcare discrimination [7,8]. One study found that individuals with Medicaid were more likely to be refused hormone replacement therapy, and more likely to lack a surgeon to perform gender-affirming surgery in their network, as compared to individuals with private insurance [7]. TGD adults who are part of other disadvantaged communities, such as being an ethnic minority or having lower socioeconomic status, face additional obstacles and higher levels of healthcare refusal [1,8]. Older LGBT adults are far more likely to have physical and mental health struggles than their non-LGBT counterparts, but older TDG adults are the most likely to have those struggles within the LGBT community [9,17]. Older TGD adults are more likely to live alone and have a community of “chosen family” instead of partners or children, which adds a layer of complexity to difficult end-of-life care decisions and increases senior care costs [9,17]. These circumstances show the need for thoughtful and individualized care for TDG individuals of all ages, necessitating competent and knowledgeable providers to navigate these sensitive topics.
Adolescence is a very stressful time in people’s lives, and recent literature shows that young TGD individuals are especially vulnerable [3,5,11]. Surveying the adolescent population directly allows researchers to analyze experiences and suggestions from youth to further improve healthcare. Currently, there is not much information on transgender youth, though the field of research has begun to grow rapidly in the past few years. In everyday life, TGD individuals are often misgendered or referred to as names that they do not identify with anymore. In the context of medical care, this leads to individuals being less likely to seek continuing care. Even without malicious intent, these actions may be incredibly damaging to the TGD individual. In a medical setting, misgendering patients may foster unspoken feelings of distrust and alienation between the patient and their doctors. This is critical because transgender individuals are less likely to continue seeking routine and specialized healthcare if they feel uncomfortable in the medical environment [2,3]. In order for healthcare professionals to serve this population, practices must be as friendly as possible. Requesting and consistently using the individual’s pronouns and preferred name is a critical first step [2-3,5]. Surveyed youth suggested that healthcare providers should ask all individuals these questions, instead of only those known or assumed to be LGBTQ+ [2]. This will lead to the subpopulation not being immediately singled out in a healthcare environment, as well as creating a welcoming space for patients who may not otherwise volunteer this information. Another suggestion was healthcare providers using gender-neutral decor in exam rooms [4]. In settings such as a gynecologist office, traditionally feminime or masculine imagery and furnishings can further alienate TGD individuals and reduce the likelihood of patient continuation. The language used in medical forms should be adjusted to encompass diverse gender expressions. Given the fact that many TGD individuals identify outside of the gender binary, medical records should allow patients to write in their identity rather than check one of two boxes [3]. The gender binary is essentially the rigid classification system of two genders, male or female, a system which is commonly rejected by members of the LGBTQ+ community and their allies. Since gender identity and the language that individuals use to express their personal sense of self is incredibly varied, giving patients more freedom to define and communicate their gender identity would allow them a greater sense of expression. This may also require reform of electronic healthcare systems to include this information, which is currently not common practice. In one study, the vast majority (79%) of TGD youth indicate they would appreciate the professional record of preferred name and pronouns [5].
A common method of surveying the adolescent population is in-depth interviews of a small sample size. These thorough accounts of real experiences are very useful, as researchers can gain a more holistic insight into the individual’s life and experiences. The downside of this research approach is the small sample size, which may lead to results that are not as applicable to larger audiences as would be the case with a larger sample size. In order to best reach this population, researchers target LGBTQ+ programs, but for many reasons, a large subset of the TGD population cannot safely participate in those programs, and therefore are not included in reviews such as this. Voices of closeted LGBTQ+ community members in general are rarely heard, meaning this subset of the population is almost always left out.
Research also suggests that preferences regarding the inclusion of gender identity information in medical records differ greatly if the patient is closeted or “out” [2]. There are factors that should be taken into account with medical records disclosing transgender identity. For instance, a TGD minor may privately disclose their gender identity or preferred pronouns to their healthcare provider. If this TGD youth was not “out” to their parents, and the healthcare provider made a note, their parents might find this while viewing their medical records. This could potentially be damaging or even dangerous to the patient, so healthcare providers should be careful with handling such delicate information. Additionally, TGD care—especially for patients that are in the process of transitioning—involves many aspects of healthcare; a team of culturally competent therapists, physicians, specialists, nurses, and staff must all be properly informed to contribute to a holistically supportive team.
Sexual Health Needs
Research into sexual health needs of young transgender people demonstrates TGD youth have unique sexual health needs that are not currently being met by their healthcare providers. Healthcare providers tend to be less knowledgeable about TGD-specific health issues, which differ from cisgender individuals [3,13,15]. Distinct aspects of TGD individuals include hormone replacement therapy (HRT), gender-affirming surgery (GAS), reversible puberty blockers, and same-sex STI transmission. Compared to previous generations, youth today are more likely to come out as transgender at a younger age, but many healthcare providers are not properly relaying healthcare information to their patients [3]. When providers fail to relay crucial information to their patients, it poses risk to the patients that could otherwise be avoided. For instance, a doctor who is unknowledgeable on STI transmission among two people that were assigned the same sex at birth, or even a doctor with personal prejudices against TGD patients, might not inform patients of essential sexual health information, thus putting the patients at higher risk. Sexual education information for teenagers is lacking, and this issue is amplified for TGD youth, many of whom receive absolutely no relevant information from professionals and alternatively turn to unvetted online sources. Healthcare providers need to stay up to date on the current literature for LGBTQ+ patients and have an obligation to confirm their patients receive adequate and age-appropriate information on topics of sexual health.
Transgender men or non-binary individuals who have been prescribed testosterone, a gender-affirming hormone replacement therapy, may suddenly experience an ovulatory event after a long period of time [15]. Testosterone can stop ovulation by suppressing the hypothalamic-pituitary-adrenal axis, but this research study is the first to show that after an extended period of time, such as several years, some individuals may “overcome” these suppressed hormones and suddenly ovulate [15]. This is important for healthcare professionals to be aware of because their patients may not be on contraceptives and will likely not expect this after suppressed ovulation. Unplanned pregnancy may result among patients who partake in sexual intercourse with sperm-producing individuals. Healthcare providers have an obligation to inform their patients of medical issues such as this, as pregnancy for a transitioning TGD individual can be an extremely emotionally stressful event, especially in the face of body and gender dysphoria.
An emerging branch of literature involves TGD patients and gynecological care. TGD patients are less likely to seek this type of care, and when they do, healthcare providers may have personal biases against treating transgender patients [4,6-7]. Transgender men or transmasculine individuals were found less likely to seek cervical cancer screenings, the main preventative test against cervical cancer. This is because of a variety of barriers on both a personal level and a wider institutional level. On a personal level, traumatic experiences with past healthcare, misgendering, and overall gender dysphoria contribute to transgender men not seeking cervical cancer screenings [4]. Institutionally, research suggests incompetent provider education is a primary barrier to accessing satisfactory healthcare. This leads to a reduced number of transgender men or transmasculine individuals continuing cervical cancer screening [4].. Healthcare professionals should focus on ways to retain transgender men as patients throughout their transition and changing gender identity, as well as providing culturally competent healthcare to this population.
In a study on gynecological health of transmasculine people, healthcare professionals were surveyed on their willingness to provide healthcare to TGD individuals. It was found that personal biases and attitudes against TGD individuals were the greatest barriers [6]. This contradicts other studies, which indicate healthcare providers’ lack of knowledge to be the biggest obstacle to accessing safe healthcare. Professional training should account for transphobic beliefs among healthcare professionals [6].
Much of the research on TGD populations are groundbreaking pilot studies, and conducting more large scale clinical studies and research is highly recommended for improving healthcare for transgender individuals [2,5,17]. Another recommendation is to standardize inclusive and informed education on transgender topics in medical school curricula and continuing education programs [3,5,8]. Informed and supportive healthcare professionals are absolutely vital in addressing health and continued patient retention among TGD individuals. More research must be done to determine the extent of additional training needed to properly serve this population.
HRT and Physiological Differences
Literature has begun to explore and emphasize that physiological differences exist between transgender individuals who are undergoing gender-affirming hormone replacement therapy (HRT) and cisgender individuals [11-17]. Hormone replacement therapy is suggested to be gender-affirming to a patient with gender dysphoria by helping their body match their preferred gender identity, and has been found to be correlated with better body- and self-perception, as well as lower sexual distress [13]. This is incredibly important in increasing the holistic wellness of a transgender patient. Limited available research suggests that transitioned TGD individuals are at greater risk for certain cardiovascular diseases, such as heart attacks, compared to the general population [16]. When researching the impact of HRT on adolescents, one pilot study found key body composition differences in regards to cardiovascular health, suggesting this population has unique cardiometabolic needs that differ from both cisgender males and cisgender females [11,16]. Similarly, in regards to resting state network, individuals on HRT were found to have “intermediate” levels of physiological values unique and distinct from cisgender male or female individuals [11,16]. For the purpose of this paper, we can think of resting state networks as networks and patterns of activity between spatially separated areas in the brain, which are helpful in analysing organization, when the brain is not processing a specific task.. This information is preliminary—and it is important to keep up with developing research—but it suggests the extreme importance of larger repeat studies. Questions for further research include long-term effects of HRT on adolescents. Additionally, research should be conducted on the distinct physiological values of individuals on HRT. In particular, do these values (the intermediate state) change the longer the individual is on HRT? If a patient were to stop HRT, would this “intermediate” state revert to values similar to their gender assigned at birth?
Another question to consider would be whether or not this intermediate state is reversible if the patient were to stop HRT for a period of time. However, such a question would bring up many ethical concerns for the psychological well-being of the study participants, as well as physical concerns of abruptly stopping medical therapy. One longitudinal pilot study found that transgender individuals on HRT had altered resting state functional connectivity in emotional, cognition, and sensorimotor ways after undergoing gender-affirming surgery [15]. These studies suggest that the brains of TGD individuals have the ability to form altered synaptic connections in a way that is different from cisgender people. Much more research is required in order to pinpoint any major connections and the implications of treating this population. These medical differences could be very important in areas such as proper drug dosage. Healthcare professionals must recognize these differences, and continue to push for more research to ensure transgender patients receive the competent care they need. Much of this research contributes to some sense of a gender binary, given that this “intermediate” state is defined as being between “the two” genders; furthermore, a TGD individual may not aspire to follow a binary gender, and providers should be thoughtful and individualized in the language they use with patients. The majority of these studies were composed of very few individuals. These results suggest that healthcare professionals must stay informed with research findings in order to keep their patients updated.
CONCLUSION
Transgender individuals face discrimination in everyday life, as well as in the medical world. This is a large problem because transgender patients have specific healthcare needs that differ from cisgender patients and must be approached and treated differently. Many of these studies are pilot studies and were only published in the last several years. Several recent studies have attempted to classify barriers transgender individuals face, specific health differences, and what steps healthcare providers need to be taking. As research in transgender healthcare continues, it is important to note that not all transgender people can be grouped under one umbrella. Subpopulations exist within the TGD community, each with their own healthcare concerns, physiological health differences, and types of care they seek and receive. In order to better treat these populations, healthcare professionals cannot treat every transgender person with identical care. This emerging research, especially on topics of physiological differences, should not be used to discourage TGD individuals from their necessary transitional therapies. Rather, a more comprehensive understanding should help healthcare providers give their patients stronger, evidence-backed information about their medical choices. In addition, there are barriers that this discussion barely touched on, such as cost, insurance issues, and overall accessibility. Many more studies are required to identify the best ways to combat transgender barriers to healthcare access in order to address the physiological differences between TGD and cisgender individuals.
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Among Virions
By Jordan Chen, Biochemical Engineering ‘24
What are viruses? Miniscule packages of protein and genetic material, smaller than all but the smallest cells, relatively simple structures on the boundaries of what we consider living. Undetectable to the human eye, these invisible contagions are rarely on the minds of the average person, occupying a semantic space in public consciousness more often than they are understood for their material reality. Stories are more likely to be described as “viral” than an actual virus, yet when the COVID-19 pandemic washed over the world at the end of 2019, the public suddenly had to confront that which was seemingly abiotic, simple, and small. However, the impact of the COVID-19 pandemic exceeded that unassuming material reality. With the shuttering of the global economy, mass death, political crisis, confusion, hysteria, and science without immediate answers, it’s become clear that the sum of COVID-19’s viral components is much more than the whole.
To emphasize this idea in the piece, coronavirus virions are depicted as massive and detailed larger than earth bodies, in a vital bloody red, surrounding and overwhelming the relatively simply shaded globe. What was formerly small, simple, and nonliving, can now be dramatically understood as larger than life, having created complex predicaments, and having taken on a life of its own in its assault against the world. This digital artwork was created in Blender.