Written by: Ken Estrellas
Written By: Rebecca Tweedell
The Gist of It:
In 2015 and 2016, the world was closely following the outbreak of a mosquito-borne virus called Zika. While these outbreaks have since faded from the forefront, with very few if any cases being detected currently, there are still a lot of unknowns about this virus. One thing we do know is that Zika virus can cause fetal microcephaly, or swelling of the brain in unborn babies. To do this, the virus must somehow be able to get to the brain. This is especially challenging because there are 2 key lines of defense working to keep viruses out, the placenta and the blood-brain barrier. The placenta is formed during pregnancy and allows oxygen and nutrients to be passed from the mother to the baby while blocking the passage of toxic materials and viruses. The blood-brain barrier exists in both babies and adults and serves as the boundary to keep the circulating blood (and any bad things that might be circulating with it) away from the brain. Since we know that the virus can get into the brain, we know these barriers must be failing, but how this happens is still a big question. Researchers at National Yang-Ming University in Taiwan recently took a major step toward solving this mystery. They studied how the virus passed through placental barrier cells and brain-derived barrier cells in the lab. They discovered that the virus actually crosses these barriers in different ways. Zika virus disrupted the ability of the placental cells to form strong junctions between themselves, weakening the physical structure of the barrier. This was not the case with the brain-derived cells, which maintained the physical structure of the barrier in spite of the virus. But the virus was still able to move through both types of barriers by going directly through the cells through a process known as transcytosis (which literally means being transported across the interior of the cell). Excitingly, blocking transcytosis with inhibitors was able to keep Zika virus from getting through the barriers. Understanding how the virus is able to get through these barriers is important for being able to stop it, and this study helped us take a big step in the right direction.
The Nitty Gritty:
Chiu et al. used the human placenta trophoblast cells JEG-3 and human brain-derived endothelial cells hCMEC/D3 to create an in vitro model to study interactions with Zika virus. They found that Zika virus could infect both of these cell types when the cells were grown in a monolayer. Using a transwell barrier assay, with a monolayer of JEG-3 or hCMEC/D3 cells in the insert and Vero cells in the bottom chamber, the researchers found that Zika virus was able to pass through the barrier formed by the cells, moving from the portion of the well above the JEG-3 or hCMEC/D3 cells to infect the Vero cells below. When passing through the JEG-3 placental cells, Zika virus damaged the integrity of the membrane, allowing FITC-dextran to pass through. However, this did not occur with the hCMEC/D3 cells. This was likely due to tight junction disruption; the researchers found that the expression of ZO-1 and occludin was decreased in JEG-3 cells following exposure to Zika virus, while this did not occur in hCMEC/D3 cells. After treatment with a proteasome inhibitor MG132, the expression of ZO-1 and occludin was rescued, suggesting that the disruption of tight junctions occurs through the proteasomal degradation pathway. Finally, the authors sought to understand whether Zika virus can use transcytosis to traverse the placental and brain-derived barrier cells. Using a fluorescently labeled virus, they measured the fluorescence in the basal chamber of a transwell following culture of the virus with the monolayer barrier in the insert at 4°C and 37°C, as transcytosis is inhibited at 4°C. They found that the amount of virus that traversed the barrier at 4°C was significantly reduced compared to the amount that traversed at 37°C, while there was no difference in the amount of FITC-dextran that could traverse. Based on the finding that the virus is able to traverse the cells by transcytosis, the authors tried to block the virus using inhibitors of endocytosis and intracellular trafficking (Nystatin, chlorpromazine, dimethyl amiloride, and colchicine). Each inhibitor reduced the amount of virus that made it through the monolayer barrier. Overall, Chiu et al. found that Zika virus crosses placental and brain-derived barrier cells in culture by transcytosis and that the virus disrupts the integrity of tight junctions to weaken the placental cell barrier in the transwell system. These findings pave the way for discovering ways to inhibit viral traversal and prevent fetal infection and encephalitis.
Original Research Article: Chiu, C.-F., et al. “The mechanism of the Zika virus crossing the placental barrier and the blood-brain barrier.” Front Microbiol (2020).
Written By: Kaitlyn Sadtler
Original Article: Davidson et al. Acta Biomaterialia 2020
The Gist of It:
Just like we take in information from our environment and change our behavior (like putting a coat on when it’s cold outside), cells take in information from their environment – the extracellular matrix. The cues they receive can cause them to change their environment by secreting different proteins, making the matrix stiffer or softer (just like we can turn on the A/C or put another log on the fire to make it cooler or warmer). Stiffening and densifying of the extracellular matrix is called fibrosis, and this process contributes to numerous disorders, including liver cirrhosis, cardiac dysfunction, skin scarring, and lung fibrosis. Understanding how our cells sense changes in the stiffness of the matrix around them and how that affects disease progression is necessary for the development of new therapeutics. Researchers from the University of Michigan Ann Arbor have developed a new material and platform to study this phenomenon in the laboratory. Davidson and colleagues synthesized a fibrous biologic matrix out of dextran vinyl sulfone (DexVS). The researchers were able to spin these fibers onto a plate to create a sort of cellular hammock, where the cells would feel just the stiffness of the material and not the plastic bottom of a petri dish. With these fibers, they were able to look in a highly controllable manner at how cells interacted with stiffer and softer matrices. In contrast to what had been seen when the interaction between cells and the matrix was studied using other methods, like looking at solid synthetic gels (jello-like), they found that stiffer materials made cells less fibrotic than softer materials did. Using these models, scientists will be able to probe interactions between cells and fiber-based matrices that are more similar to the natural extracellular matrix and compare to previous studies which used fully synthetic hydrogels that do not have fibers. Moving forward, these scientists have developed ways to modify these materials with different proteins and change the way that the cells interact with the matrix. Further research like this could expand our understanding of what happens to our cells in stiff environments, which could lead to therapeutics for fibrotic diseases.
The Nitty Gritty:
Previously, researchers had developed an electrospun methacrylated dextran (DexMA) to study matrix stiffness in vivo. However, DexMA fibers degraded in vitro due to hydrolytic cleavage of the ester bonds in the polymeric matrix within days of exposure to cell culture media. Here, the researchers presented a dextran vinyl sulfone (DexVS) that lacks the sensitive ester bonds and can still be functionalized via Michael-type addition and crosslinked with a photoinitiator (in this case, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP). They tested the stiffness of these materials by changing the concentrations of photoinitiator or duration of UV exposure. They further functionalized these materials with cyclic RGD (cRGD) for cell attachment or with methacrylated heparin to promote association of cell-secreted matrix with the synthetic polymer fibers. DexVS fibers were spun onto a multi-well plate to create a suspended surface with mechanical properties independent of the tissue culture plastic. Culturing normal human lung fibroblasts (NHLFs) on stiffer and softer matrices revealed that softer fibrous matrices, in the presence of pro-fibrotic soluble transforming growth factor beta-1 (TGFβ1), induced higher levels of myofibroblast-associated alpha smooth muscle actin (αSMA) than did stiffer matrices. This is contradictory to studies performed with solid hydrogels, such as poly(ethylene glycol) (PEG)-based hydrogels, that suggested stiffer matrices induce more of the fibrotic myofibroblast phenotype. Further research using multiple model systems in vitro and comparison to in vivo samples will hone in vitro modeling to pave the way for understanding mechanistic biology, modeling disease, and screening novel therapeutics.
Original Research Article: Davidson, C.D., et al. “Myofibroblast activation in synthetic fibrous matrices composed of dextran vinyl sulfone.” Acta Biomaterialia (2020).
Written by: Sayan Roychowdhury
Original Article: Zhu et al. PLOS Computational Biology 2020
The Gist of it:
Autonomous aerial vehicles, such as drones, are carefully programmed and trained to move based on their surroundings. However, these controls sometimes react poorly when introduced to new or unknown environments. So, how can safe and reliable flying strategies be developed for use in unfamiliar territory? To answer this question, a group of scientists from Boston University and the University of Washington turned to hawkmoths for inspiration. Previous research shows that hawkmoths navigate largely based on visual cues just as drones do, making the moths’ flight decision-making very applicable to drones. In this study, the researchers gathered physical flight data from hawkmoths through a virtual forest projected on a screen. The researchers then created a moth decision-making model based on this data; this model could simulate how a hawkmoth would react to seeing a certain visual scenario. The researchers tested this model on a wide variety of forest arrangements differing in size and density to examine how well a hawkmoth would perform when encountering new surroundings. They also compared the performance of the hawkmoth to an optimal flight pattern for each virtual forest.
The researchers found that for each case, the moth decision-making model performed very well, suggesting that the moths’ strategy is quite versatile, even for unfamiliar terrain. In particular, the moth model worked best in dense forest scenarios, possibly indicating that hawkmoths have developed flight strategies best suited to the types of forests they encounter in their natural habitat. These results are exciting because researchers can study biologically evolved flight patterns to help program autonomous vehicles to perform better in new and unfamiliar environments.
The Nitty Gritty:
To study navigational patterns, the researchers connected eight hawkmoths (Manduca sexta) to a metal rod and placed them in front of a large screen displaying a virtual forest moving at a constant speed of 2 m/s. Each moth’s motion in the horizontal direction was measured through a torque meter, capturing changes in direction based on the view. Data were collected for each moth at 5 varying levels of fog density to simulate varying visibility conditions. The virtual forest and the moth’s flight trajectory were discretized in time and space, and a Markov Decision Process was used to model the flight. Logistic regression was used to estimate the parameters of the moth control policy. This policy was further refined using a reward function based on distance to trees – minimizing distance to simulate hiding from predators in the foliage, while maintaining enough distance to avoid collision. The original moth policy and an optimized one were then compared on a new set of virtual forests with varying size and density, where the optimal policy was tuned on a per forest basis. The original moth control policy was very robust and was within 30% of the performance of the refined policy for all of the dense forests. Thus, this work shows that hawkmoths can serve as a reliable starting point for successful bio-inspired control and decision rules for autonomous aerial vehicles exposed to new terrains.
Original Research Article: Zhu, H., et al. “Learning from animals: How to navigate complex terrains.” PLoS Comput Biol 16.1 (2020): e1007452.
Written by: Sravya Kotaru
Original article: Liu et al. BioMed Research International 2020
The Gist of It:
Paraquat is a common herbicide used to kill weeds in croplands. It is poisonous if consumed, breathed, or touched. Severe poisoning can cause multiple organ failure including lungs, kidneys, and liver, and can be fatal. Even mild poisoning leads to long-term respiratory issues from lung damage and scarring. The usage of paraquat is highly debated; it is banned in Europe but is still widely used in the United States and other parts of the world. The people at most risk of exposure are those using the herbicide and those living in areas near where it is used, but it is also possible for paraquat to contaminate food and water sources and reach consumers. Once paraquat enters the human body, it produces harmful molecules called reactive oxygen species (ROS), which cause inflammation and tissue damage. Specifically, in the lungs, paraquat damages the tiny air sacs called alveoli, which are like small balloons that fill with air during each breath. In the alveoli, oxygen from the air diffuses into the bloodstream. When paraquat is breathed in and reaches these lung alveoli, it causes inflammation and the build-up of scar tissue around these air sacs. This scarring restricts the alveoli’s ability to expand with each breath, causing shortness of breath and lung disease. So far, there is no antidote or treatment for paraquat poisoning. Excitingly, scientists at North Sichuan Medical College in China recently discovered more details about how paraquat causes lung damage, allowing them to identify a potential target for treatment. They found that the ROS produced by paraquat lung poisoning in rats damaged mitochondria, the energy factories in alveolar air sacs. This damage turns on two proteins called PINK1 and Parkin that are responsible for removal of the damaged mitochondria and cause scar tissue to develop around damaged alveoli, thus causing lung injury. The researchers also found that deleting the protein PINK1 from lung cells grown in the laboratory prevented the production of fibronectin and collagen, two molecules that form scar tissue. These results are good news because they suggest that PINK1 could be targeted to develop a paraquat antidote and prevent lung damage. This possibility must be further explored.
The Nitty Gritty:
Paraquat, a common herbicide, is a known oxidant that generates ROS and causes mitochondrial damage in alveolar epithelial cells, resulting in alveolitis and pulmonary fibrosis. Independently, it is known that damaged mitochondria accumulate the kinase PINK1 on their outer membrane; PINK1 in turn recruits Parkin to initiate mitophagy. The authors investigated whether PINK1-Parkin signaling and mitophagy are involved in paraquat-mediated poisoning. They observed that in rat models, paraquat exposure resulted in loss of alveolar architecture and a build-up of fibrous extracellular matrix in lungs over time, indicative of fibrosing alveolitis. The poisoned rat lungs also showed a time-dependent increase in the expression of the extracellular matrix fibers fibronectin (FN) and collagen (COL-1), which contribute to the formation of fibrous scar tissue. In addition, there was a time-dependent increase in PINK1 and Parkin expression and loss of mitochondrial membrane potential, indicative of mitophagy. The researchers observed similar results in the cultured A549 lung cancer cell line, where paraquat induced a time- and dose-dependent loss in cell viability and increase in the expression of PINK1, Parkin, FN, and COL-1. Moreover, silencing PINK1 expression in A549 cells negated the effects of paraquat-mediated upregulation of Parkin, FN, and COL-1 expression. Thus, mitophagy and fibrosis due to paraquat poisoning appear to be PINK1-dependent, and PINK1 is a possible molecular target for the development of a paraquat-specific antidote.
Original Research Article: Liu, K., et al. “Paraquat exposure induces pulmonary cell mitophagy by enhancing the PINK1/Parkin signaling.” BioMed Res Int 6 (2020): 1–6. doi: 10.1155/2020/7103105.
Written by: Abby Stahl
Original Article: Al Amir Dache et al. The FASEB Journal 2020
The Gist of It
You may have heard that mitochondria are the “powerhouse” of the cell, but what is DNA from these tiny organelles doing circulating in the bloodstream? That was the question a team of scientists at the Institut Regional du Cancer de Montpellier in France asked in response to several studies showing that DNA from mitochondria could be found in circulation.
Mitochondria are important producers of cellular energy that evolved millions of years ago through a symbiotic relationship between primitive eukaryotic cells (our ancestors) and free-living prokaryotes (bacteria). Mitochondria have retained many of their bacterial features today, including having their own DNA. The fact that mitochondrial DNA is in the bloodstream has been known by doctors for years, and its presence can even be used to help diagnose diseases and conditions such as diabetes, cancer, and heart disease. Surprisingly, this team of researchers found that there are 50,000 times more copies of mitochondrial DNA in the bloodstream than nuclear DNA, or the genomic DNA at the “core” of the cell. However, the purpose of this mitochondrial cell-free DNA (McfDNA) was still unknown.
By collecting blood plasma from healthy volunteers, the scientists found that McfDNA was longer and more stable than nuclear cell-free DNA (NcfDNA), suggesting the McfDNA might be protected or encapsulated by some larger structure. The researchers went on to find that spinning the plasma at a high speed caused sedimentation of most of the McfDNA. Spinning at high speed can also cause intact mitochondria to be spun down. In fact, using a dye that labels live mitochondria, they found an abundance of the organelles within the centrifuged blood plasma. More powerful electron microscopy confirmed that these mitochondria existed without a cell around them, and metabolic studies showed that these mitochondria were functional at consuming oxygen, an important step in energy production.
These research studies are the first to report the presence of intact and functional mitochondria in blood from healthy individuals. The scientists estimate that there are between 200,000 and 3.7 million cell-free, intact mitochondria per milliliter of blood plasma and that these mitochondria might have crucial biological roles still to be discovered!
The Nitty Gritty
In this study, the authors began by comparing the stability of DNA found in the blood of healthy individuals. They hypothesized that NcfDNA would be more stable than McfDNA due to the presence of supporting histone proteins that mitochondrial DNA lacks. To their surprise, the McfDNA had a greater DNA Integrity Index, meaning the McfDNA had a mean size greater than 300 base pairs when assessed from 13 blood samples. Sequencing of cell-free DNA from 80 additional individuals showed that McfDNA contained two populations of DNA – small fragments between 30–300 base pairs (approximately 20%) and large pieces greater than 1000 base pairs (approximately 80%); these populations could not be sequenced due to limits of sequencing technology.
To further determine the structure of this stable McfDNA, the researchers centrifuged blood samples at increasing speeds to sediment different components. Interestingly, the McfDNA sedimented at 16,000 × g, whereas NcfDNA was retained in the blood plasma, suggesting McfDNA was larger and denser. Importantly, the researchers also verified these findings in cells that had been cultured in media to confirm that the McfDNA was not being released by platelets during the experimental handling of blood samples, showing an abundance of McfDNA pelleted at 16,000 × g in cell cultures. In all cases, the McfDNA was too large to pass through a 0.22 mm filter, suggesting it might be retained within mitochondria organelles themselves, which are typically 0.4–1.5 mm in size and are known to sediment at 7,000–20,000 × g .
To confirm these suspicions, the scientists used MitoTracker Green dye to visualize live mitochondria and found them in serum samples and cell media supernatants. Western blot analysis also confirmed the presence of membrane transport proteins, TOM22 and TIM23, in these McfDNA fractions. Finally, the researchers used electron microscopy and identified structures with a double membrane and similar size to mitochondria in the samples, although they noted some morphological differences.
After determining that mitochondria were present and surrounding the McfDNA in blood and cell samples, the scientists asked whether these mitochondria were metabolically functional and able to produce energy, a process known as respiration. Using a Seahorse XF flux analyzer, the researchers found that the isolated mitochondria consumed oxygen and were sensitive to inhibition and activation by known regulators of respiration.
These studies show that human blood contains a previously unknown and significant population of mitochondria that can be useful for diagnostics; however, the physiological roles of these cell-free organelles (if any) remain a mystery for now.
Original Research Article: Al Amir Dache Z, A., et al. “Blood contains circulating cell‐free respiratory competent mitochondria.” FASEB J (2020). doi: 10.1096/fj.201901917RR. [Epub ahead of print].
Written by: Ken Estrellas