1,451 Matching Annotations
  1. Apr 2024
    1. long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution

      The authors cultivated and observed extremely small populations of bacteria over an extended period of time. In this study specifically, the authors were able to study and monitor individual cells and their growth dynamics.

    2. Next, the segment is flushed with sterile growth medium to completely rinse out the lysis buffer.

      After cleaning the segment with lysis buffer to remove wall-adhering cells, the segment must then be flushed with sterile growth medium in order to remove any remaining buffer. This is important as it allows the authors to maintain a constant environment within the segment.

    3. Using the ability to monitor individual cells in the microchemostat cultures, we observed that the oscillations in cell density correlated with specific cell morphologies

      Here the authors noticed that changes in cell density correlated with their appearance, or morphology. For instance, when the bacteria had a small population, they were small and cylindrical (healthy-looking), and their population grew rapidly without problems. However, as their numbers increased, they looked worse and started expressing more killer proteins that led to a decline in density.

    4. To demonstrate the ability of the microchemostat to facilitate analysis of complex growth dynamics, we used it to monitor the dynamics of cell populations containing a synthetic “population control” circuit

      Engineered bacteria with a synthetic population control circuit are used to study the growth and behavior of bacterial populations in controlled environments.

    5. We created a chip-based bioreactor that uses microfluidic plumbing networks to actively prevent biofilm formation. This device allows semicontinuous, planktonic growth in six independent 16-nanoliter reactors with no observable wall growth

      As depicted in Figure 1A, the authors developed a device to grow bacteria without forming biofilms, which are communities that adhere to surfaces and produce a protective slime layer. They achieved this by isolating a section of the bioreactor, eliminating and cleansing the cells within, and then reopening the section.

    6. The microchemostat is operated in one of two alternating states: (i) continuous circulation, and (ii) cleaning and dilution. During continuous circulation, the peristaltic pump moves the microculture around the growth loop at a linear velocity of ∼250 μm s–1

      The microchemostat was primarily operated in the continuous circulation mode, but it required cleaning and dilution to remove any cells adhering to the walls. During the cleaning and dilution process, mixing is stopped and the segment is isolated from the rest of the reactor and cleaned.

    7. interfere with continuous bioreactor operation

      Biofilms impacted the performance of the bioreactor, leading to a decrease in its efficiency.

    8. passive treatment

      Multiple types of passive treatment such as polyethylene glycol, ethylenediaminetetraacetic acid, polyoxyethylene sorbitan monolaurate, and bovine serum albumin were tested to see if they would prevent biofilm formation. These surface coatings were used as a passive treatment, or a treatment in which the authors would not need to actively intervene. After testing, none of the treatments proved to be effective and the authors decided they had to "actively" kill the bacteria to prevent biofilm formation.

    9. We performed more than 40 growth experiments with Escherichia coli MG1655 cells in five different chips, using a variety of growth media ([MOPS EZ Rich (Teknova, Inc.)] and LB broth with various concentrations of glucose and bacto-tryptone) at 21°C and 32°C.

      Nutrient-rich environments allow for rapid growth of bacterial cultures. This is due to the fact that nutrient-rich environments provide many different types of organic and inorganic compounds which bacteria use as a source of energy.

      Temperature growth can have a significant impact on bacterial growth. Optimal temperature allows for the metabolic processes within bacteria to perform at an effective rate, allowing for an increase in bacterial growth. When the temperature of growth medium is below or above optimal, bacterial growth decreases and cell death may occur.

      Please check out the following video on engineered bacteria and synthetic genetic circuits: Syntheic Biology: Programming Living Bacteria - Christopher Voigt https://youtu.be/lNttxYdGHs4

    10. near-constant environment

      The authors aimed for a consistent environment with fixed temperature and consistent nutrient levels to conduct a controlled study. This approach enabled them to observe cell growth while maintaining a stable environmental condition.

    11. In theory, the small population size in the microchemostat (∼102 to ∼104 cells versus ∼109 cells in macroscale cultures) should reduce the overall rate at which mutants may occur and take over a population.

      The smaller the population, the less likely mutations are to occur and spread. Conversely, maintaining a large population increases mutation probability. The authors were puzzled by the loss of regulation in the cells, as smaller populations should theoretically minimize the impact of mutations on the synthetic gene circuit, thus preventing loss of regulation.

    1. systematically sweeping the PFP and N2 flow rates over the same range as in Fig. 3A reveal the effect of molar flow rates on resultant nerve temperature

      The device in this study functions by circulating PFP through the microfluidic system, absorbing heat from its surroundings and evaporating. Dry N2, which is very cold, maintains PFP in its liquid phase, enhancing its heat absorption capacity before evaporation. Figure 3B illustrates that neither a high PFP nor N2 amount alone effectively cools the system, highlighting the necessity of both for successful nerve cooling. As depicted in figure 3C, an optimal PFP molar fraction of around 0.13 yields the most effective cooling outcome.

    2. the effect of PFP flow rate on nerve-temperature cooling rate.

      Increasing the PFP flow rate, as shown in Figure 3D, enhances the cooling rate of nerve tissue. This occurs because higher PFP flow pushes out warmed PFP more rapidly, allowing fresh, cooler PFP to absorb more heat in a shorter time. Adjusting PFP flow rate allows for controlling the tissue cooling rates.

    3. For a nerve blood flow of 50 μl/min (Fig. 3H), the temperature of the nerve increases by 2.0°C (from 3.5° to 5.5°C).

      In nerve tissue, heat is primarily transferred through direct contact, a process known as conduction. However, there's also convective heat transfer facilitated by surrounding biofluids and blood flow around the nerves. In the study, comparisons between environments where only conduction occurs (such as a hydrogel at 37°C) and those with convective effects (like water at 37°C) help illustrate these phenomena (Fig. 3G). Additionally, when the authors simulated the impact of blood flows through the targeted nerve, they found out that it contributes to a temperature increase of 2.0°C, highlighting the impact of perfusion on nerve temperature (Fig. 3H).

    4. Nerve coolers embedded in a thermochromic tissue mimic in three different configurations serve as models for experimental study of temperature gradients in radial (Fig. 4A) and longitudinal (Fig. 4B) views along the nerve and across the surface of an uncurled, planar device (Fig. 4C).

      Different orientations of the microfluidic cooling device enable precise cooling over specific regions, as the cooling effect is localized to the tissue in direct contact with the coils, rather than affecting the entire surrounding area. The cooling area within the layers of the hydrogel remains confined to the shape of the device on the surface.

    5. Thermal three-dimensional finite element analysis confirms that the cooling effect is largely confined radially (Fig. 4G) and longitudinally (Fig. 4H) within the extent of the cooling cuff and above a flat cooler (Fig. 4I). The temperature of the vapor remains confined inside the microfluidic channel, as indicated by the cold region that extends radially down and to the right in the z = 0 mm plane for Fig. 4G.

      The authors utilized finite element analysis (FEA) to demonstrate that the cooling effect of the device remains localized and does not extend beyond the applied area. As distance from the tissue surface increases, the cooling effect diminishes.

    6. In devices constructed for multiday experiments, transcutaneous connections to a bioresorbable microfluidic evaporative cooler and temperature sensor mounted to the sciatic nerve (Fig. 5C) route subcutaneously along the spine to a headcap (Fig. 5D).

      The cooling cuff is attached to the injured sciatic nerve and then routed along the spine to a headcap. This headcap is placed on the rat's head and allows for further monitoring and control of the device. Figures 5C-D illustrate how the device interfaces with the rat's sciatic nerve and the pathway of the connections to the headcap. The injured nervous tissue in the rat causes a pain response when touched, which is measured each time contact is made. By applying cooling with the device, this pain response should decrease and eventually be eliminated as the temperature decreases.

    7. Electromyography (EMG) of the tibialis anterior muscle indicates a 92% reduction in EMG magnitude and 64% increase in signal latency of neuromuscular activity during cooling from 31° to 5°C over a period of 8 min (Fig. 5A).

      Electromyography is the measurement of muscle response or electrical activity in muscle tissue when stimulated by nerves. Here, they used electromyography while simultaneously cooling the nervous tissue responsible for activity in the tibialis anterior, a large muscle in the lower half of the leg. They perfomed it over various cooling temperatures from 31°C down to 5°C , and found that as the decreased the temperature, the electrical activity in the tibialis anterior decreased.

    8. A serpentine magnesium trace with a width and length of 25 μm and 72 mm, respectively, provides temperature feedback through the temperature coefficient of resistance of Mg

      The resistance-based temperature sensor detects temperature changes by measuring variations in electrical resistance of the serpentine magnesium channel with changes in temperature.

    9. The technology consists of a hybrid microfluidic and electronic system for cooling and simultaneously measuring the temperature of a peripheral nerve

      The term microfluidic refers to the behavior and movement of fluids through micro-sized channels. In this device, perfluoropentane (PFP) flows through the microfluidic channels, and as it evaporates, it cools the surrounding system. The electronic system functions to measure the temperature of the surrounding nerves. The resistance-based temperature sensor detects temperature changes by measuring variations in electrical resistance with changes in temperature.

    10. aberrant neural signals are well defined in select anatomical regions, (ii) nerves carrying aberrant neural signals are already isolated, and (iii) a need for opioid therapy exists after operation

      The authors intend for this device to be used for pain management in cases where, after a patient undergoes a surgical operation, (i) the pain signals are in specific, contained parts of the body, (ii) the signals travel along specific nerves, ensuring that only the affected nerves are targeted while leaving others unaffected, and (iii) a non-addictive method of pain reduction is needed to avoid harmful long-term affects.

  2. Mar 2024
    1. In Top10F′ cells, more complete induction of the circuit was achieved

      Here, they use another strain of E. Coli which is more tightly regulated, leading to stronger growth regulation (i.e. does not escape the circuit regulation). However, from the cultures that were turned on at various time points (Curves 2 through 6), only culture 4 was able to sustain the oscillatory growth behavior. This observation suggests potential malfunctions within the synthetic circuit.

    2. For example, upon inoculation, culture 3 (Fig. 3A, point a) was composed of healthy (small and cylindrical) cells.

      When the bacteria were first added to the growth media, they appeared healthy. This changed as the population grew.

    3. This process is repeated sequentially on different growth chamber segments, thus eliminating biofilm formation and enabling pseudocontinuous operation.

      The process of flushing the fluidic channels and replacing the once existing biofilms with sterile medium is used by the authors to allow the bioreactor function properly. Flushing these fluidic channels must be performed actively, as the passive approached showed no effect on removing biofilms.

    4. wall-adhering cells

      Wall-adhering cells are cells that grow directly on the sides or walls of the chamber they are growing in. Wall-adhering cells create biofilms and the authors use a lysis buffer to flush these cells. At which point the sterile medium is then reconnected with the growth chamber.

    5. inoculation

      Inoculation is the process of introducing something into an environment suitable for growth. Inoculation was used to add E.coli MG1655 cells to five different chips.

    1. The microfluidic system includes transcutaneous colinear interconnects that deliver liquid coolant [perfluoropentane (PFP)] and dry N2 to a serpentine evaporation chamber

      The system includes tubes that insert through the skin and connect to the device's cooling system along the same direction. The PFP functions as a coolant by absorbing the heat of the surrounding tissue, causing it to evaporate. The N2 is quite cold relative to PFP, so it keeps the PFP in a liquid state. The more N2, the more heat the system can absorb before the PFP evaporates, so changing the amount of N2 allows for control of the temperature change.

    2. The mass flow rates of the PFP and N2 and the geometry of the evaporation chamber govern the magnitude and localization of the cooling effect. At low PFP molar flow ratios (XPFP = 0.1), the PFP fully evaporates after passing through three serpentines, with marginal liquid PFP buildup at the corners of the microchannels (Fig. 2C). At high molar flow rates (XPFP = 0.5), PFP proceeds through annular flow and passes along the sidewalls of the microchannels

      The dry N2 is used to initially keep the PFP in a liquid state. For this experiment, the molar flow ratio is the ratio between the liquid nitrogen, N2, and the coolant gas, PFP. The authors conducted two experiments, the molar flow ratio at 0.1 and 0.5, respectively. They observed that at 0.1, PFP only made it through a fraction of the system before absorbing the heat of the surroundings caused it to completely evaporate, as there is not enough N2 to keep it in liquid form. At 0.5, they observed some PFP remaining in liquid form throughout the entire system, meaning the heat of the surroundings was not enough to overcome to cooling effect of the N2.

    3. The simultaneous initiation of PFP and N2 flows into this structure prompts evaporation of PFP at the microfluidic junction between the PFP and N2 channels and along the serpentine chamber. PFP, which boils near room temperature (28° to 30°C), is bioinert and compatible with nonfluorinated elastomers.

      The location and strength of the cooling effect by the device is controlled by how much of the liquid coolant, perfluoropentane (PFP), is supplied to the serpentine system, as well as the amount of liquid nitrogen, N2. The liquid nitrogen is very cold and keeps the PFP in a liquid state. The cooling is achieved when the PFP absorbs the heat of its surroundings, causing it to evaporate. This process is known as evaporative cooling.

  3. Feb 2024
    1. shows devices wrapped around a silicone phantom nerve and submerged in phosphate-buffered saline (PBS) (pH 7.4) at 75°C, as an accelerated aging test. The results show that the materials largely dissolve within 20 days and that elimination of residues occurs after 50 days under these conditions

      In this experiment, the authors wrapped the devices around a model silicone nerve and submerged them in a solution called phosphate-buffered saline (PBS) at a high temperature (75 C). PBS is a standard aqueous solution that mimics the chemical composition and pH of bodily fluids, such as blood and interstitial fluid. The high temperature simulates an accelerated aging process, allowing them to see how quickly the materials break down over time. The results revealed that the materials mostly dissolved within 20 days, and any remaining residues disappeared after 50 days under these conditions. This helps researchers understand how the devices will behave in the body over time and ensures they are safe and biocompatible.

    2. soft, stretchable mechanics at the device level

      The device was constructed with soft materials that can change shape and direction so that it can conform to the tissue without causing damage or blocking of important processes in the body. Furthermore, the functional components of the device were constructed so that they can continue to serve their purpose and remain intact while being stretched.

    3. A bioresorbable elastomer, poly(octanediol citrate) (POC), forms the microfluidic system

      The backbone of the microfluidic device is an elastic, biocompatible, rubber-like material on which the rest of the components of the device are attached.

    4. constructed entirely with water-soluble constituent materials that controllably dissolve to biocompatible end products in the biofluids that are contained in subcutaneous tissues

      The device in this paper was constructed exclusively with materials that dissolve at a predictable rate when exposed to the fluids contained at the deepest layer of the skin, and such that both the intact device and the products of the dissolving process are harmless and processable for the human body.

    1. optical microscopy

      The authors examine a sample of bacteria cells and determine individual bacterial cells within the microchemostat using optical microscopy in figure 1. The authors display six microchemostats that operate in parallel on a single chip like a coin of 18 mm in diameter. Various inputs have been loaded with food dyes to visualize channels and sub-elements of the microchemostats. Authors demonstrate a single microchemostat and its main components. By this process authors track cell growth, density, and morphological changes over extended periods. Therefore, Optical Microscopy allows authors to observe individual cells and their responses to the synthetic circuit, shedding light on cellular behavior and interactions.

  4. May 2023
    1. colocalization of Lst1-3xGFP with Atg40-2xmCherry

      To examine the association of two proteins in the cell, colocalization of two fusion proteins with different florescence tagged fused with two proteins are utilized to be visualized under the confocal/florescence microscope, to test whether these two signals (red and green florescence in this study) could be overlapping in the cell.

      LST1 fused with GFP, the green florescence protein. ATG40 fused with mCherry, the red florescence protein.

    2. Because increased levels of autophagy receptors increase ER-phagy and the number of ER-containing autophagosomes (11, 12), ER-phagy receptors are thought to play a role in packaging ER into autophagosomes (12). To address if Lst1 works with Atg40 to perform this function, we induced Atg40 expression with rapamycin (11) and asked if this promotes an association with Lst1

      To investigate how the role of ER-phagy receptor, ATG40, involved in packaging the cargo for recycling in ER specifically, the authors made the connection of observation and results obtained in ATG40, and associated with LST1 to hypothesize that LST1 could be associated with the ER-phagy receptor, ATG40.

    3. Rapamycin also induced the expression of the nucleophagy receptor Atg39 (11) but did not induce the colocalization of Lst1 with Atg39 (fig. S8, C and D). Furthermore, Lst1 did not contribute to the degradation of the nuclear ER marker Hmg1 (fig. S8, E to H).

      In yeast, the ER consists of 3 subdomains, the cytoplasmic ER, the cortical ER, and the perinuclear ER (also called nuclear envelope).

      Atg39 specifically localizes to the nuclear envelope. Therefore, the Atg39-dependent pathway in selective autophagy terms as nucleophagy.

      Conversely, Atg40 predominantly localizes to the cytoplasmic ER and the cortical ER, and loads fragments (ER tubules or sheets) of these ER subdomains into the phagophore. Thus, Atg40 is exclusively involved in ER-phagy.

      To exclude the possibilities that LST1 is localized to the nuclear envelope and do not involve in nucleophagy, the authors examine the colocalization of Atg39 and Lst1, and rule out the possibility that the Lst1 is localized in nuclear envelope and do not reside on the nuclear envelope and its marker, Hmg1.

    4. Atg8-Lst1 colocalization in wild-type (WT) but not atg40Δ cells

      Atg8 also plays an important role in cargo recognition for selective autophagy by interacting with the receptor protein. When Atg40 is mutant, Lst1 is failed to interact and colocalize with Atg40, indicating that Lst1 is acting together with Atg40 to form autophagosome. And Sec23 is partially required with Atg40 to form autophagosome.

    5. we performed binding studies with lysates prepared from untreated and rapamycin-treated cells expressing Atg40-3xFLAG.

      This experiment is an in vitro binding assay by purifying the GST fusion of target coat proteins of the COPII and incubated with Atg40 fusion protein with FLAG epitope tagged expressed in the yeast

    6. Representative electron micrographs of WT and pep4Δ strains treated for 12 hours with rapamycin

      pep4Δ strain is a vacuolar protease-deficient yeast strain that lost protease activity wihle fail to degrade the autophagic cargo and served as negative control when compared with the wild-type original strain.

    7. atg14Δ mutant

      autophagy-deficient yeast strain which fail to form the autophagosomes to degrade and recycle substances.

    8. we examined pathway induction by flow cytometry in cells carrying an integrated UPR-regulated GFP reporter

      To induce the UPR (unfolded protein response), DTT (dithiothreitol) is added to induce protein misfolding in the ER by blocking protein disulfide bond formation, thus activate and induce the UPR.

      The relative intensity of GFP fluorescence signal in the cells was measured using the flow cytometry.

    9. SEC24C

      SEC24C in mammalian cells is the homolog for Lst1 in yeast.

    10. Torin2 (TOR inhibitor2)

      Torin 2 is a potent and selective mTOR inhibitor, which decreases cell viability and induces autophagy and apoptosis.

    11. ULK1, a component of the autophagosome biogenesis machinery

      ULK1 is one of two mammalian homologues of the yeast ATG1 kinase, known for its role in autophagy initiation

    12. How sites on the ER are targeted for ER-phagy is unclear. We reasoned that the cytosolic machinery that recognizes and binds to ER-phagy receptors may play a role in marking specific sites on the ER where autophagy will occur. Because COPII coat subunits are known to participate in membrane-budding events at the ER (1), we investigated whether coat subunits play a role in sequestering ER domains into autophagosomes during ER-phagy.

      Key question raised by the authors, how the dispersed site throughout the ER are recognized by the selective autophagy receptors to deliver the cargo for degradation.

      The authors hypothesized that, COP11 coat proteins which involved in membrane-budding at the ER, could involve in ER-phagy.

    13. FAM134B

      The mammals homolog of Atg40 in yeast. To verify the evolutionarily conserved function the ER-phagy receptor in higher order and complex organism such as in mammal cell, FAM134B, the homolog protein of Atg40 in mammals, was used to test its functional similarity.

    14. To determine whether any of the SEC24 isoforms are required for ER-phagy in mammalian cells, we knocked down each of the four isoforms by small interfering RNA (siRNA) in U2OS cells

      The authors tested whether or not the defeated of Lst1 in yeast will have the similar observation in mammalian cells by looking at the homologs of the isoforms of Sec24 in mammals.

    15. To determine whether ATZ aggregates in the absence of Atg40 or Lst1, microsomal membrane fractions prepared from WT, atg14Δ, atg40Δ, and lst1Δ cells were analyzed on sucrose gradients. Soluble ATZ was primarily found at the top of the WT gradient, whereas ATZ from mutant lysates was largely in the pellet

      ATZ-pYES2 is a vector natively expressing the ATZ, a misfolded protein, in yeast. ATZ-pYES2 used to observe a as substrate targeted for autophagy degradation.

      Wild type, and mutants of the atg40 and lst1 were transformed with ATZ-pYES2 to observe whether loss-of-functions of atg40 and lst1 will lead to the accumulation of aggregated ATZ protein in the ER, which indicates that Atg40 and Lst1 are essential ER-phagy.

  5. Apr 2023
    1. This mechanical force–induced increase in nanoparticle translocation was reduced significantly when the cells were incubated with the antioxidant NAC (Fig. 4J). This finding implies that elevated intracellular production of ROS due to a combination of mechanical strain and nanoparticle exposure might be responsible for the observed increase in barrier permeability to these nanoparticles. We also found that preconditioning of endothelial cells with physiological levels of shear stress (15 dyne/cm2) slightly increased the rate of nanoparticle translocation in the presence of mechanical stretch (fig. S11), presumably due to a shear-induced increase in endothelial permeability, as previously demonstrated by others (39).

      The mechanical force due to breathing increases the movement of nanoparticles across the alveolar-capillary barrier. This suggests that a combination of mechanical force and nanoparticle exposure may lead to an increase in the release of toxic reactive oxygen species (ROS) within the cells, which could explain why more nanoparticles were able to pass through the cell barrier.

    2. To determine the physiological relevance of these observations, we conducted similar studies in a whole mouse lung ventilation-perfusion model, which enables intratracheal injection of nebulized nanoparticles and monitoring of nanoparticle uptake into the pulmonary vasculature ex vivo (fig. S10). When 20-nm nanoparticles were injected into whole breathing mouse lung, they were delivered into the deep lung, and histological analysis revealed that they reached the surface of the alveolar epithelium, as well as the underlying interstitial space and microvasculature (Fig. 4H).

      The researchers used a mouse lung model to inject tiny particles and see how they were taken up by the lung's blood vessels. They found that when 20-nm particles were injected into a whole breathing mouse lung, they went deep into the lungs and were found on the tissue lining the surface of the alveoli, within small blood vessels, and within fluid-filled spaces that exists outside of blood vessels and lymphatic vessels.

    3. When the ultrafine (12 nm) silica nanoparticles were added to the alveolar epithelium in the absence of mechanical distortion, there was little or no ROS production. However, when the cells were subjected to physiological levels of cyclic strain (10% at 0.2 Hz), the same nanoparticles induced a steady increase in ROS production that increased by a factor of more than 4 within 2 hours (Fig. 4B), and this response could be inhibited with the free radical scavenger, N-acetylcysteine (NAC) (fig. S5). ROS levels in the underlying endothelium also increased by a factor of almost 3 over 2 hours, but initiation of this response was delayed by about 1 hour compared with the epithelium (fig. S5). Experiments with carboxylated Cd/Se quantum dots (16 nm) produced similar results (fig. S6), whereas cyclic strain alone had no effect on ROS even when applied for 24 hours (fig. S7). Nanomaterial-induced ROS production also increased in direct proportion to the level of applied strain (Fig. 4C). In contrast, exposure of alveolar epithelial cells to 50-nm superparamagnetic iron nanoparticles under the same conditions only exhibited a small transient increase in ROS production (Fig. 4D).

      The authors exposed the cells to various nanoparticles (silica nanoparticles, carboxylated Cd/Se quantum dots, 50-nm superparamagnetic iron nanoparticles) and monitored the oxidative stress on the artificial lung tissue due to toxic effects of the nanoparticles. The authors quantified the reactive oxygen species produced by the cells as a measure of oxidative stress. They found out that mechanical strain applied to the lung-on-a-chip increases the toxic effects of nanoparticles on cells.

    4. Moreover, the low level of protein permeability (2.1%/hour for fluorescently labeled albumin) exhibited by cells cultured at the air-liquid interface (Fig. 2D) closely approximated that observed in vivo (1 to ~2%/hour) (24).

      The authors introduced the fluorescently labeled albumin into the microvascular channel and monitored its diffusion across the alveolar-capillary interface. By measuring the amount of albumin that crossed the interface over a certain period of time, they could determine the permeability of the barrier.

    5. Similar studies carried out with Transwell systems that represent the state of the art for in vitro analysis of tissue barrier permeability

      The transwell migration assay is a commonly used technique for studying the migration of particles across a membrane (like the alveolar membrane). Unlike the lung-on-a-chip, which simulated particle translocation with mechanical strain experienced during breathing (figure 4), the transwell system only accounted for the movement of particles over time. This means that the transwell assay is limited in its ability to mimic particle uptake in the alveoli, as it does not accurately mimic the mechanical strain observed in human lung tissues.

  6. Mar 2023
    1. When we introduced fluorescent nanoparticles (20 nm in diameter) into the alveolar microchannel and monitored nanoparticle translocation across the alveolar-capillary barrier in air-liquid interface culture by measuring the fraction of particles retrieved from the underlying microvascular channel by continuous fluid flow (Fig. 4F), we observed only a low level of nanoparticle absorption under static conditions.

      The authors introduced fluorescent nanoparticles in the alveolar microchannel and observed the movement of these nanoparticles across the membrane, from alveolar side to the capillary side. When there was no mechanical strain, the authors observed minimal nanoparticle movement across the membrane.

    2. Because cytokines are produced by cells of the lung parenchyma, we simulated this process by introducing medium containing the potent proinflammatory mediator, tumor necrosis factor–α (TNF-α), into the alveolar microchannel in the presence of physiological mechanical strain and examined activation of the underlying microvascular endothelium by measuring ICAM-1 expression. TNF-α stimulation of the epithelium substantially increased endothelial expression of ICAM-1 within 5 hours after addition (Fig. 3A and fig. S3), whereas physiological cyclic strain had no effect on ICAM-1 expression. The activated endothelium also promoted firm adhesion of fluorescently labeled human neutrophils flowing in the vascular microchannel, which do not adhere to the endothelium without TNF-α stimulation (Fig. 3B and movies S5 and S6).

      Expression of ICAM-1 and adhesion of neutrophils are an indication of inflammatory response. Here, the authors mimicked lung inflammation by adding TNF-alpha to the epithelial (alveolar) side and measured ICAM-1 expression and neutrophil adhesion on the epithelial (capillary) side using the lung mimic device. The authors observed that TNF-alpha greatly increased ICAM-1 expression and neutrophil adhesion.

    3. The level of applied strain ranged from 5% to 15% to match normal levels of strain observed in alveoli within whole lung in vivo, as previously described (25). Vacuum application generated uniform, unidirectional mechanical strain across the channel length, as demonstrated by measured displacements of fluorescent quantum dots immobilized on the PDMS membrane (Fig. 2E and movie S2). Membrane stretching also resulted in cell shape distortion, as visualized by concomitant increases in the projected area and length of the adherent cells in the direction of applied tension (Fig. 2F and movie S3).

      To mimic the strain applied in human lungs, the researchers stretch the artificial lung tissue by 5% to 15% in one direction using vacuum. They put quantum dots (nanoscopic fluorescent particles) on the PDMS membrane to visualize the displacement and quantify membrane stretching.

    4. This was accomplished by microfabricating a microfluidic system containing two closely apposed microchannels separated by a thin (10 μm), porous, flexible

      In the alveolar regions of the human lung, layers of cells coexist and interact to help the body perform gas exchange, maintain tissue structure/function, and keep infections at bay. In this work, the authors account for the distribution of alveolar cell layers by growing two different types of human cells on opposite sides of a membrane: epithelial cells (that line the inner portion of the alveoli and accept fresh air during inhalation) and microvascular endothelial cells (that line the outside of the alveolar space and exchange carbon dioxide waste during exhalation). This membrane is special because it can be stretched out, mimicking the expansion and relaxation of an alveolar sack during breathing! In a variety of tests, the authors confirmed their lung-on-a-chip device to be a viable and reproducible imitation of the human lung.

    5. Application of physiological cyclic strain (10% at 0.2 Hz) also induced cell alignment in the endothelial cells in the lower compartment (fig. S2 and movie S4) and, hence, mimicked physiological responses previously observed in cultured endothelium and in living blood vessels in vivo (26, 27). Cyclic stretching caused some pulsatility in the fluid flow, but this unsteady effect was negligible due to the small channel size and low stretching frequency (see SOM text).

      The cyclic strain of 10% at 0.2Hz mimics inhaling and exhaling because 10% is the average strain on the lungs when breathing and 0.2Hz corresponds to the rate of breathing. It is noted that the periodic variations in flow were negligible.

    6. We mimicked this subatmospheric, pressure-driven stretching by incorporating two larger, lateral microchambers into the device design. When vacuum is applied to these chambers, it produces elastic deformation of the thin wall that separates the cell-containing microchannels from the side chambers; this causes stretching of the attached PDMS membrane and the adherent tissue layers (Fig. 1A, right versus left). When the vacuum is released, elastic recoil of PDMS causes the membrane and adherent cells to relax to their original size. This design replicates dynamic mechanical distortion of the alveolar-capillary interface caused by breathing movements.

      The two chambers on each side of the device allow for the application of a vacuum that stretches the membrane mimicking the alveolar sack, thereby mimicking how lung cells are exposed to mechanical stretching while breathing.

    1. pep4Δ strains

      vacuolar protease-deficient yeast strain that lost protease activity wihle fail to degrade the autophagic cargo and served as negative control when compared with the wild-type original strain.

    2. ubiquitin-like protein Atg8

      Atg8 protein is a marker protein to observe autophagosome formation. When Atg8 protein engineered with the red florescence protein (RFP), RFP-Atg8, RFP-Atg8 protein will be conjugated to the lipids and enable the membrane fusion to localize on the autophagosome.

    3. green fluorescent protein (GFP)–autophagy-related protein 8 (Atg8)

      In short, GFP-ATG8. GFP is a protein in the jellyfish Aequorea Victori that exhibits bright green fluorescence when excited at a wavelength of 488nm and has an emission peak at about 507nm ( blue to ultraviolet range).

      GFP is served as biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression.

      GFP consists of 238 amino acid with 27 kilo Dalton of the protein size. When ATG8 fused to GFP (GFP-ATG8), the ATG8 here as a protein of interest (ATG8 is a ubiquitin-like protein required for the formation of autophagosomal membranes) to carry the GFP which used as a reporter and exhibit green signal.

      GFP-ATG8 will serve as a protein visualized marker gene which localized on the double membrane vesicle, the autophosome.

    4. Atg8-Lst1 colocalization in wild-type (WT) but not atg40Δ cells

      Atg8 also plays an important role in cargo recognition for selective autophagy by interacting with the receptor protein. When Atg40 is mutant, Lst1 is failed to interact and colocalize with Atg40, indicating that Lst1 is acting together with Atg40 to form autophagosome. And Sec23 is partially required with Atg40 to form autophagosome.

  7. Feb 2023
    1. A 1-cm-thick soil column (connecting two air-filled chambers) was either left empty (control) or filled with uncompacted soil (1.1 g cm–3) or compacted soil (1.6 g cm–3) (Fig. 3I and fig. S25B). Ethylene was injected into the upper chamber (an increase in pressure was avoided) and ethylene concentrations were subsequently measured over time in the lower chamber until an equilibrium was reached between the chambers.

      In this experiment, the authors used a vertical equipment consisting of a glass chamber at the top, a glass chamber at the bottom, and a column between the two chambers connecting them. The column is filled with uncompacted or compacted soil, or left empty. Ethylene was injected to the upper chamber to a certain initial concentration, and then was allowed to diffuse through the column to the bottom chamber. Samples were taken from the upper and the bottom chambers for measurement of ethylene concentration.

    2. Mathematical modeling

      The authors constructed mathematical models to predict the efficiency of ethylene diffusion in uncompacted or compacted soil.

    3. We used transgenic Arabidopsis and rice expressing either an ethylene biosensor featuring EIN3 (9) or OsEIL1 sequences fused with GFP

      In this experiment, the authors grew Arabidopsis or rice roots expressing GFP-based ethylene-responsive reporters, 35S:EIN3-GFP (Arabidopsis) and proOsEIL:OsEIL1-GFP (rice) in uncompacted or compacted soil. They measured ethylene response by visulizing the intensity of green signals released by the reporters.

    4. ethylene treatment

      In this experiment, the authors treated wild-type rice roots with or without ethylene. They measured root width and cap shape.

    5. we compared their impact on root tip shape

      In this experiment, the authors aimed to determine if mechanical imdedance plays a role in affecting root growth in compacted soil, in addition to the effects caused by ethylene. The authors grew wild-type rice and ethylene-insensitive rice ein2 mutant in compacted or uncompacted soil. Supposedly, ein2 mutant should also show some degrees of root growth change if mechanical impedance plays a role. They imaged roots with microscope and measured the area of root cap.

    6. this growth response also occurred in other classes of roots

      In this experiment, the authors grew different Arabidopsis lines in compacted or uncompacted soil. Arabidopsis lines used included wild-type (in which the ethylene response functions normally) and lines defective in ethylene response (etr1). They used microscope to image root tips and measure epidermal cell length and cortical cell diameter.

    7. we grew rice lines in columns entirely filled with either uncompacted soil (1.1 g cm–3) or highly compacted soil (1.6 g cm–3, with a 1-cm top layer packed at 1.1 g cm–3 to help establish seedling root growth).

      In this experiment, the authors grew different rice lines in compacted or uncompacted soil. Rice lines used included wild-type (in which the ethylene response functions normally) and lines defective in ethylene response (osein2 and oseil1). They used computed tomography to generate images of roots and measured root length.

      The first author showed a video demostrating the facility for growing plants and the imaging system: https://www.youtube.com/watch?v=pk5_7cA5qLo

    8. Roots exposed to elevated levels of ethylene exhibited growth inhibition

      In this experiment, the authors did two sets of experiments. First, they grew plants with or without exposure to high levels of ethylene and measured root length and root diameter. Second, they grew plants in uncompacted or compacted soil and did the same measurements.

    9. we treated roots expressing

      In this experiment, the authors applied the gas barrier to the tips of roots expressing GFP-based hypoxia-responsive reporters, pPCO1:GFP-GUS, pPCO2:GFP-GUS and RAP2.12-GFP. They measured hypoxia response by visulizing the intensity of green signals released by the reporters.

    10. examined the effect of covering root tips with a gas-impermeable barrier

      In this experiment, the authors applied a type of grease (gas barrier) to root tips to inhibit gas diffusion. They measured ethylene response by visulizing the intensity of green signals released by the ethylene-responsive EIN3-GFP marker.

    1. We also demonstrated that this system could mimic the innate cellular response to pulmonary infection of bacterial origin. Living Escherichia coli bacteria constitutively expressing green fluorescent protein (GFP) were added to the alveolar microchannel. The presence of these pathogens on the apical surface of the alveolar epithelium for 5 hours was sufficient to activate the underlying endothelium, as indicated by capture of circulating neutrophils and their transmigration into the alveolar microchannel. Upon reaching the alveolar surface, the neutrophils displayed directional movement toward the bacteria, which they then engulfed over a period of a few minutes (Fig. 3E and movies S8 and S9), and the phagocytic activity of the neutrophils continued until most bacteria were cleared from the observation area.

      They placed E. coli. cells on the top (alveolar) side of the membrane to mimic a lung infection. The neutrophils on the bottom (capillary) side of the membrane migrated across the membrane, captured and engulfed the bacteria on the epithelial surface. This process is similar to how our body fights lung infections.

    2. Real-time, high-resolution, fluorescent microscopic visualization revealed that soon after adhering, the neutrophils flattened (fig. S3) and migrated over the apical surface of the endothelium until they found cell-cell junctions, where they underwent diapedesis and transmigrated across the capillary-alveolar barrier through the membrane pores over the period of several minutes (Fig. 3C and movie S7). Phase-contrast microscopic visualization on the opposite side of the membrane revealed neutrophils crawling up through the spaces between neighboring cells and emerging on the surface of the overlying alveolar epithelium (Fig. 3D), where they remained adherent despite active fluid flow and cyclic stretching.

      Upon exposure to inflammatory molecules such as TNF, the immune cells (neutrophils) initially adhered to the endothelial cells on the bottom of the membrane. Then, they migrated across the cell-coated membrane over several minutes. They remained on top of the alveolar epithelium in the presence of fluid flow and breathing movements. This behavior of the immune cells (neutrophils) is similar to the immune response of our body to a lung infection.

    3. Addition of air to the upper channel resulted in increased surfactant production by the epithelium (Fig. 2B and fig. S1), which stabilizes the thin liquid layer in vitro as it does in whole lung in vivo, such that no drying was observed. This was also accompanied by an increase in electrical resistance across the tissue layers (Fig. 2C) and enhanced molecular barrier function relative to cells cultured under liquid medium (Fig. 2D).

      The authors used three methods to check whether their microdevice mimics the lung tissue. 1) Surfactant production by epithelial cells, 2) Electrical resistance measurements across the membrane, 3) Transport of a small protein across the membrane. The authors confirmed that the cell-coated membrane closely emulates alveolar sacks after two weeks of culture, as they produced surfactants (Fig 2b), and displayed high electrical resistance (Fig. 2c) and did not allow transport of small proteins (albumin) across the membrane (Fig 2d).

    4. When human alveolar epithelial cells and microvascular endothelial cells were introduced into their respective channels, they attached to opposite surfaces of the ECM-coated membrane and formed intact monolayers composed of cells linked by continuous junctional complexes containing the epithelial and endothelial junctional proteins, occludin and vascular endothelial cadherin (VE-cadherin), respectively (Fig. 2A). These cells remained viable for prolonged periods (>2 weeks) after air was introduced into the epithelial microchannel and the alveolar cells were maintained at an air-liquid interface (fig. S1).

      The human alveolar epithelial cells were grown and formed a uniform layer of cells on the top side of the membrane mimicking the lung surface. Similarly, the microvascular endothelial cells were grown and formed a uniform layer of cells on the bottom side of the membrane mimicking the capillary surface. The authors checked whether an intact, continuous cell layer was formed on each side of the membrane by imaging the proteins (such as occludin, VE-cadherin) that help connect cells to each other to form leak-free junctions. The cells stayed alive for over 2 weeks in an air-liquid interface.

    5. To mimic delivery of airborne nanoparticles into the lung using our microdevice, we injected nanoparticle solution into the alveolar microchannel and then gently aspirated the solution to leave a thin liquid layer containing nanoparticles covering the epithelial surface. When alveolar epithelial cells were exposed for 5 hours to 12-nm silica nanoparticles that are commonly used to model the toxic effects of ultrafine airborne particles (34–36), the underlying endothelium in the microvascular channel became activated and exhibited high levels of ICAM-1 expression (Fig. 4A).

      Authors put a nanoparticle solution in the microchannel and withdrew enough of the solution to leave a thin layer on the epithelial surface. The endothelium displayed ICAM-1 expression meaning that there was an immune response.

    6. Fabrication begins with alignment and permanent bonding of a 10-μm-thick porous PDMS membrane (containing 10-μm-wide pentagonal pores) and two PDMS layers containing recessed microchannels (Fig. 1C). A PDMS etching solution composed of tetrabutylammonium fluoride and N-methylpyrrolidinone is then pumped through the side channels (Fig. 1D). Within a few minutes, PDMS etchant completely dissolves away portions of the membrane in the side channels, creating two large chambers directly adjacent to the culture microchannels.

      A porous PDMS layer is sandwiched between two PDMS layers with three open, large sections and microchannels. After they are permanently attached, a chemical is used to dissolve the porous PDMS layer from the two outer sections.

    1. atg14Δ mutant

      autophagy-deficient yeast strain which fail to form the autophagosomes to degrade and recycle substances.

    2. SEC24C

      SEC24C in mammalian cells is the homolog for Lst1 in yeast.

    3. we examined pathway induction by flow cytometry in cells carrying an integrated UPR-regulated GFP reporter

      To induce the UPR (unfolded protein response), DTT (dithiothreitol) is added to induce protein misfolding in the ER by blocking protein disulfide bond formation, thus activate and induce the UPR.

      The relative intensity of GFP fluorescence signal in the cells was measured using the flow cytometry.

    4. Torin2 (TOR inhibitor2)

      Torin 2 is a potent and selective mTOR inhibitor, which decreases cell viability and induces autophagy and apoptosis.

    5. ULK1, a component of the autophagosome biogenesis machinery

      ULK1 is one of two mammalian homologues of the yeast ATG1 kinase, known for its role in autophagy initiation

    6. cleavage of green fluorescent protein (GFP)–autophagy-related protein 8 (Atg8) in the vacuole

      A semi-quantitative GFP-Atg8 cleavage assay to observe the vacuolar delivery of GFP-Atg8 by bulk autophagy assessed by Western blotting using an anti-GFP antibody.

    7. To determine whether Lst1 functions in bulk autophagy, we measured the activation of vacuolar alkaline phosphatase (Pho8Δ60) 4 hours after starvation

      To measure the bulk autophagic flux from the cytosol into the vacuole, the Pho8Δ60 assay measures the enzymatic activity resulting from delivery to the vacuole from cytosol. The genetic engineered Pho8 truncated of 60 amino acid of the N-terminal fragment will remain in the cytosol and is delivered to the vacuole only through bulk autophagy.

      A journal article to explain assays to monitor autophagy in yeast. https://doi.org/10.3390/cells6030023

    8. lst1Δ, iss1Δ, and sec24-3A mutants,

      Two other yeast SEC24 paralogs, lst1 and issI, are also cargo protein of COPII complexes. Mammalian cells have four SEC24 homologues.

      The loss of function of the SEC24 paralogs in yeast, lst1Δ, iss1Δ, were used to determine which subunit of the SEC24 coat protein is involved specifically in the selective ER-phagy, but not the non-selective bulk autophagy.

      sec24-3A mutant is the phosphorylation site mutated mutant from changed from Threonine to Alanine amino acid (Sec24-T324A/T325A/T328A = Sec24-3A mutant).

    9. The long incubation period with rapamycin to induce ER-phagy

      Rapamycin is a drug to induce ER stress by inhibiting the mTOR pathway in the autophagy process. Long incubation time is refer to 24hrs.

    10. How sites on the ER are targeted for ER-phagy is unclear. We reasoned that the cytosolic machinery that recognizes and binds to ER-phagy receptors may play a role in marking specific sites on the ER where autophagy will occur. Because COPII coat subunits are known to participate in membrane-budding events at the ER (1), we investigated whether coat subunits play a role in sequestering ER domains into autophagosomes during ER-phagy.

      Key question raised by the authors, how the dispersed site throughout the ER are recognized by the selective autophagy receptors to deliver the cargo for degradation.

      The authors hypothesized that, COP11 coat proteins which involved in membrane-budding at the ER, could involve in ER-phagy.

    11. Because increased levels of autophagy receptors increase ER-phagy and the number of ER-containing autophagosomes (11, 12), ER-phagy receptors are thought to play a role in packaging ER into autophagosomes (12). To address if Lst1 works with Atg40 to perform this function, we induced Atg40 expression with rapamycin (11) and asked if this promotes an association with Lst1.

      To investigate how the role of ER-phagy receptor, ATG40, involved in packaging the cargo for recycling in ER specifically, the authors made the connection of observation and results obtained in ATG40, and associated with LST1 to hypothesize that LST1 could be associated with the ER-phagy receptor, ATG40.

    12. colocalization of Lst1-3xGFP with Atg40-2xmCherry

      To examine the association of two proteins in the cell, colocalization of two fusion proteins with different florescence tagged fused with two proteins are utilized to be visualized under the confocal/florescence microscope, to test whether these two signals (red and green florescence in this study) could be overlapping in the cell.

      LST1 fused with GFP, the green florescence protein. ATG40 fused with mCherry, the red florescence protein.

    13. Rapamycin also induced the expression of the nucleophagy receptor Atg39 (11) but did not induce the colocalization of Lst1 with Atg39 (fig. S8, C and D). Furthermore, Lst1 did not contribute to the degradation of the nuclear ER marker Hmg1 (fig. S8, E to H).

      In yeast, the ER consists of 3 subdomains, the cytoplasmic ER, the cortical ER, and the perinuclear ER (also called nuclear envelope).

      Atg39 specifically localizes to the nuclear envelope. Therefore, the Atg39-dependent pathway in selective autophagy terms as nucleophagy.

      Conversely, Atg40 predominantly localizes to the cytoplasmic ER and the cortical ER, and loads fragments (ER tubules or sheets) of these ER subdomains into the phagophore. Thus, Atg40 is exclusively involved in ER-phagy.

      To exclude the possibilities that LST1 is localized to the nuclear envelope and do not involve in nucleophagy, the authors examine the colocalization of Atg39 and Lst1, and rule out the possibility that the Lst1 is localized in nuclear envelope and do not reside on the nuclear envelope and its marker, Hmg1.

    14. Lst1 interacts with Atg40, we performed binding studies with lysates prepared from untreated and rapamycin-treated cells expressing Atg40-3xFLAG.

      This experiment is an in vitro binding assay by purifying the GST fusion of target coat proteins of the COPII and incubated with Atg40 fusion protein with FLAG epitope tagged expressed in the yeast.

  8. Dec 2022
    1. We fractionated a crude cytoplasmic extract (2, 3) (fig. S3A) and verified by immunoblotting that the resulting cytosol was largely devoid of mitochondria, as indicated by the absence of a mitochondrial marker protein, the voltage-dependent anion channel (fig. S3B).

      Here, the authors wanted to test how the speed of apoptotic trigger waves are affected in the absence of the mitochondria. As a result, they obtained the cytosolic extract by fractionation and checked for the absence of the mitochondria using a marker protein by immunoblotting.

      The marker protein resides and functions in the mitochondria and hence the absence of this protein can be used as an indicator for the absence of the mitochondria in the extract.

      Immunoblotting (also called western blotting) is a technique used to detect the presence and levels of a specific protein in biological samples.

      Here is a video illustrating how immunoblotting is performed in the lab: https://www.youtube.com/watch?v=OkH8u84t84M

    2. To further test this possibility, we added recombinant GST–Bcl-2 protein to cytoplasmic extracts to see whether trigger waves were affected. Bcl-2 is a stoichiometric inhibitor of the pro-apoptotic truncated Bid (tBid) protein and of the pore-forming Bak and Bax proteins, and so the expectation was that GST–Bcl-2 would slow the trigger waves

      Here, the authors wanted to know if Bax and Bak contributed to the generation and speed of apoptotic trigger waves.

      To test this they blocked the activity of Bax and Bak by adding Bcl-2 (known inhibitor of Bax and Bak) to the extract and tested the speed of the apoptotic trigger waves as before.

  9. Nov 2022
    1. Fig. 1. RECS1 induces cell death through the mitochondrial pathway of apoptosis. (A) Left: MEFs expressing doxycycline-inducible Flag-RECS1 were stimulated with doxycycline (DOX), and the levels of RECS1 were determined by Western blot. Right: Cell death was determined by PI and SYTO16 costaining followed by automated microscopy (n = 7). Mw, weight-average molecular weight; NT, not treated. (B) MEFs were transiently transfected with the indicated concentrations of MYC-RECS1 followed by Western blot analysis. (C) Left: Bright-field (BF) and PI fluorescent images of MEFs transfected with 0.4 μg of MYC-RECS1 for 24 hours. Right: Cell death was determined by PI staining followed by FACS (n = 3). (D) Left: Images of MEF Flag-RECS1 cells cultured with doxycycline in the presence or absence of the caspase inhibitor QvD-OPh (QvD; 40 μM) for 24 hours (red, PI staining). Right: Corresponding cell death kinetics (n = 3). (E) PI staining and FACS of cells treated as in (D) (n = 4). (F) MEF Flag-RECS1 wild-type (WT) or BAX and BAK (BB) double-knockout (DKO) cells were treated with doxycycline, and indicated proteins were assessed by Western blot. (G) Indicated cells were cultured with doxycycline for 38 hours, and cell death was determined by FACS (n = 3). (H) Images (left) and quantification (right) of RECS1 (Flag) colocalization with indicated organelle markers in Flag-RECS1 MEFs treated with doxycycline. GA, Golgi apparatus. Data represent means ± SD (A, C, and D) or means ± SEM. Statistically significant differences were determined comparing the best individual fit for each curve using the extra-sum-of-squares F test (A and D) or two-way analysis of variance (ANOVA) followed by Holm-Sidak’s multiple comparisons test (C, E, and G). *P < 0.05; **P < 0.01; ****P < 0.0001).

      Question:

      Through which pathway does RECS1 induce cell death

      Western blot:

      A technique to analyze/detect the levels of a specific protein in the sample.

      Wild type (WT) Cells:

      These are normal cells with all expected qualities and properties.

      BAX and BAK (BB) double-knockout (DKO) cells: Cells in which both BAX and BAK have been artificially made non-functional.

      1A

      Doxycycline was used to control (induce) expression of RECS1. (left) RECS1 expression increased over 24 hours as measured by western blot using anti-FLAG antibody. (Right) Induction of expression of RECS1 by doxycycline alone caused about 20% increase in cell death after 28 hours. Cell death was estimated from the number of cells with propidium iodide stain.

      1B

      Increased concentration of plasmid result in more RECS1 in cells. Cells were transformed with MYC-RECS1, and the level of this protein was detected by western blot.

      1C.

      Cell death increased at higher concentration (overexpression) of RECS1. Here, MYC-RECS1 plasmid was introduced into cells and then treated with PI. Cells that stain red are dead and are more when 0.4ug of MYC-RECS1 is applied, compared to when 0.1ug is applied.

      1D & E.

      QvD-OPh (QvD) is an inhibitor of BAX and BAK proteins. These are caspases that mediate cell death through the mitochondria. Inhibiting these proteins reduced apoptosis.

      1F.

      Overexpressing RECS1 in cells lacking BAX and BAK prevents cell death. Therefore, RECS1 induces cell death through BAK and BAX i.e., though the mitochondrial apoptosis pathway.

      1H

      RECS1 colocalizes with lysosome markers LAMP1, LAMP2 and GM130 but not the ER marker, ERp72. Here, it regulates the susceptibility of cells to lysosomal stress.

    2. Fig. 9. RECS1-like/CG9722 modulates viability in response to intracellular stress in D. melanogaster. (A) One copy of dRECS1 was overexpressed in the wing imaginal discs of animals fed with control food or food supplemented with CQ (100 μg/ml) or Tm (10 μg/ml). The phenotype of adult male (right) and female (left) wings was quantified (n = 3 to 7, >80 wings per condition). (B) Control (yw) and mutant (dRECS1KO) male and female flies were treated with Tm (10 μg/ml), and the animal death rate was quantified daily (n = 2 to 3). (C) WT, dRECS1KO, or dRECS1IR pupae were treated with Tm, and the percentage of eclosion was quantified (n = 2 to 4). (D) WT and dRECS1KO female flies were fed a normal diet or starved, and the death rate was daily quantified (n = 3). (E) WT and dRECS1KO knockout male flies were fed a normal diet or starved, and the death rate was daily quantified (n = 2 to 3). Data represent means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****P < 0.0001.

      Figure 9

      9A

      In the presence of lysosomal stress inducers, TM and CQ, overexpression of dRECS1 amplified abnormal wing development in flies.

      9B & C

      Flies with reduced dRECS1 expression exhibit longevity in ER stress-inducing conditions

      9D & E

      dRECS1KO female flies (flies without RECS1 expression) are more resistant to nutrient starvation. CG9722 (dRECS1) regulates ER and lysosomal stress-induced apoptosis in flies

    3. Fig. 7. RECS1 is a pH-dependent calcium and sodium channel. (A) Alignment showing the conservation of the critical Arg60 residue from BsYetJ. Right: Comparison of the conserved di-aspartyl sensor. (B) Tridimensional structural model of the human RECS1 in closed and open conformations. Zoom: Key conserved C-terminal residues involved in pH sensing and channel opening. (C) X. laevis oocytes were injected with mRNA for Flag-RECS1 WT or D295Q, and ionic single-channel currents were recorded in the cell-attached mode using patch clamp at pH 7 or 6.5. Inset: Amplified plots depicting three opening states for RECS1 (O1, O2, and O3). (D) Quantification of the probability to find the channel in the open state (NPo) for RECS1 WT and D295Q (left). The NPo for open states O1 (middle) and O2 (right) is also shown [n = 4 (RECS1 WT) or n = 3 (RECS1 D295Q)]. (E) Images of X. laevis oocytes injected with different concentrations of Flag-RECS1 (top) and oocyte survival (bottom). Arrowhead indicates morphological alterations (n = 3). NI, not injected. (F) Recordings of macroscopic currents using the cut-open technique. (G) Permeability ratios with respect to K+ for indicated cations (n = 3 to 7). (H) Top: Indicated MEF Flag-RECS1 cells were treated with doxycycline, and protein levels were monitored by Western blot. Dashed lines indicate Western blot splicing. Bottom: Cells were treated with doxycycline and 25 μM CQ for 24 hours. Cell death was determined by FACS and normalized to WT (100%) (n = 3). Data represent means ± SEM. Statistical differences were determined by two-way (D, E, and G) or one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

      Figure 7

      Driving question: What is the possible biochemical role or nature of RECS1?

      7A & B

      Multiple sequence alignment of proteins related to RECS1, based on a critically conserved Arg 60 residue of a closely related bacteria BsYetJ (7A). MODELLER platform was used to construct a 3-D structural model of RECS1 in both the open and closed states. Model suggests RECS1 to be an ion channel, particularly Asp 295 as a critical residue in this function (senses pH changes and regulates opening). An equivalent Asp residue is critical in other related proteins.

      7C & D

      Testing the electrophysiological properties of RECS1. Does RECS1 allow ionic movements? Under what conditions? At a neutral pH, 7 and constant voltage, both RECS1 WT and mutant cells exhibited similar spontaneous current spikes, which reduced at lower pH (6.5) in three open states (01-03), suggesting that RECS1 is pH-dependent channel. Probability of finding RECS1 in an open state also reduces with pH, confirming that RECS1 activity is pH-dependent.

      7E

      Frog larvae (Oocytes) overexpressing RECS1 die in RECS1-dose dependent manner, consistent with the role of RECS1 a death regulator.

      7F & G

      Ca2+ ions are more permeable that Na+ ions in RECS1-dependent manner (F), while mutation of RECS1 at Asp295 hinders influx of Ca2+ and Na+ but not Ba+ or Cs2+ (G).

      7H

      Overexpression of D295Q (RECS1 mutant) blocks lysosomal stress induced MEF cell death, thus functional RECS1 is required for lysosomal stress-induced cell death regulation.

    4. Fig. 5. Inhibition of the endogenous RECS1 protects cells against ER stress. (A) Knockdown of the endogenous mouse RECS1 using two different siRNA pools was confirmed by quantitative polymerase chain reaction (qPCR). (B) Left: Cells transfected with RECS1 siRNAs were treated with Tm (50 ng/ml), and cell death was determined by SYTOX green staining. A representative experiment from four independent experiments is shown. Right: Quantification of cell death at 24 hours (n = 4). (C) Cell death kinetics of cells transfected as in (B) and treated with 25 nM Tg. Left: Representative kinetics. Right: Quantification at 24 hours (n = 3). Data are shown as means ± SD. Statistical differences were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

      What is the function of native RECS1 in cell death regulation and control of ER stress.

      5A

      RNA interference was used to transiently reduce expression of RECS1 in MEFs. No effect on basal cell viability was observed.

      5B & C

      Silencing RECS1 reduced the effect of Tm and Tg- induced ER stress on MEF cells.

    5. Fig. 4. RECS1 sensitizes cells to ER stress. (A) Annexin V versus PI FACS density plots from MEF Flag-RECS1 cells cultured with doxycycline for 16 hours and then treated with Tm (100 ng/ml) for 16 hours followed by FACS (n = 9). (B) Experimental design for cell death kinetic experiments. (C) Cell death kinetic analyses of MEFs treated as in (A) (n = 6). (D) Cell death of MEF Flag-RECS1 cells incubated with doxycycline and then treated with 100 nM Tg for 24 hours (n = 3). (E) Corresponding cell death kinetic analysis (n = 3). (F) Clonal assay of MEF Flag-RECS1 cells treated with doxycycline alone or in the presence of Tm (50 ng/ml) or 25 nM Tg. (G) Cell death kinetics of MEF Flag-RECS1 cells cultured with doxycycline and then treated with Tm (100 ng/ml) in the presence or absence of 40 μM QvD (n = 3). (H) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and treated with Tm (100 ng/ml) or 50 nM Tg for 24 hours. Cell death was determined by FACS (n = 2 to 3). Data are shown as means ± SD (C, E, and G) or means ± SEM. Statistical differences were determined by the extra-sum-of-squares F test (C, E, and G) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      What is the effect of RECS1 over expression on ER- induced cell death

      4A

      Overexpression of RECS1 in ER stressed cells (TM treatment) triggers cell death. Dead cells are shown by annexin V and PI.

      4B & C

      Cell death correlates with ER stress induction and progression in RECS1 over expressing MEF cells

      4D & E

      Cell death correlates with ER stress induction and progression in RECS1 over expressing cells treated with calcium pump inhibitor, Thapsigargin (Tg).

      4F

      Cells over expressing RECS1 have reduced long term survival and reduced growth under ER stress

      4G & H

      QvD and Tm (G), BAX and BAK deficiency inhibited RECS1-triggered cell death under ER stress as expected.

    6. Fig. 3. RECS1 causes LMP in cells undergoing stress. (A) HeLa Flag-RECS1 cells were cultured with doxycycline and then treated with indicated concentrations of CQ, QvD, or both for 16 hours. Immunofluorescence against galectin-1 was performed. (B) Quantification of galectin-1 puncta per field (n = 2; 6 to 27 fields in total). (C) MEF Flag-RECS1 cells were cultured with doxycycline and treated with 50 μM CQ for indicated time points. The enzymatic activities of cathepsin D, acid phosphatase, and hexosaminidase were assessed from cytosolic, lysosomal-free fractions (n = 3). (D) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and then treated with indicated concentrations of CQ for 24 hours. Cell death was determined by PI staining followed by FACS (n = 3). (E) MEF Flag-RECS1 cells were cultured with doxycycline and treated with CQ for 16 hours. Immunofluorescence against LAMP-2 (green), BAX N-terminal domain (red, BAX 6A7 antibody), and Flag-RECS1 (blue, anti-Flag antibody) was performed. The triple colocalization of Flag-RECS1, LAMP-2, and active BAX is visualized as white color. (F) Representative immunofluorescence images of Flag-RECS1 MEF cells treated as in (E). Bottom panels correspond to zoomed white boxes (marked as z) from the top panels. (G) Intensity profiles for LAMP-2, BAX, and Flag-RECS1 signals along the line shown with an L1 (left) or an L2 (right) in (F). Data are shown as means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Effect of RECS1 expression on Lysosome membrane permeabilization

      3A & B

      After LMP, the cytosolic proteins LGALS1/galectin-1 and LGALS3/galectin 3 relocate to the lysosomal glycocalyx and form punctate structures. Cells overexpressing RECS1 had increased LGALS1/galectin-1 puncta at high chloroquine doses.

      3C

      The activity of lysosome enzymes, notably cathepsin D, acid phosphatase and hexosaminidase is increased in RECS1 overexpressing cells under chloroquine treatment.

      3D.

      The resistance of BAK and BAX DKO cells to lysosomal stress-induced cell death confirms that these caspases are required in this process.

      3E & G

      BAX colocalizes with RECS1 in in LAMP2 containing vesicles and redistributes to larger LAMP2 puncta, specifically in RECS1-positive lysosomes upon CQ treatment in a RECS1-dpemdndent manner.

    7. Fig. 2. RECS1 triggers cell death upon lysosomal stress. (A) Flag-RECS1 MEFs were stimulated with doxycycline for 16 hours and treated with a library of 77 cytotoxic compounds for 24 hours. Cell death was determined by PI staining using an ImageXpress Micro XL automated microscope. ETO, etoposide; NVB, vinorelbine tartrate; DTX, docetaxel. (B) Heatmap of the top 10 sensitizing compounds in (A). For each compound, heatmap colors depict doxycycline-treated PI values after the subtraction of cell death induced by doxycycline alone and by the compound in the absence of doxycycline. *CQ and HCQ were used at 50 μM. **Cisplatin (CDDP) was used at 150 μM. (C) Left: Representative images of MEF Flag-RECS1 cells pretreated with doxycycline and treated with 50 μM CQ for 24 hours (red, PI staining). Right: Cell death kinetic analyses of cells treated as in (B) (n = 3). (D) Cells were cultured as in (C) and then treated with indicated concentrations of bafilomycin A1 for 24 hours. Cell death was determined by FACS (n = 4). (E) Left: Representative images of HeLa Flag-RECS1 cells treated as in (C). Right: Corresponding cell death kinetic analyses (n = 3). (F) HeLa Flag-RECS1 cells were treated as in (E), and cell death was assessed by FACS (n = 3). Data are shown as means ± SD (C and E) or means ± SEM (A, D, and F). Statistically significant differences were determined using the extra-sum-of-squares F test (C and E) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001.

      Role of RECS1 in stress-induced cell death

      2A & B

      Cell death was triggered in RECS1-overexpressing cells treated with a subset of lysosomal stress-inducing drugs but not other drugs. Chloroquine and Hydroxychloroquine treatments were associated with the highest cell death

      2C & D

      Treatment of RECS1 overexpressing MEF cells with Chloroquine (lysosomal stressor) or Vacuolar ATPase inhibitor, bafilomycin resulted in over 80% cell death in 24 hours, suggesting that RESC1 mediates cell death triggered by lysosomal stress.

      2E & F

      Treatment of RECS1 overexpressing Hela cells with Chloroquine or bafilomycin resulted in over 60% cell death in 24 hours.

    8. In the open state, this latch becomes dislodged, and the second transmembrane is displaced out, leading to the opening of the channel (Fig. 7B, magenta). Our model suggests that RECS1, as demonstrated for other proteins of the LFG and BI-1 families (11, 12, 25), could operate as an ion channel. Our bioinformatic analysis also suggests that Asp295 (D295) is a strong candidate to regulate the channel activity of RECS1 and was therefore selected for mutagenesis and functional studies.

      The authors think that Asp295 is very important in regulating the activity of this predicted ion channel. They then make this residue non-functional to study its function in cell death

    9. Fig. 6. RECS1 increases lysosomal acidification and calcium content. (A) Lysosomal pH of HeLa Flag-RECS1 loaded with FuraDx and OGDx and cultured in the absence or presence of doxycycline for 24 hours (n = 5, 15 to 16 total coverslips). (B) Corresponding lysosomal calcium concentration. (C) Left: Cytosolic calcium levels were determined in HeLa Flag-RECS1 cells treated with doxycycline. Cells were treated with 5 μM Tg to stimulate ER calcium release. Right: Maximum ER calcium peak (n = 4, 10 to 11 coverslips). ns, not significant. (D) MEF WT and BAX and BAK DKO Flag-RECS1 cells were loaded as in (A), and lysosomal pH was determined after 24 hours of doxycycline treatment (n = 6, 12 to 17 coverslips per condition). (E) Lysosomal calcium measurements (n = 6). (F) Knockdown of the endogenous mouse RECS1 by siRNA was confirmed by qPCR after 48 hours of transient transfection. (G) Cells transfected as described in (F) were loaded as described in (A), and lysosomal pH was determined after 48 hours of transfection (n = 5, 14 to 15 coverslips). (H) Lysosomal calcium measurements (n = 5, 14 to 15 coverslips). (I) MEF Flag-RECS1 WT and BAX and BAK DKO cells were treated with doxycycline for 16 hours and analyzed by TEM. Image magnification, ×4000 (n = 13 to 17 cells per condition). (J) Diameter of primary and secondary lysosomes (n = 8 to 13 cells per condition). Data represent means ± SEM. Statistically significant differences were detected using ratio-paired two-tailed t test (A, B, D, E, G, and H) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

      Role of RECS1 in lysosomal pH regulation and calcium accumulation.

      6A, B & C

      Lysosomal pH was reduced in RECS1 overexpressing HeLa cells (A), while calcium concentration increased about 2-fold under these conditions (B), but no effect on ER calcium concentration (C).

      6D & E

      The reduction in lysosomal pH in MEFs is dependent on BAX and BAK expression and a over three-fold increase in calcium content was observed in cells overexpressing RECS1.

      6F, G & H

      RECS1 silenced cells have increased lysosomal pH (F, G), whereas the luminal pH was only slightly reduced when RECS1 was inhibited (H).

      6 I & J

      Number of secondary lysosomes but not size increased in cells over expressing RECS1 in BAX/BAK expression-dependent manner, suggesting an increased catabolic.

    10. pH (OGDx)

      Fluorescence probe used for measuring lysosomal pH. Find more information about this probe at the following link: https://journals.biologists.com/jcs/article/115/3/599/34970/pH-dependent-regulation-of-lysosomal-calcium-in

    11. (siRecs1#1 and siRecs1#2)

      Two siRNA would degrade/break down RECS1 and prevent its expression

    12. mobility shift

      A technique used in biology to detect proteins bound to DNA based on their rate of movement in a gel. DNA-free protein appear smaller that the same protein bound to DNA.

    13. ip/Grp78 and Chop/Gadd153 (fig. S5H), two sensitive markers of ER stress (13, 15).

      Proteins whose expression changes during ER stress.

    14. During LMP, lysosomal intraluminal cathepsins and hydrolases leak into the cytosol, where they act as cell death proteases

      LMP results in the escape of protein-degrading proteins from the lysosomes to the cytoplasm, leading to initiation of cell death

    15. BAX- and BAK-dependent manne

      These are required for RECS1 to cause cell death

    16. Specific structural and functional criteria are required to validate a BH3 motif, since definitive bioinformatic procedures that rely purely on sequence analyses are currently lacking (28). To this end, we synthesized two peptides: RECS1280–299 and RECS1296–311, based on the N- and C-terminal BH3-like motifs, respectively.

      Two synthetic (non-natural) amino acid sequences were made . These amino acid sequences resemble the BH3-like motifs. These helped in determining the structure and function of RECS1

    17. deletion mutants: RECS1Δ910, lacking the C-terminal BH3 motif, and RECS1Δ868, lacking both

      Sequences of RECS1 with some nucleotides at the C-terminal removed. These targeted the two BH3-like segments.

    18. RECS1 has been traditionally classified within the TMBIM superfamily (8). However, detailed sequence and phylogenetic analyses suggest that RECS1, together with GRINA, LFG, GAAP, and Tmbim1b, forms a distinct and evolutionarily conserved protein family, termed the “LFG family,” characterized by a shared common ancestry and the lack of the signature BI-1 motif (fig. S1A) (7, 26). Previous sequence comparison using the BH3 motif consensus from Prosite (PS01259) identified one putative BH3 motif on the C-terminal domain of the human RECS1 and LFG proteins (22). The BH3-like motif in the C-terminal region of RECS1 contains the characteristic minimal Leu-X3-Gly-Asp/Glu sequence, but no additional BH3-like motifs of this kind were found in the other members of the LFG and BI-1 protein families (fig. S1B). However, an additional conserved BH3-like sequence consisting of Leu-X4-Asp is also present in an adjacent sequence of RECS1, in addition to GRINA and LFG (fig. S1B). These putative “BH3 motifs” are conserved in humans and in several species of non-human primates (fig. S1, C and D). However, only one such motif is present in rodents, suggesting that they represent a late evolutionary event.

      RECS1 sequence contains a short segment on the C-terminal that is thought to be involved in positive regulation of cell death. This short sequence is also found in other related proteins in other organisms.

    19. BAX and BAK double-knockout (DKO) background (Fig. 1F)

      Cells in which both BAX and BAK are non-functional

    20. transient transfection

      Temporary expression of foreign plasmid DNA in cells. The foreign DNA does not get integrated into the cell genome.

    21. Together, these results suggest that the C-terminal region of RECS1 is essential to induce cell death, but this sequence does not bear functional bona fide BH3 motifs.

      Is there a minimum peptide sequence length within the C-terminal below which the C-terminal is irrelevant in RECS1-regulated cell death?

    22. itochondrial potential (ΔΨm)–sensitive dye JC-1

      JC-1 changes when mitochondria charge changes

    23. o investigate the role of RECS1 on the control of cell death in vivo in a vertebrate model, we overexpressed the full-length human RECS1 in zebrafish by injecting in vitro synthetized mRNA to one-cell stage embryos, followed by the analysis of animal viability and morphology 24 hours post-fertilization (hpf)

      The authors expressed a high dose of human RECS1 in Zebrafish embryo. It is expected that the human RECS1 will have similar function in Zebrafish, as this protein function is conserved across different organisms.

  10. Oct 2022
    1. If the MOMP–caspase–BH3 protein loop contributed to the generation of trigger waves, inhibition of the executioner caspases would be expected to slow or block the waves (Fig. 1A). To test this, we added the caspase-3 and -7 inhibitor N-acetyl–Asp-Glu-Val-Asp–aldehyde (Ac-DEVD-CHO) to the reconstituted extracts. Because high concentrations of the inhibitor make it difficult to monitor trigger waves with the fluorogenic caspase substrate Z-DEVD-R110, we used an additional probe, tetramethylrhodamine ethyl ester (TMRE), a red fluorescent dye that responds to changes in mitochondrial membrane potential

      Here, the authors inhibited caspase activity in reconstituted cytoplasmic extracts and tested its effect on the speed of apoptotic trigger waves by monitoring the caspase activity and mitochondrial membrane potential (mark of an active mitochondria).

  11. May 2022
    1. In this case, the asymmetry of the trace of fluorescence intensity as a function of time measured near the end of the channel (green trace inFig. 4B) indicates that the band is still broadening rapidly as it reaches the end of the channel:

      Figure 4B shows the spread of a fluorescent plug introduced into a straight channel. As the plug moves along the channel, it spreads out due to a process called axial dispersion. As a result its length increases and its intensity decreases. Axial dispersion is often an undesired phenomenon observed in microfluidic devices.

    2. we use the leading edge of the fluorescent region to measure the angular displacement of the fluid in the cross section, Δφ. 

      Here, the authors captured optical images of the leading edge of the fluorescent solution to monitor and quantify the rotation of the fluid due to chaotic mixing by the herringbone ridge structures. The images are analyzed to determine the angular displacement Δφ as shown in Figure 1C.

    3. Figure 3D shows the evolution of σ for flows of different Pe in the SHM (open symbols), in a simple channel as in Fig. 3A (▴), and in a channel with straight ridges as in Fig. 3B (•).

      The authors measured the variation in fluorescence intensity across the channel cross-section throughout its length and plotted the standard deviation as a function of the channel length. Minimal variation in fluorescence signal (σ values near zero) represents effective mixing. The staggered herringbone induces the most effective mixing because the σ values reach zero within a short distance along the channel.

    1. The fabrication process for the tactile sensor based on Co AW and the air gap structure is shown in fig. S3.

      The authors chose to use a co-based amorphous wire (Co AW) for its strong magnetic properties. The researchers were able to use a B-H Loop Tracer machine to test the permeability of the wire. This test showed that as increasing frequency was applied, the wire was able to maintain a high permeability and impedance.

      Additionally, the authors determined the correct thickness of the PDMS ring and free standing membrane that would allow a sufficient air gap to be present between the magnetic particles and inductor. The air gap allows for pressure-induced deformations in the membrane which is detected by the changes in the magnetic flux through the inductor.

    2. recorded from the sensor were converted into digital-frequency signals by using an LC oscillation circuit composed of a tactile sensor and a capacitor of 100 nF

      In this experiment, the authors tested the ability to convert the analog signals from the sensor into digital-frequency signals. They loaded the sensor with 50, 113, and 1000 Pa shown in fig. 4B. These are applied pressures which are commonly experienced by humans throughout the day. The authors found that the number of pulse waveforms increased with increased loading, which is similar to human responses to force stimuli.

    1. For a 10-mm-long robot, as shown in fig. S9 (A and B), we observed noticeable motion even under an AC drive voltage as low as 8 V peak to peak (movie S6), which is a relatively low voltage requirement among insect-scale piezoelectric actuators (45).

      The attached video demonstrates the locomotion of a 10 mm-long prototype in the controlled transparent quartz tube environment. Specifically, the relatively low voltage requirement is demonstrated by the noticeable motion at 8V.

      https://www.youtube.com/watch?v=msQS1Ks1nI0

    2. We then fabricated prototype robots with different lengths ranging from 10 to 30 mm at an interval of 5 mm using the map of λ/L and β/π of 0.1 and 0.4 for guidance.

      The authors chose to test different lengths of robots to find which length was most efficient for movement. They began at 10mm and moved up 5mm till they reached 30mm. This yielded 5 tests of different length robots. Given the results in Fig. 3, the 10mm robot was deemed most efficient in locomotion.

  12. Apr 2022
    1. The micrographs in Fig. 3, A and B, show that for flows with high Péclet number (Pe = 2 × 105), there is negligible mixing in a simple channel (Fig. 3A) and incomplete mixing in a channel with straight ridges (Fig. 3B) after the flow has proceeded 3 cm—the typical dimension of a microfluidic chip—down the channel.

      Here the authors describe how they compared the mixing performance of 1) a straight microchannel, 2) a microchannel with straight ridges, and 3) a microchannel with staggered herringbone ridges to prove that their design (staggered herringbone ridges) had the best mixing performance.

    2. 26. Recent experimental results confirm that ridges in the floor of a channel do generate transverse components in electroosmotic flows (28).

      "Here, the authors note that the ridge structures result in mixing not only in pressure driven flows but also in electroosmotic flows."

    3. 22. We have evaluated the extent of the chaotic region in the cross section by numerically integrating an approximate two-dimensional representation of the transverse flow to generate a Poincaré map. The map is dense (no islands) everywhere in the cross section except in a narrow band (<10% of height) at the top of the channel. We have not systematically optimized the design of the mixer with respect to these Δφm and p values.

      Here the authors describe the mathematical process of analyzing the data presented in Fig. 2B to determine the extent of the chaotic region.

    4. 29. Labeled polymers were prepared by allowing poly(ethylenimine) (molecular weight ∼500,000) to react with fluorocein isothiocyanate. The product was dialyzed for several days. Diffusivities were calculated based on the broadening of fluorescent streams of the dye in confocal images flows of known speed: D = 4 × 10−6 cm2/s in water and D = 2 × 10−8 cm2/s in 80% glycerol/20% water. Flow speeds were measured by weighing the fluid collected at the outlet of the channel. Viscosity of the glycerol/water solution was estimated to be 0.67 g/cm·s by comparing the flow rate to that of water through the same channel with the same applied pressure.

      Here the authors provide a description of the fluorescent streams used in the first 3 figures and how they were prepared.

    5. Within the limits of our simple model of mixing (13), we estimate from the linear portion of the plot in Fig. 3E that λ is on the order of a few millimeters; the average width of the filaments of unmixed fluid decreases by a factor of ∼3 as the fluid travels this axial distance. This estimate agrees qualitatively with the evolution seen in Fig. 3C.

      When possible it is important to compare the data gathered experimentally with the expected result (determined logically or mathematically) to make sure that the experiments are valid and accurate within an acceptable range of error. Here the authors state that their experimental data qualitatively agrees with their mathematical model.

    1. A major regional change in the doubled CO2 experiment with our three-dimensional model (6, 8) was the creation of hot, dry conditions in much of the western two-thirds of the United States and Canada and in large parts of central Asia.

      Since the authors' model produces estimates across the globe, it is able to reveal stark regional changes in addition to overall averages.

    2. Volcanic eruptions of the size of Krakatoa or Agung may slow the warming, but barring an unusual coincidence of eruptions, the delay will not exceed several years.

      This model predicts that even large changes in the energy balance that counteract warming due to greenhouse gases will only have a partial effect. These predictions generally agree with observations recorded since the time of publication.

    3. Projected global warming for fast growth is 3° to 4.5°C at the end of the next century, depending on the proportion of depleted oil and gas replaced by synfuels (Fig. 6).

      The authors' predictions of warming due to greenhouse gas emissions are generally in line with observed warming since this paper's publication (1981) as well as modern simulations.

    4. The key fuel choice is between coal and alternatives that do not increase atmospheric CO2.

      The authors use the model to compare energy scenarios in which we use varying amounts of coal, oil, and gas (which emit greenhouse gases) to meet future electricity demand. These scenarios include replacement by either nuclear energy or alternative synthetic fuels. The scenarios also vary in how quickly fossil fuels are removed from the energy mix.

    5. Thus we consider fast growth (~3 percent per year, specifically 4 percent per year in 1980 to 2020, 3 percent per year in 2020 to 2060, and 2 percent per year in 2060 to 2100), slow growth (half of fast growth), and no growth as representative energy growth rates.

      It is difficult to predict future trends in energy consumption, so the authors decide on several scenarios to show the range of possible outcomes in the temperature trend.

    6. Prediction of the climate effect of CO2 requires projections of the amount of atmospheric CO2, which we specify by (i) the energy growth rate and (ii) the fossil fuel proportion of energy use.

      Since the authors' model now agrees well with observation, they use it to generate predictions about future temperature trends due to increasing carbon dioxide emissions. To estimate future conditions, they primarily consider emissions due to the energy sector.

    7. Radiative forcing by CO2 plus volcanoes accounts for 75 percent of the variance in the 5-year smoothed global temperature

      Most of the variation in temperature is found to be the result of changes in the energy balance by carbon dioxide concentration and aerosol emissions from volcanic eruptions. This indicates these two factors are the most important in modeling temperature trends.

    8. The main uncertainties in the climate model-that is, its "tuning knobs"-are (i) the equilibrium sensitivity and (ii) the rate of heat exchange with the ocean beneath the mixed layer.

      By varying the unknown parameters and observing when the model best fits the empirical results, the authors find fitted values for those parameters.

    9. Radiative forcing by CO2 plus volcanoes and forcing by CO2 plus volcanoes plus the sun both yield a temperature trend with a strong similarity to the observed trend of the past century (Fig. 5), which we quantify below.

      By including the best possible values for the parameters in their equation for heat flow, the authors model the temperature trend for the previous century and compare it to the reconstructed global temperature history. The agreement between these results is a good indicator of the reliability of the model.

    10. We developed an empirical equation that fits the heat flux into the earth's surface calculated with the l-D RC climate model (model 4):

      The authors combine the model results into a single equation which relates the flow of heat into the earth's surface with dependence on solar radiation output, aerosol concentrations and properties, and temperature change.

    11. Years later (Fig. 4c) the surface temperature has increased 2.8°C.

      The ocean takes several years to come into balance with the atmosphere and ground once carbon dioxide concentrations increase. This causes a substantial time delay in the warming of the air due to the greenhouse effect.

    12. A few months after the CO2 doubling (Fig. 4b) the stratospheric temperature has cooled by ~5°C.

      When carbon dioxide concentration increases, the model predicts that less radiation is emitted to space. Therefore, after a short increment of time, the stratosphere cools. The troposphere remains roughly the same temperature because the ocean is still absorbing much of the warming.

    13. To relate these empirical tests to the CO2 greenhouse effect, we illustrate the flux changes in the I-D RC model when CO2 is doubled.

      The authors look at how each component of the energy balance changes when carbon dioxide concentration changes.

    14. Phenomena that alter the regional radiation balance provide another model test.

      The authors observe the impact of regional phenomena on the global energy balance. They observe the model's response to seasonal changes in temperature and solar irradiation of the surface. This test shows agreement between the model and real observations.

    15. A primary lesson from the Mount Agung test is the damping of temperature change by the mixed layer's heat capacity, without which the cooling would have exceeded 1.1°C (Fig. 2).

      The slow response time of the ocean to changes in the energy balance reduces the impact of short-term phenomena, like cooling due to the emission of aerosols from volcanic eruptions.

    16. We reexamined the Mount Agung case for comparison with the present global temperature record, using our model with sensitivity ~2.8°C.

      The authors look at a specific volcanic eruption to verify that the observed effect of the resulting aerosols (suspended solid particles in air, here composed of ash and soot) is consistent with the effect predicted by their model. They confirm that the expected cooling is consistent between measurements and their predictions.

    17. Another conclusion is that global surface air temperature rose ~0.4°C in the past century, roughly consistent with calculated CO2 warming.

      The reconstructed temperature record agrees with the overall expected warming due to carbon dioxide emissions. However, other factors clearly contribute to the year-to-year variations.

    18. Northern latitudes warmed ~0.8°C between the 1880's and 1940, then cooled ~0.5C between 1940 and 1970, in agreement with other analyses (9, 43).

      The authors verify that their reconstructed temperature record is in agreement with records produced by other methods to show that the chosen formula is reliable.

    19. The temperature trends in Fig. 3 are smoothed with a 5-year running mean to make the trends readily visible.

      There is substantial variation in individual weather records as air fronts move and interact with one another. The authors average the data for each subsequent five-year period so that short-term trends interfere less with the visibility of long-term trends.

    20. We combined these temperature records with a method designed to extract mean temperature trends.

      The authors attempt to reconstruct the global temperature history by taking records from weather stations and applying them to a grid of cells dividing the planet's surface. This helps account for the uneven distribution of weather measurements and provides general trends for the previous century.

    21. The net impact of measured trace gases has thus been an equilibrium warming of 0.1°C or slightly larger.

      The effect of greenhouse gases other than carbon dioxide is significant for warming. Although carbon dioxide is the largest contributor, gases like methane have a much higher effect per amount released.

    22. A global surface albedo change of 0.015, equivalent to a change of 0.05 over land areas, would affect global temperature by 1.3°C.

      Increasing albedo (light reflected by the land) reduces warming by causing solar radiation to be reflected back out to space rather than absorbed by the surface.

    23. Based on model calculations, stratospheric aerosols that persist for 1 to 3 years after large volcanic eruptions can cause substantial cooling of surface air (Fig. 2).

      Stratospheric aerosols can reduce warming by blocking incoming solar radiation.

    24. A 1 percent increase of solar luminosity would warm the earth 1.6°C at equilibrium (Fig. 2) on the basis of model 4, which we employ for all radiative perturbations to provide a uniform comparison.

      The authors introduce a number of factors other than carbon dioxide to the model to observe their impact on warming. These components all affect the earth's energy balance in some way: increases in solar output and other greenhouse gases increase warming, whereas stratospheric aerosols and increased surface reflectance reduce warming.

    25. The mixed-layer model and thermocline model bracket the likely CO2 warming.

      Overall global warming depends on the ability of the ocean to diffuse heat from the surface to deeper water. By varying the ocean's ability to mix, the authors obtain upper and lower bounds for the likely warming as carbon dioxide concentrations increase.

    26. The heat capacity of just the mixed layer reduces this to 0.4°C, a direct effect of the mixed layer's 6-year thermal response time.

      If the ocean has a higher heat capacity (it can store more heat with a lower change in temperature), it can partially buffer overall surface warming due to carbon dioxide.

    27. This increase in thermal response time is readily understandable, because feedbacks come into play only gradually after some warming occurs.

      The feedback mechanisms increase the amount of warming but not the rate of heat flow, so it will take the oceans longer to reach the increased temperature.

    28. Table 1 shows that the initial rate of heat storage in the ocean is independent of feedbacks.

      Because the models all predict the same flow of heat into the ground (column F in Table 1) if the surface temperature is held constant, this means that the heat flow into the ocean must also not change.

    29. This sensitivity (i) refers to perturbations about today's climate and (ii) does not include feedback mechanisms effective only on long time scales, such as changes of ice sheets or ocean chemistry.

      The change in temperature as carbon dioxide levels grow is only calculated for changes relative to the early 1980s (when this paper was published). The authors also note that effects occurring over decades or centuries are not considered, as they are assumed to contribute little to the timescale of the projections in this article.

    30. The sensitivity of the climate model we use is thus ΔTs ~ 2.8°C for doubled CO2, similar to the sensitivity of three-dimensional climate models (6-8).

      The change in temperature with respect to changes in carbon dioxide concentration in the authors' chosen model is judged to be reliable, partially because it is similar to values generated by more complex calculations.

    31. Model 4 has our estimate of appropriate model sensitivity.

      Model 4 is chosen as the best model for estimating temperature sensitivity to carbon dioxide levels, as the moist adiabatic lapse rate introduced in Model 3 underestimates the temperature response at high latitudes. Furthermore, the overestimation of the effect of cloud temperature is judged to roughly balance the effects of underestimating feedback from snow/ice and vegetation.

    32. The vegetation albedo feedback was obtained

      In order to estimate how much temperature and reflection of light by plants feed into one another, the authors compare current vegetation levels to models of a previous ice age. During an ice age, one would expect vegetation to be at a minimum, so the difference between climate then and now should be partially due to the difference in plant cover.

    33. Models 5 and 6 illustrate snow/ice and vegetation albedo feedbacks (19, 20).

      The final two models incorporate important feedback mechanisms: as the climate warms, snow and ice melt, reducing the amount of light that is reflected out to space. This causes warming to increase even further since more light is absorbed by the surface and atmosphere. The amount of the surface covered by vegetation also responds to temperature and ice cover.

    34. Model 4 has the clouds at fixed temperature levels, and thus they move to a higher altitude as the temperature increases (18).

      This model is different from the previous ones, because clouds are now allowed to move between altitudes as their temperature changes instead of remaining at a fixed level. This causes the clouds to always emit the same amount of thermal radiation, limiting the total outgoing radiation from cloudy regions.

    35. Model 3 has a moist adiabatic lapse rate in the convective region rather than a fixed lapse rate.

      This model is distinct from the previous one, in that the decrease in temperature with altitude is not a fixed value. Rather, it depends on temperature more strongly due to the consideration of how water condenses as a parcel of air rises.

    36. Fixed relative humidity is clearly more realistic than fixed absolute humidity, as indicated by physical arguments (13) and three-dimensional model results (7, 8).

      From this point forward, the authors fix relative humidity in the models (warmer air holds more water) because it causes the calculations to have better agreement with more complex models.

    37. Model 2 has fixed relative humidity, but is otherwise the same as model 1.

      The second model fixes relative humidity (the amount of water in the air, relative to how much water the air can hold at most) rather than absolute humidity (the amount of water per unit volume of air). This means that warmer air will hold more water than cooler air.

    38. This case is of special interest because it is the purely radiative-convective result, with no feedback effects.

      The simplest model shows the effects of only radiation and convection in the atmosphere. The difference between these results and a fuller, more complicated model reveals the total impact of feedback mechanisms, including reflectance off of snow and plants.

    39. inserting them individually into the model

      Many factors contribute to the energy balance of the atmosphere. The authors evaluate the effect of each by running the model, inserting the various elements one at a time. The difference in the output after each factor is added gives a measure of its importance to the climate predictions in general.

    40. Our computations include the weak CO2 bands at 8 to 12 μm, but the strong 15-μm CO2 band, which closes one side of the 7- to 20-μm H2O window, causes ≥ 90 percent of the CO2 warming.

      Carbon dioxide absorbs some frequencies more strongly than others. These calculations consider absorption from strong and weak bands of carbon dioxide's absorption spectrum, even though the strongest band is the major contributor to the gas' warming potential.

    41. Multiple scattering and overlap of gaseous absorption bands are included.

      These calculations account for light that is scattered multiple times as it propagates through air. The model also considers frequencies of light where multiple gases are capable of absorption.

    42. The term dFc/dh is the energy transport needed to prevent the temperature gradient from exceeding a preassigned limit, usually 6.5°C km-1. This limit parameterizes effects of vertical mixing and large-scale dynamics.

      Large changes in temperature over small vertical distances are unlikely to occur in the real world, so the model is constrained to avoid too steep a slope in the altitude/temperature curve.

    43. integrated over all frequencies, using the temperature profile of the previous time step and an assumed atmospheric composition.

      The amount of light thatis absorbed, emitted, and scattered by the atmosphere is different for different frequencies of light, so these calculations must be done in parallel for each frequency. The contributions to the energy balance can then be added together.

    44. The l-D RC model uses a time-marching procedure to compute the vertical temperature profile from the net radiative and convective energy fluxes:

      This model considers the dependence of variables on time by only considering time intervals of fixed duration. This permits the modeling of the atmosphere's response to stimuli without requiring more complicated calculus-based methods.

    45. Observed surface temperatures of these planets confirm the existence and order of magnitude of the predicted greenhouse effect (Eq. 3)

      Observing conditions on other planets allows us to generalize conclusions about Earth, as we cannot conduct a true experiment on the entire atmosphere of Earth to see how different factors are related.

    46. The excess, Ts − Te

      The effective radiating temperature would be the temperature of the surface if only the surface were absorbing and emitting radiation. However, gases in the atmosphere also absorb and emit, so the average altitude from which outgoing radiation originates is somewhere above the surface, and T(e) is the temperature at that altitude.

    47. The effective radiating temperature of the earth, Te

      This model assumes that the Earth emits exactly as much radiation as it absorbs over the long term.

    48. Finally, we compute the CO2 warming expected in the coming century and discuss its potential implications.

      Finding equations that accurately describe historical climate change allows the authors to generate predictions about the future. It is important to look at a variety of approaches to see how the predictions of different models vary.

    49. We first describe the greenhouse mechanism and use a simple model to compare potential radiative perturbations of climate.

      By constructing mathematical models of climate change, the authors can simulate how different atmospheric effects are related to one another. They can also observe how different models agree and disagree with each other, which allows for the selection of the best model by comparison with measurements.

  13. Mar 2022
    1. the sensitivity of the tactile sensor was investigated.

      The authors added vertical forces to the sensor and measured the impedance. This was done to show that the sensor is able to distinguish between noise and subtle pressure.

    2. frequency changes under loadings of 50, 113, and 1000 Pa

      This experiment shows the changes in frequency under different loads. The authors tested their tactile sensor under various pressure and frequency conditions that mimic the human response to force stimuli. Here, they can determine the tactile input (pressure) by the frequency (of frequency shift) measured by the sensor. The authors derived an equation relating the frequency to pressure.

    3. The height should be optimized to ensure that the inductive sensing element works best at a biased magnetic field in the range of 0 to ~6.2 Oe.

      This experiment was done in order to find the optimal height at which the inductive sensing element works within a magnetic field range of 0-6.2 Oe. They measured impedance for polymer magnets with different magnetizations approaching the sensing element. For each magnet, there is a height at which the sensor was most sensitive. For the maximum region of sensitivity, they chose the magnet with a magnetization of 0.3 electromagnetic units (emu) at a height of 1.3 mm.

    4. frequency reached 250 kHz

      Magneto-impedance is a function of frequency. In order to optimize the sensing component of the device, the authors wanted to determine which frequency yielded the highest percent change in magneto-impedance. They tested a range of frequencies from 0.5 kHz to 500 kHz. It was determined that the frequency of 250 kHz produced the largest percent change (500%) in magneto-impedance, and therefore the tactile sensor will be most sensitive when operated at this frequency.

    5. Frequency has a significant effect on the GMI ratio response under the loading.

      In this experiment, they wanted to test the sensitivity at different frequencies. The sensitivity of the device increased as the frequency increased, but decreased after the frequency exceeded 250 kHz. That means the optimal frequency is 250 kHz, which is consistent with the optimal frequency of the inductive sensing element.

    6. a subtle force applied to the polymer magnet caused displacement, and impedance was changed due to the variation in magnetic field.

      In this experiment, the authors wanted to determine the change of the magneto-impedance ratio that was caused from a force being applied to the polymer magnet. They tested four different frequencies by applying a downward force (displacement). As displacement increased, the magneto-impedance ratio decreased.

    1. In this case, a 10-mm-long prototype (0.024 g) robot was used to achieve a relative running speed up to 20 BL/s driven near its resonant frequency at 850 Hz. In comparison, under driving frequencies of 800 and 900 Hz, lower relative running speeds of 13 and 3.6 BL/s were recorded, respectively (movie S3).

      The attached video demonstrates the locomotion of a 10 mm-long prototype in the controlled transparent quartz tube environment. Three different driving frequencies were used to help characterize the relationship between driving frequency and robot speed.

      https://www.youtube.com/watch?v=iSCqbpvZqvM

    2. Experimentally, the robustness of the prototype soft robot was demonstrated by applying a 100-g mass (1500 times its own body weight) with little change in its speed after the mass was removed, as shown in movie S7. Moreover, the soft robot could continue to function (one-half of the original speed) after being stepped on by an adult human (59.5 kg), a load about 1 million times its own body weight (Fig. 5, A to C, and movie S7).

      The first 20 seconds of the attached video demonstrate the robustness of the soft robot in two different circumstances. We find that the device continues to function after the application of a 100-g mass, as well as after being stepped on.

      https://www.youtube.com/watch?v=ank3w_YvedQ

    3. To further increase the running speed, we added and attached a back leg to a 3 cm–by–5 cm prototype robot to emulate galloping-like gaits (movie S8).

      The attached video demonstrates a new type of movement pattern, galloping, where back legs have been attached to the device.

      https://www.youtube.com/watch?v=4_AGZvFOZzs

    4. With the more effective galloping-like gait mechanism, a two-legged robot achieved a running speed about three times that of a one-legged 3 cm–by–1.5 cm robot under similar driving conditions, as shown in movie S9.

      The attached video provides a comparison of the one-legged robot with the new galloping two-legged robot, with the latter traveling at three times the prior.

      https://www.youtube.com/watch?v=V1alOFHv05k

    5. One simple way to turn would be to assemble two separated electrical domains, as shown in figs. S13 and S14 and movie S10.

      The attached video demonstrates the ability for the soft robot to turn, specifically through the use of two electrical domains.

      https://www.youtube.com/watch?v=5E88Abvp2FU

    6. The slope climbing capability of the robot is demonstrated in movie S7, in which the robot reached 7 BL/s while climbing a slope with an angle of 7.5° (Fig. 5D) and 1 BL/s while climbing a slope with an angle of 15.6° (Fig. 5E). Our soft prototype robot could also carry loads equal to the weight of a peanut (0.406 g) (Fig. 5, F and G).

      The last 10 seconds of the attached video demonstrate device robustness in 2 circumstances. The robot successfully climbs inclined surfaces and carries a peanut.

      https://youtu.be/ank3w_YvedQ?t=20

    7. Using a prototype robot of 10 mm (length) by 15 mm (width) by 3 mm (height) as an example, we first selected 25 combinations (Fig. 3B, gray dots) of the above geometric parameters to fabricate prototypes and conducted experiments to plot the normalized running speed map as a function of relative leg position (λ/L) and relative leg angle (β/π) in Fig. 3B.

      The authors chose to test different angles for the device curvature to optimize speed for efficient movement. Specifically, the relative leg position (λ/L) and angle (β/π) were varied between 0.1-0.5 and 0.1-0.7 respectively.

    8. Hence, we designed the bending angle of the front leg to be less than 90° to enhance the forward movements and reduce backward movements.

      The authors chose a 90º bending angle for the front leg to optimize the forward moving speed of the robot.

    1. These stirring flows will reduce the mixing length by decreasing the average distance, Δr, over which diffusion must act in the transverse direction to homogenize unmixed volumes.

      The implementation of ridges along the microchannel floor disrupts the flow of fluid, allowing diffusion to act more quickly compared to steady flow down a smooth channel.

  14. Feb 2022
    1. The dynamic sensing was demonstrated by placing small live insects of 0.8-mg mass onto the tactile sensor.

      In this experiment, the authors created a way to test the sensor's ability to detect changing tactile information. To do this, they placed an ant on the sensor and let it walk around. The authors show the path of the ant on the sensor in fig. 3C. They found that the sensor was able to detect the ant's movements across the sensor. They were also able to detect the movement of the ant's antenna when it was still. The authors show the data from this experiment in fig. 3D.

    2. The detection limit of the tactile sensor was further investigated by dropping water of different volumes (1 μl, equal to 10 μN) onto the tactile sensor

      In this experiment, the authors created a different way to test the sensor's detection limits. They dropped different volumes of water on the sensor. The authors found that the impedance increased with increasing volumes of water. This means that the device was able to "sense" the weight of the different volumes of water. They found that the device's detection limit was 0.3 Pa. The authors show the data from this experiment in fig. 3B.

    1. To quantify the degree of mixing (convection plus diffusion) as a function of the axial distance traveled in the mixer and ofPe, we measure the standard deviation of the intensity distribution in confocal images of the cross section of the flow like those in Fig. 3, A to C: σ = 〈(I − 〈I〉)2〉1/2, whereI is the grayscale value (between 0 and 1) of a pixel, and 〈 〉 means an average over all the pixels in the image. The value of σ is 0.5 for completely segregated streams and 0 for completely mixed streams.

      The standard deviation (of fluorescence intensity) is determined by averaging the grayscale value of images taken and used as a metric for the degree of mixing present in each channel. Lower standard deviation values indicate less difference in the intensity values across an image and signify more complete mixing.

    2. We used soft lithographic methods to make the channels in poly(dimethylsiloxane) (16, 17).

      After designing the master mold, an elastic polymer known as polydimethylsiloxane (PDMS) is prepared and poured over it. This substance cures (becomes solid) over a short period of time and is removed from the mold, where it is bonded to a glass cover slip. The channel itself is comprised of the space between the glass surface and the patterned PDMS.