2.5 M butyl lithium was added to a solution of furan at -78 deg Celsius under inert atmosphere. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was cooled to -78 deg Celsius and 43 was added. Then NBS was added. The reaction was warmed to room temperature and 10 mL of water was added. Extraction with ethyl acetate and followed by vacuum evaporation afforded a residue. This was purified by silica gel column chromatography to give 48 as a colorless oil.

Copper-Catalyzed Oxidation: To a premixed 2.0 M NaOH and 30% hydrogen peroxide and 15 was added in THF at 0 deg Celsius. The mixture was stirred at room temperature for 11 hours. 1.0 M aqueous hydrochloric acid was added and reaction mixture was extracted with ethyl acetate. The organic layer was washed with brine. Volatiles were evaporated with rotary evaporator and the residue was purified by silica gel column chromatography. 45 was obtained as a yellow solid.

Suzuki Coupling Reaction: 15, boronic acid, potassium phosphate, Pd(dppf)Cl2 and dioxane were added to a vial in a glove box. Then, the vial was removed from the glove box and water was added. The reaction mixture was heated to 100 deg Celsius for 2.5 hours. Volatiles were rotary evaporated and the residue was purified by silica gel column chromatography to give 37 as a colorless oil.

Deboronation and Deuterium Exchange: To a reaction vial, 15, [Ir(cod)OMe]2 and THF were added. The vial was sealed, removed out of the dry box and D2O was syringed. The reaction mixture was heated to 80 deg Celsius for ~3 h. The volatiles were removed by rotary evaporator and the residue was purified by column chromatography. 39 was obtained as a colorless oil.

To a reaction vial, cyclopropane carboxylate, B2Pin2, 6.3 micromol of [Ir(cod)(OMe)]2, 2-mphen and cyclooctane (solvent) were added. The reaction mixture was heated at 100 degrees Celsius for 20 h. The product mixture was purified by silica gel column chromatography. CH2Br2 was added as the internal standard and the product was characetrized by H-NMR spectroscopy

Diisopropylbenzene, B2PIn2, [Ir(cod)(OMe)]2, 2.5 mg of 2-mphen and cyclooctane were added to a reaction vial. The reaction mixture was heated to 100 deg Celsius, cooled and then stirred for 20 h under inert atmosphere. The reaction progress was monitored with GC-MS. Crude product was purified by silica gel column chromatography.

Metallophotoredox Reaction: In a glove-box, 4,4’-di-tert-butyl-2-2`-bipyridine and NiCl2.DME were added to a reaction vial, followed by addition of THF. The mixture was heated on a heating plate for 10 min. The vial was taken back into the glove-box, and the volatile materials were removed, followed by addition of Ir[dFCF3ppy]2(bpy)PF6, 4-bromobenzonitrile, Cs2CO3, potassium triflluoroborate and dioxane. The vial was capped and stirred under irridation of a 34 W blue LED lamp for 48 h. The crude reaction mixture was extracted with ethyl acetate. The resulting solution was concentrated under reduced pressure, and the residue was purified by column chromatography to give the product 52 as a colorless oil.

0.250 mol of the substrate, 3 equivalents of B2Pin2, 6.3 micromol of [Ir(cod)(OMe)]2, 13 micromol of 2-mphen and 200 microliter of cycloctane were added to a vial. The reaction mixture was heated to 100 degrees Celsius for 20 hours. Product was purified by column chromatography and characterized by proton NMR spectroscopy.

Heat was applied outside of dry box in this procedure. Then, the reaction vial is taken back into the glove box for further manipulations.

To a vial, an alcohol of interest was added inside a dry box. This was followed by 1.3 equivalents of HBPin and stirred for ~ 10 minutes to convert the alcohol to the borate ester. The vial was fitted with a cap and heated to 100 degrees Celsius outside the dry box. The vial was cooled and then 2.5 mol % of [Ir(cod)(OMe)]2, B2Pin2 and cyclooctane (solvent) were added under inert conditions. Heated to 80 degree Celsius (outside dry box) to activate the catalyst. Cooled to room temperature and charged with remaining B2Pin2 and more cyclooctane under inert conditions. Reaction mixture was stirred for 20 hours. CH2Br2 was added as an internal standard. Product was analyzed by proton NMR spectroscopy.

In a nitrogen filled glove box, a vial was charged with (MsSH)IrBPin3, a ligand, B2Pin2 (bis-pinacolatodiboron) and THF. Ligands are phenanthroline derived. Dodecane was used as the internal standard. The reaction vial was sealed with a Teflon cap. The mixture was heated to 100 degrees Celsius. Reaction was monitored by GC.

The same reaction was repeated replacing THF with diethyl ether.

The ligands used are 2-methylphananthroline (mphen), 2,9-dimethylphenanthroline (2,9-dmphen) and 3,4,7,8-tetramethylphanathroline (tmphen).

In a glove box under inert atmosphere, catalyst [Ir[Cod][OMe]2], 2-mphen and B2Pin2 were added. A suitable alkane (substrate) and cyclooctanol (solvent) were added and the vial sealed with a Teflon cap. Reaction was heated for 100 degrees Celsius for 20 hours. The reaction was cooled. Product was isolated and purified by column chromatography. Product was analyzed by proton NMR spectroscopy.

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.

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.

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.

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

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.

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.

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

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.

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

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.

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.

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.

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.

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).

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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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

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.

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.

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.

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.

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.

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).

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.

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.

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.

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

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

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.

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

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

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.

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

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).

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

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.

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.

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.

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.

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.

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

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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

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

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.

Proteins whose expression changes during ER stress.

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

These are required for RECS1 to cause cell death

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

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

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.

Cells in which both BAX and BAK are non-functional

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

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

JC-1 changes when mitochondria charge changes

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.

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).

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.

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.

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.

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.

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.

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

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.

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.

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

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

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

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.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

Stratospheric aerosols can reduce warming by blocking incoming solar radiation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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

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

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