- Mar 2023
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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.
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- Feb 2023
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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.
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- Dec 2022
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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.
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- Sep 2021
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we stained sections with antibodies for CRF, SST2/4R, and D2R
The experimenters used fluorescent antibodies specific for binding to CRF to ensure that they are examining CRF neurons, as previous work indicates that SST and dopaminergic interneurons send their transmitters to CRF neurons. The experimenters also perform staining with fluorescent antibodies specific for SST2/4R, the somatostatin receptor, and D2R, the dopamine receptor.
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immunostained for tyrosine hydroxylase (TH), an enzyme in the dopamine synthetic pathway
By staining brain tissue sections for an enzyme in the dopamine synthetic pathway, the experimenters can determine how much dopamine is being produced.
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- Apr 2021
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After the temperature was raised to room temperature, the chip components were pick-and-placed on the predefined contact pads. The placement accuracy was ensured using an optical microscope. Pouring polyimine solution on top and curing at room temperature for 48 hours, the LM circuitry and chip components were encapsulated.
After the polyimine film was heated up, the chips could be placed and almost fused with the polyimine to keep them in place.
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To synthesize polyimine, terephthalaldehyde (1.5 g, 11.18 mmol), 3,3′-diamino-N-methyldipropylamine (1.251 g, 8.61 mmol), and tris(2-aminoethyl)amine (0.252 g, 1.72 mmol) were mixed in methanol (20 ml), as shown in fig. S2A.
The specific polymers that are used to make up this polyimine.
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This process represents totally six different configurations. Optical microscope images in fig. S1C prove robust connection between the LM circuitry and the rigid chip components under extreme deformation.
The authors look at the connection between the circuitry and the electronic parts to make sure that under this stress the parts are still communicating with each other.
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amine in methanol, the excessive amine monomers can react with the imine bonds inside the polyimine network
When the electronic circuit is coated in this acid, the polyimine separates from the parts of the circuit. This allows the author to repair or replace any part of the circuit board instead of replacing the whole thing.
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. To further demonstrate the self-healing performance, the cutting and self-healing process at the same location of a polyimine encapsulated LM conductor was repeated five times.
The author's cut the electronic circuit 5 times to prove that it can self heal through even being cut. The electronic circuit will repair itself after being cut.
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LM circuitry does not have fatigue issues, encapsulation does not affect the mechanical performance, and there is no stress concentration in the conductor at the interfaces between the rigid chip components and the soft substrate (fig. S1C).
The authors test to see if the electronic circuit can keep up with the mechanical stress that is placed on the polyimine.
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To test biocompatibility, fig. S4 shows optical images comparing the skin conditions before (top left) and after wearing a polyimine film for 12 hours (bottom left) and 24 hours (bottom right), respectively. No noticeable adverse effects on the skin can be observed.
The authors took pictures before and after wearing the sensor to see if the skin was affected by the polymer.
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Screen printing method is adopted to brush EGaIn LM against the mask, and peeling off of the mask leaves patterned LM traces on the polyimine substrate.
Screen printing is a technique in which a mesh is used to transfer ink onto a substrate. In this case EGaln LM is used to transfer LM onto the polyimine.
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- Mar 2021
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As demonstrated in Fig. 4A, when the LM circuit is cut broken by a razor blade (top frame), the ECG signal is lost (Fig. 4B, top). The top inset in Fig. 4A illustrates a magnified view of the broken LM conductor. After slightly pressing the two sides of the cut location, the LM conductor is self-healed, and the ECG sensor regains sensing capability
The authors physically cut the LM circuit to determine that the EKG signal would be lost. Afterwards the two cut regions were slightly pressed together, and the LM material was self-healed. This allowed the ECG sensor to regain its sensing capability.
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The polyimine and LM were modeled as Neo-Hookean hyperelastic material using three-dimensional (3D) hybrid stress elements (C3D8H), and the chip components were modeled using elastic isotropic material with 3D stress elements (C3D8).
The Polyimine and LM were 3D modeled on the software Abaqus. This software allowed the authors to determine the young's modulus and the Poisson ratio for each of the materials. These are important mechanical properties that allow the authors to model the mechanical behavior further.
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- Feb 2021
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We took advantage of the regular and symmetrical shape of the quadruped to illustrate camouflage strategies that can break up recognizable silhouettes
By using a common geometrical shape the authors were able to better camouflage the robot by using the idea that the robot fit in with the normal geometry found in nature. If the robot was designed in a way that caused it to have an abnormal shape it would have a harder time hiding in specific environments where there was more repeated normal patterns.
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Simple image analysis performed on the camouflaged and uncamouflaged robot
Through the different experiments preformed on the robot grayscale thresholding was used as a way of determining which if the specific intensities of the the color layers on the robot matched those of the background. Also infrared thermal imaging was used on the robot to note how color change in the color layers affect the camouflage of the robot in the IR spectrum
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The color layers are easily fabricated
Color layers were fabricated using soft lithograpy of Ecoflex in combination with a thin film. They were then coated with a liquid and attached to the robot
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Figure S5B shows the loading and unloading stress-strain curves of polyimine under cyclic loading test, which exhibits good elasticity with slight hysteresis
The cyclic loading test is the repeated application of stress and strain to determine fatigue stress. The cyclic test for this experiment test the self-healing capacitates of the material, can the material go back to its original shape when stretched out. In this case, the polyimine had good elasticity, but exhibited some hysteresis. Hysteresis, in this case, is when the polyimine was stretched, but then had a small lag when returning to its original shape.
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A model reaction as shown in fig. S2B indicates that the imine bond exchange reaction is efficient at both room temperature and 80°C.
Bond exchanges often are affected by temperature, and often times when bonds are heated up they can denature. It was important to test if hotter temperatures would affect the imine bond exchange reaction. The bonds were tested at room temperature and then 80 °C, the temperature did not affect the nature and effectiveness of the bond exchange reaction.
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As shown in fig. S2C, the high similarity between the FTIR spectra of the original and recycled polyimine supports the notion that these two films are chemically identical.
FTIR stands for Fourier-transformed infrared spectroscopy, this technique is used to obtain absorption or emission of infrared light. In this case FTIR was used to determine if the original and recycled polyimine was chemically identical. If the length of the infrared waves are significantly different then that is a clear indicator that the substances have different chemical components. In this case, the length of the infrared waves are similar, meaning that this is the first indicator that the two materials are chemically similar.
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- Feb 2020
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bite rates
Divers would follow prey fish during the daytime and visually observe the number of times that the tracked fish would bite at the plants upon which they grazed within a five-minute window of time. This data was collected as a measurement of prey activity, the timing of which could be compared with the periods of activity for the blacktip reef sharks
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bio-logging
Devices were attached externally or internally to captured sharks, which were then released and tracked. The attached devices would record measurements of several data types periodically. Devices either transmit information as it is being collected, or scientists must retrieve a logging device from the shark to access the data at the end of the experiment.
Bio-logging allows scientists to observe some aspects of the behavior of animals in their natural environment, rather than within the constraints of a laboratory setting that may alter the behavior of the animal.
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- Feb 2019
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LL=∆MM+∆nn∆LL=∆MM+∆nn<math xmlns="http://www.w3.org/1998/Math/MathML"><mfrac><mrow><mo>∆</mo><mi>L</mi></mrow><mi>L</mi></mfrac><mo>=</mo><mfrac><mrow><mo>∆</mo><mi>M</mi></mrow><mi>M</mi></mfrac><mo>+</mo><mfrac><mrow><mo>∆</mo><mi>n</mi></mrow><mi>n</mi></mfrac></math>(1
This equation is basically saying that the changes in crop losses due to pests can be determined by adding up the change in insect metabolism and the change in where the insects are located. If the metabolic needs of insects increases or the area that they live in increases, then crop losses will also go up.
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- Dec 2018
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One may now ask, How much must the carbonic acid vary according to our figures, in order that the temperature should attain the same values as in the Tertiary and Ice ages respectively?
This is a concise statement of the question Arrhenius sought to answer with his model.
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However, can marine reserves also benefit large, roving reef predators that are potentially mobile throughout their life?
This question sheds light on a topic regarding the suitability of marine reserves not only as a permanent safety harbor for recovery and expansion of the species but also the temporary inhibition of the space by species that are mobile, whether they would use the space to breed for protection or for a stable source of food and shelter.
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Energy intake and thermal excess were positively correlated with body size as measured by the curved fork length (CFL) of tagged tunas
A big tuna (large body size) requires more energy which means it needs to feed from a higher amounts of prey or big preys. Consequently, the heat production is higher provoking a thermal excess.
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Here, we quantify energy intake in 144 wild Pacific bluefin tuna in the CCLME on over 39,000 days (fig. S1), using HIF measurements from implanted archival tags. We estimate energy intake using a model developed with laboratory data collected from similar-sized bluefin tuna (22) at a range of ambient temperatures.
By analyzing the temperatures of digestion in bluefin tuna, the author can draw conclusions as to what the tuna is eating, how much energy is gained from the meal, and how often the tuna eats. HIF means heat increment of feeding.
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- Sep 2018
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In our experiment, we deliberately abandoned sequence-specific switching and used electrical fields to move the components of a DNA machine with respect to each other.
This is the hypothesis and plan in response to the authors' goal to design an improved moveable robotic system that is faster and more efficient than previous systems.
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- Aug 2018
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mosquitoes surviving transient knockdown of Yob in nonsexed embryos
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Transfection experiments in larvae
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- Dec 2017
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Collections along the two coasts and adjacent islands of central Panama at depths less than 5 m
Their collection may have been affected. It was mentioned by the author that she and her colleagues collected from muddy environments since the sites were sewage dump locations. Most of the shrimps were covered in mud, therefore hard to distinguish. Also, the author mentioned transit ships traveling between the Atlantic and Pacific sides of Isthmus. These ships would migrate the shrimps away from their homes; may affect how the team paired shrimps based on their mtDNA. ~J.D.A.
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conventional starch gel electrophoresis
Gel Electrophoresis is a method of separating macromolecules such as DNA, RNA, or proteins. The separation is based on the molecules size and charge. The shorter molecules will travel further in the gel agarose than longer molecules. ~ S.Z.
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Dividing the values of allozyme and mtDNA sequence divergence for pairs P1-Cl, P2-C2, P3-C3, and P4-C4 by the estimate for time since final closure of the Panama seaway of 3.0 to 3.5 million years ago (Ma) (6, 7) yields an approximate rate of divergence of 0.03 to 0.04 for Nei's D and 2.2 to 2.6% for mtDNA sequence per 106 years (21).
Rate of divergence for the first four pairs of shrimp was calculated by dividing the allozyme and mtDNA divergence values by the time elapsed since final closure. ~S.Z.
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Kimura's corrected percent sequence divergence
Kimura is a Japanese biologist whose evolutionary theory was based at the molecular level and believed that most genetic differences between species were neutral and not driven by natural selection.
Kimura's corrected percent sequence divergence is an analysis that compares the genomes of species and allows to determine the divergence between their genetic code which can help conclude moment of speciation. (DV)
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Similarly, T205 (and, less so, S199 and S396) were phosphorylated in p38γCA transgenic mice (fig. S19). pT205 increased after PTZ in p38γ+/+ animals but was virtually abolished in p38γ−/− mice, whereas pS199, pS396, and pS404 were induced in both p38γ+/+ and p38γ−/− mice (fig. S19).
This experiment wanted to see if the mutant sites were phosphorylated in the APP23.p38γ−/− and APP23.p38γ+/+ mice.
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Although p38γ hyperphosphorylates tau during long-term in vitro kinase assays (25), the temporal profile of p38γ-induced tau phosphorylation in acute signaling remains unknown. Short-term in vitro kinase reactions using phosphorylation site–specific tau antibodies revealed phosphorylation at Ser199 (S199), Thr205 (T205), S396, and S404 (fig. S17). Mass spectrometric analysis confirmed these and 14 additional, though low-abundant, sites (figs. S17C and S18 and table S4).
The author set up different assays to test which variants were being phosphorylated by tau.
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- Nov 2017
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A long-standing hypothesis has linked escalation in plant defense that allows escape from insect herbivores to range expansion and speciation (Ehrlich and Raven 1964). Such escalation can include increases in the diversity of defense strategy (novel defense types), increases in the total amount of defense investment, or both (Agrawal et al. 2009). Range expansion, or merely an imperfect match between the distribution of plants and their natural enemies, may confront plants with different herbivore assemblages and/or variation in habitat resources across their range (Thompson 2005, Züst et al. 2012). This variation, in turn, may accelerate the evolution of differing defense strategies across habitats. Alternatively, natural enemies may not be major selective agents driving habitat specialization. In this case, we would predict that there would be few qualitative and quantitative defense differences between habitats, especially when diverging lineages of host plants occur in close proximity and also experience some gene flow across the habitat boundary.
The main point the author is trying to make here is that there is a direct correlation between increase in plant defense and increase in land covered by plants as well as speciation of plants. He also explains that due to the expansion, rapid evolution of defenses begin to take place. The herbivores eating these plants do differ in tactics from region to region, increasing the amount of different defense strategies in the plants. He explains an alternative viewpoint stating that predators do not influence speciation much, therefore defenses are similar amongst most plants.
-Otniel Gonzalez
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The high levels of genetic differentiation detected within C. jimenezii raise questions whether these two populations can be treated as different varieties/subspecies within this taxon or if indeed they may represent two different species.
In this study, the researchers found some questions as to whether the two species of C. jimenezii could be grouped with the same taxa.
Upon meeting with the author, it was stated that the molecular tools used, found a large amount of differentiation. This was not what the authors hoped and in turn was concluded that they were to remain in separate taxa.
RA
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Therefore we recommend not translocating material between these two populations for genetic conservation or ecological restoration programs until the taxonomy of this species within Coccothrinax is further studied.
Genetic conservation of this population is important due to the few individuals left in Haiti and the Dominican Republic.
Upon meeting with the author, the difficulty of finding the purebred species was discussed as were the methods.
RA
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not been previously assessed in response to human-influenced stressors
Although this experiment focuses on microbial and fungal effects on streams, humans sometimes also have an impact.
These effects may include the leaking of septic tanks into bodies of water that increase human waste and phosphorus levels. Fishing and polluting also affect stream ecosystems.
Read more in the attached link.
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flow-proportional nitrogen (N) and phosphorus (P)
An irrigation line was used along the 70-150 meter streams to pump in liquid nutrients. The nutrients were pumped proportional to the flow of the water.
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Our results generally support the use of litterbags to measure larger-scale C dynamics
It is often necessary to use a simpler model version of a larger system because it is easier to observe.
It is difficult to observe the effects on a whole stream, but the litter bags are observed and represent the carbon loss in streams.
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Higher C loss rates at the litterbag scale than the reach scale are expected, because litterbags track distinct parcels of C, whereas reaches receive additional C inputs over time.
Litter in the litter bags have a higher concentration of carbon than the stream, which has carbon more spread out. The litter will have higher loss of carbon but generally represents reach-scales.
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pools of benthic fine and coarse POC declined
Benthic fine and coarse particulate organic carbon can be used as a measurement when observing loss of carbon caused by detrivores.
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consideration of differences among litter species and potential divergence in rates due to the degree of biological processing (2, 14).
In this experiment, leaf litter from different types of trees were used. The results are in Figure 3.
The structure and chemistry of leaves are different and this determines how fast they may decompose.
For example, tulip poplar decomposes the quickest and has a low carbon to nitrogen ratio. Comparatively, oak decomposes slowly and has a higher carbon to nitrogen ratio.
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(i.e., litterbag)
The litterbag technique can be used to measure the density lost due to composition by microbes and fungi.
A known type and amount of litter is stuffed in the bag and is left for a certain amount of time in the stream. The bag is then later taken out and weight wet. The bag is allowed to dry and then the dry weight is taken.
The link included shows the method for finding the difference in mass. Additionally, the methods of this paper include grinding the litter and finding the composition.
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We measured the response of terrestrial C loss rates in whole 70- to 150-m stream reaches (tables S1 and S2).
The experiments were conducted on streams in the Appalachian Mountains of North Carolina.
Because of the elevation, there are little to no fish influencing the data. Mainly microbes and fungi influence the nutrient levels of the streams.
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We conducted two manipulative experiments at large spatial and temporal scales and focused our measurements on forest-derived leaf litter, because it is the most biologically active pool of terrestrial C in forest streams and is renewed annually (7). After a pretreatment year, we enriched one stream with N and P at a set ratio for 5 years in a paired watershed design (N+P experiment; a second stream acted as a control) and used expanded N and P gradients in a second experiment in five other streams for 2 years after a pretreatment year (N×P experiment) (table S1).
Two experiments were done separately to test the effects of nitrogen to phosphorus ratios on streams.
Pre-treatment of streams include recording the levels of nutrients and typical conditions throughout a year.
The first experiment had two watersheds with one being the control and the other having an addition of nitrogen and phosphorus to match a ratio that was decided by the scientists.
The second experiment was done on five streams with different combinations of the nitrogen and phosphorus ratio.
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Dividing the values of allozyme and mtDNA sequence divergence for pairs P1-Cl, P2-C2, P3-C3, and P4-C4 by the estimate for time since final closure of the Panama seaway of 3.0 to 3.5 million years ago (Ma) (6, 7) yields an approximate rate of divergence of 0.03 to 0.04 for Nei's D and 2.2 to 2.6% for mtDNA sequence per 106 years (21).
Rate of divergence for the first four pairs of shrimp was calculated by dividing the allozyme and mtDNA divergence values by the time elapsed since final closure. ~S.Z.
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conventional starch gel electrophoresis
Gel Electrophoresis is a method of separating macromolecules such as DNA, RNA, or proteins. The separation is based on the molecules size and charge. The shorter molecules will travel further in the gel agarose than longer molecules. ~ S.Z.
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Collections along the two coasts and adjacent islands of central Panama at depths less than 5 m
Their collection may have been affected. It was mentioned by the author that she and her colleagues collected from muddy environments since the sites were sewage dump locations. Most of the shrimps were covered in mud, therefore hard to distinguish. Also, the author mentioned transit ships traveling between the Atlantic and Pacific sides of Isthmus. These ships would migrate the shrimps away from their homes; may affect how the team paired shrimps based on their mtDNA. ~J.D.A.
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Kimura's corrected percent sequence divergence
Kimura is a Japanese biologist whose evolutionary theory was based at the molecular level and believed that most genetic differences between species were neutral and not driven by natural selection.
Kimura's corrected percent sequence divergence is an analysis that compares the genomes of species and allows to determine the divergence between their genetic code which can help conclude moment of speciation. (DV)
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in human skin grafted to severe combined immunodeficiency disease mice,
In this experiment, skin from a human was grafted onto mice with depressed immune systems. This means a piece of human skin was transplanted onto mice who's immune systems could not protect them from diseases.
(ABE)
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The treatment of mouse NC cells carrying Kitl mutations with Edn3
In this study, the cells from the neural crest of mice that carried the specific mutation Kitl were treated with endothelin 3.
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murine NC cultures
This study was done on the neural crests taken from murine cultures, so it is also an in vitro study of the effects of endothelin on the development on melanocytes but this time on a different species.
(ABE)
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Treatment of quail NC cultures with Edn3
The study described here consisted of treating quail (type of bird) neural crest cultures with Edn3. This study was in vitro, meaning the embryonic neural crest was removed and treated outside of the organism. This study showed the role that Edn3 plays in development as stated in the paper.
(ABE)
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Here, we quantify energy intake in 144 wild Pacific bluefin tuna in the CCLME on over 39,000 days (fig. S1), using HIF measurements from implanted archival tags. We estimate energy intake using a model developed with laboratory data collected from similar-sized bluefin tuna (22) at a range of ambient temperatures.
By analyzing the temperatures of digestion in bluefin tuna, the author can draw conclusions as to what the tuna is eating, how much energy is gained from the meal, and how often the tuna eats. HIF means heat increment of feeding.
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- Oct 2017
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PAUP
PAUP stands for Phylogenetic Analysis Using Parsimony. This is a computational phylogenetics program that is used to develop phylogenetic trees. PAUP implements parsimony into its calculations, meaning that the simplest explanation is the most preferred. (JP)
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Nei's D for allozymes
Nei's D is a formula that measures genetic distance. This measure assumes that genetic differences are caused by mutation and genetic drift. Genetic distance is a measure of the genetic divergence between species.
The changes in structure or protein sequences of allozymes was used in conjunction with this formula to find the genetic distance between different species of snapping shrimp. (JP)
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We evaluated shark residency in the pass using acoustic telemetry
This was used to determine if the sharks were regularly leaving the pass to forage.
- D.N.B.
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enabling us to determine whether sharks are feeding, rather than only resting
allowing us to find out whether sharks are feeding or are just resting.
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- Sep 2017
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We first constructed a high-resolution bathymetry map using a multibeam sonar system to characterize the study site (Figure 1) and georeferenced the aggregation of gray reef sharks, Carcharhinus amblyrhynchos, using a towed-diver methodology. Video-assisted underwater visual census surveys (n = 13), conducted as drift transects across the entire shark school, were used to provide a precise total shark census within the pass (June–December 2014).
A combination of the data collected from the sea floor structure (topography) and video surveys through transect lines were used in order to try to find a correlation between the habitat and the shark density. YS & WT
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- Aug 2017
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We constructed an E. coli strain SX4
The authors built a specific strain of E. coli that they could easily trace throughout these next experiments.
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Therefore, we designed a fusion protein consisting of Venus and a membrane protein, Tsr, as the reporter for monitoring lac promoter activity.
To construct the E. coli strain expressing the Tsr-Venus fusion protein, they used the previously published λRED recombination system, in which the chimeric tsr-venus gene had the coding sequence of tsr gene fused to the N-terminus of venus and a kanamycin drug resistance marker. The chimeric gene was incorporated into the lacZ locus.
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we demonstrate probing protein expression in individual Escherichia coli cells under the control of a repressed lac promotor, one molecule at a time (23)
Some authors like to state exactly what they have shown early on in the paper. In this case, the authors prepare the reader for a methods type paper.
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- Aug 2016
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We experimentally tested our hypothesis that mimicry of target species alarms increases the intensity of target responses by playing four different call types at 20-min intervals [equivalent to natural alarm rates (7)] to individual pied babblers (n = 20 babblers in 10 groups) experimentally provisioned with a food item.
In the current work, the authors demonstrate that they themselves are also heartless.
Melissa can edit as SitC
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- Aug 2015
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optical microscope micrographs of the solution
This optical microscope, commonly referred to as a light microscope, uses light to magnify images.
The resolution power of an optical microscope is smaller than that of a TEM.
Optical microscope images were taken to confirm/support the starlike supermicelles seen in the TEM (which were several micrometers in diameter). The authors stated since the supermicelles were approximately 3 micrometers in diameter, the optical microscope could resolve the starlike strucutres. However, if the supermicelles were much smaller than 3 micrometers then resolution using an optical microscope would not be possible.
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Transmission electron microscopy (TEM) micrographs of a drop-cast sample of the solution showed the formation of large starlike structures
TEM is a microscopy technique where a beam of electrons passes through a thin-film sample. As the electrons interact with the sample, an image is produced. Samples that have atoms with high atomic numbers can produce darker images/provide better contrast.
The TEM samples were prepared on a carbon-coated grid. A drop of the micelle suspension was placed on the grid and then the excess solvent was removed using filter paper.
Using TEM the authors can verify they have achieved unidirectional growth. Additionally, they were able to see larger starlike micellar aggregates formed from the target noncentrosymmetric micelles.
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to remove the central M(PFS60-b-PDMS660) micelle block from the XLM(PI1424-b-PFS63)-b-M(PFS60-b-PDMS660)-b-XLM(PI1424-b-PFS63) triblock co-micelles to release the XLM(PI1424-b-PFS63) daughter micelles
Figure 1B depicts the synthetic strategy used to make micelles seeds capable of unidirectional growth:
After the cross-linking step, a solvent is chosen to dissolve the middle block (PFS-b-PDMS) of the triblock co-micelle. By doing this, the remaining PI-B-PFS micelle blocks can be used as seeds for elongation from the non-crosslinked end.
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creation of a sample of triblock co-micelles with two cross-linkable corona domains located on the end blocks and a non–cross-linkable corona domain as the central block.
Figure 1A depicts the synthetic strategy used to make triblock co-micelles:
1) The first step was to make micelles with crystalline cores. They did this by making a suspension of poly (ferrocenyl dimethylsilane)-b-poly(dimethylsiloxane) in hexane at 60C°. The solution was allowed to age at room temperature. They then sonicated the suspension in order to break up the micelles into smaller micelle seeds.
2) The next step was to add a second block copolymer to give bidirectional growth to form a triblock co-micelle. This was performed using a solution of unimers in THF which was added to the micelle suspension and allowed to age in order to form a longer micelle.
3) Finally, the polyisoprene end of the block co-micelle is cross-linked to prevent further growth using Karstedt's catalyst.
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Karstedt’s catalyst–promoted hydrosilylation with tetramethyldisiloxane was then used to cross-link the PI corona of the micelles to generate XLM(PI1424-b-PFS63) cylinders
Karstedt's catalyst is a platinum compound capable of forming bonds between carbon and silicon atoms.
In a separate publication, ref. 31 (DOI: 10.1021/ja206370k), the authors discovered the catalyst's ability to cross-link the double bonds in polyisoprene in the absences of silicon-containing molecules.
In this paper, Karstedt's catalyst was used to prevent block elongation from one end of the exposed cylindrical core to achieve unidirectional growth.
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- Jul 2015
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Finally, we established that dynein, myosin II, MTs, and actin were also involved in uncoating when IAV entered by endocytosis.
Because the host cell entry comprises multiple steps, the authors investigated which one was affected by the depletion of HDAC6. They noticed that HDAC6 has a function specifically during the uncoating and the vRNPs import.
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Using RNAi, we found that uncoating after PM fusion was decreased in myosin 10–depleted but not in myosin 9–depleted A549 cells (to 42% of control) (Fig. 3C). Inhibition of type II myosins with ML-9 and blebbistatin reduced uncoating to 38 and 48%, respectively
Authors investigated the role of myosin 10 and 9, which belong to type II myosin, in IAV uncoating after plasma membrane fusion. They blocked separately the gene expression for both Myosin 9 and 10 and observed that uncoating was reduced only in myosin 10-depleted cells. Additionally, two different inhibitors of type II myosins inhibited the uncoating.
Often, when more than one technique is available for exploring an hypothesis (i.e. involvement of myosin in IAV uncoating), scientists use them all to confirm their observations. By getting rid of myosins, authors can understand how important it is in the uncoating. Two ways were used to do so: blockage of the expression of the corresponding genes and inhibition of myosins by using drugs.
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we found that
By depleting dynactin 2 via RNAi and inhibiting dynein via treatment with CilioD (dynein inhibitor), the authors found out that viral uncoating was reduced in A549 cells. Also, uncoating is decreased in MEFs expressing HDAC6 that lacks the dynein-binding domain.
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it was observed using indirect immunofluorescence that after ammonium chloride (NH4Cl) washout to allow penetration of capsids from LAMP1-positive endosomes, the distribution of HDAC6 changed (Fig. 2E). Instead of a diffuse cytosolic staining, HDAC6 now associated with M1-containing vacuoles, where it gave a distinct rim-staining
This experiment confirmed that HDAC6 associates with M1. Authors highlighted HDAC6 associated to M1-containing vacuoles by using indirect immunofluorescence, in which one antibody binds to the target protein (HDAC6 in this experiment) and a second antibody, bearing a fluorophore, binds to the first antibody.
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Super-resolution microscopy of purified IAV using the same antibody showed free Ub staining in more than half of virus particles
The authors verified that what observed by immunoblotting was ubiquitin by using the super-resolution microscopy technique.
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we used HDAC6 KO MEFs that had been rescued either with WT HDAC6 (WTr) or with HDAC6s with point mutations either in the two deacetylase domains (HDm)
Following the observation that HDAC6 was implicated in the viral uncoating, next question that the authors asked was: knowing that HDAC6 has deacetylase activity domains as well as ubiquitin binding domain, what HDAC6 function is critical in the uncoating? To study these functions separately, the authors re-introduced WT HDAC6 or HDAC6 point mutated for etheir deacetylase or ubiquitin binding domain in HDAC6 knock-out MEFs
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we induced fusion of the virus directly with the plasma membrane (PM), a process that allows delivery of viral capsids into the cytosol without endocytosis
Authors ran a second experiment to confirm the involvement of HDAC6 after the fusion step. In this experiment, they bypassed the endocytosis process by means of pH change.
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However, capsid uncoating and nuclear import of vRNPs were reduced to 22 and 17%, respectively (Fig. 1A). In HDAC6-depleted A549 cells, uncoating was reduced to 21% compared with controls, with no effect on HA acidification or fusion
Because the host cell entry comprises multiple steps, the authors investigated which one was affected by the depletion of HDAC6. They noticed that HDAC6 has a function specifically during the uncoating and the vRNPs import.
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infection was reduced to 30%, and viral titers to 48%, compared with wild-type (WT) MEFs
To determine whether HDAC6 was involved with virus infection, the author examined mutant cells in which HDAC6 expression was silenced. The author observed that when HDAC6 is absent, viral infection and concentration were reduced.
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We generated com-plete and partial mitochondrial genomes from18 prehistoric canids and 20 modern wolves ofEurasian and American origin (Table 1 and tableS2) by performing DNA capture followed by high-throughput sequencing
This technique allows the authors to determine the precise order of nucleotides within specific regions of a DNA sample.
high-throughout means that large volumes of DNA can be sequenced quickly.
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e generated com-plete and partial mitochondrial genomes from18 prehistoric canids and 20 modern wolves ofEurasian and American origin (Table 1 and tableS2) by performing DNA capture followed by high-throughput sequencing
This technique allows the authors to determine the precise order of nucleotides within specific regions of a DNA sample.
high-throughout means that large volumes of DNA can be sequenced quickly.
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Local file Local file
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To track the dynamics of the cytoskeletonwithout introducing invasive probes, we specif-ically targeted short SWNTs (~100 to 300 nm;fig. S2) to the endogenous kinesin-1 motor Kif5cin cultured COS-7 cells (see supplementary ma-terials).
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