286 Matching Annotations
  1. Jan 2024
    1. s 4 U

      4-Thiouridine is an atypical nucleotide formed with the 4-thiouracil base found in transfer RNA (tRNA). 4-Thiouridine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. "plays a role in protecting cells from near-UV killing"

    2. imeLapse-seq

    Annotators

    1. s4U

      4-Thiouridine is an atypical nucleotide formed with the 4-thiouracil base found in transfer RNA (tRNA). 4-Thiouridine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. "plays a role in protecting cells from near-UV killing"

    1. exposed on the upper face of the DDT

      for IgG binding

    2. d) EM micrographs, class averages and models (top, middle, bottom, resp.) of DDT1-6. Scale bars represent 10 nm.

      direct measurement of the DDT structures

    3. central hub of the design was modified to generate individual tiles

      so the different number of DDT arms used to control antigen pattern

    4. nanoscale shapes that are chemically accessible,

      accessible to IgG, controlling oligomerisation and complex valency

    5. Formation of these hexameric platforms is reliant upon the ability of the IgG monomers to bind to antigens at a spacing that provides the means to then form Fc-mediated complexes.

      so spacing and availability of antigens on a membrane affecting the type of IgG complex that is forming Controlling the antigen patterns enables manipulation of IgG valency------ see how this is checked/measured?

    6. complement-mediated MAC pore formation.

      using the degree/amount of membrane rupture to assess complement activation levels----- and the link to Ab valency

  2. Nov 2023
    1. TWISTER
    2. Fmoc solid-phase peptide synthesis

      possibly an older technique?

    3. supercoil

      A superhelix is a molecular structure in which a helix is itself coiled into a helix

    4. Sequence-to-structure relationships inproteins are immensely complex and are not fully understood.

      look at more recent research

    5. the components of such toolkits shouldbe fully characterized in all respects from sequence through tostructure, function, and interactions

      THIS!!! A GOOD STATEMENT!! One thing that I didn't like about previous projects was limitation on the investigation- what was needed to progress. I was interested in continuing to analyse a protein, but this was not seen as important. One attractive thing about this research is the comprehensive nature of investigation into the peptide functioning

    6. heptad repeat

      The heptad repeat is an example of a structural motif that consists of a repeating pattern of seven amino acids: a b c d e f g H P P H C P C

    Annotators

  3. May 2023
    1. COX= cyctochrome C oxidase in the IMM

    Annotators

    1. Aside from playing a rolein mitochondrial fusion, CL also interacts with DRP1.

      cardiolipin

    Annotators

  4. Apr 2023
    1. Size exclusion chromatography:a real example

      SEC will show peaks for different complex organisations, especially depending on the buffer used (does it cause dissociation, e.g. by addition of Tris). This can be used to identify if proteins form dimers/ tetromers (etc.) by the number of peaks on the chromatograph.

    2. Size exclusion chromatography:

      Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix

      SO larger molecules are unable to fit into most of the pores of the matrix meaning that they pass through much more quickly than smaller molcules. The smaller the molecule, the longer the path/distance travelled in the column due to the level of indirectness. Small molcules will enter into pores and be slowed down.

      Therefore the faster the elution, the larger the molecule--- these appear in the lower volume fractions as they leave the column first. This is also why columns have specific ranges of molcule size as to enable sufficient separation between molcules.

    3. Atomic force microscopy

      Atomic force microscopy (AFM) is a three-dimensional topographic technique with a high atomic resolution to measure surface roughness. AFM is a kind of scanning probe microscope, and its near-field technique is based on the interaction between a sharp tip and the atoms of the sample surface

    Annotators

    1. isoproterenol or vehicle

      so then the channels were activated to see how the biotinylation proteome changes

    2. containing biotin phenol, followed by exposure to hydrogen peroxide, induced robust biotinylation of proteins in a striated z-disk pattern, coinciding with the pattern of transgenic α1C and β2B subunits

      so bitotin needed for labelling (endogenous not enough and can affect other cellular processes) and HP to activate the APEX-

    3. isoproterenol.

      agonist of the calcium channels?

    1. 5 d

      this is a very long period in vivo- not as fast as previously said for in vitro... improving this is important for use of prox labelling in vivo settings

    1. It is still possible that some of these proteins are not directAurora substrates, but secondary targets

      this is where the on paper notes explain further experiments are required to show direct p-p interaction between the kinase and possible substrates in vivo and in vitro!!

    2. suggesting that there are additional Aurora substrates not coveredby our analysis

      ah so it agrees that its not full coverage

    3. alleviated this effect by including a technical replicate for each exper-iment

      hmmmm still not necessarily detecting everything? search mass spec coverage?

    Annotators

    1. A combination of both mechanisms, trappingand pulling, may best explain the dynamic activity of thismultisubunit motor

      the more recent 2017 paper continues this 'mixed mechanism' theory, so this is still believed

    2. the preproteins interact withthe intermembrane space tail of the receptor Tom22

      So the presequences interact with Tom22 on both the cytosolic and IMS side of the OM.

    3. inser-tion of α-helical proteins into the outer membrane prob-ably involves several other pathways that have only beenpartly characterized

      is there a more recent paper on this?

    Annotators

    1. The Acid chainhypothesis

      paper on word doc on this... simple not well established

    Annotators

    1. This delay is due to post-transcriptional processes, because quantitative reverse transcription-PCR (qRT–PCR) detected VENUS messenger RNA minutes after auxin treatment

      MATURATION OF THE FLUORPHORES BEING KEY!! this is the major disadventage of expression reporters

    1. doi: 10.1073/pnas.1706379114

      hypothesis annotated

    2. Alternative splicing increases proteome diversity and transcriptome plasticity

      Phytochrome controls alternative splicing to mediate light responses in Arabidopsis. 2014. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284612/

      hyp

    3. Leivar and Quail, 2011, TIPS
    4. Bourbousse et al., 2012,PLOs Genetics
    5. Imaging nuclear dynamics: a new era

      has been used in plants, but not yet applied to chromatin density measurements.... GOOGLE

    6. Transcription Factories

      HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions. 2023. HEAT RESPONSE THO https://doi.org/10.1038/s41467-023-36227-3

      pretty sure this is correct... should use this paper for reference!

      cannot find anything in photoreceptors

    7. Investigating the hierarchy of the 3D genome

      HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions. 2023. HEAT RESPONSE THO https://doi.org/10.1038/s41467-023-36227-3

      same as previous... is this what is being referred to?

    8. Light and temperature induce changes in nuclear architecture components

      Chromatin-associated SUMOylation controls the transcriptional switch between plant development and heat stress responses. https://doi.org/10.1016/j.xplc.2020.100091 This paper from the dissertation... good examples of chromatin changes in response to temp...

    9. doi:10.1016/j.pbi.2018.05.018
    10. Feng et al., 2014, Nature Commin.. 154
    11. Light induced gene re-positioning in Arabidopsis

      opposite effect for cold-induced repositioning

    12. nuclear periphery

      to allow quick translation following transcription

    13. Light Signaling Components Control Nuclear Architecture Reorganization DuringCotyledon Development

      https://doi.org/10.1073/pnas.1503512112

      hypoth

      'Upon light perception, master regulators of the light signaling pathway trigger rapid and massive nuclear expansion, condensation of large chromatin domains, and increased transcriptional activity of genes.'

    14. Cry2 mediated reversible changes in chromatin condensation regulated by light

      BL does not directly cause a change in compaction, but cry2 plays a role is decreasing chromatin density. In response to BL cry2 is degraded, reducing this activity. Therefore after a while, the chromatin densityincreases again? check paper?

    15. Chromocentres

      Chromocenters form from the clustered aggregation of pericentromeric regions from two or more individual chromosomes and create a heterochromatic hub that may influence a wider environment for transcriptional silencing.

    Annotators

    1. This study indicates that the protein components of the plant NE are important for regulating chromatin organization and activity

      nuclear envelope (NE)

    1. conventional FISH does not efficiently label DNA regions of 10 kb or less;

      THE PAPER ON FRET-FISH??? for specific gene loci (in fluoro proteins doc)

    1. Nuclear condensates for landscaping the chromatin structure

      are any of these proteins interacting with photoreceptors?? could google some if necessary

    1. It can still be a labor-intensiveand time-consuming task to optimize the system toproduce enough protein for crystallization trials.

      ALSO: overexpression can cause aggregation. If not expressed in the native envionment, conformation and the different complexes formed may differ.

    Annotators

    1. RECENT

      remember, this is NOT a recent paper

    2. average over many particles images

      this is both useful, and a disadvantage!

    3. projection-slice theorem

      'The projection slice theorem states that by collecting a group of one-dimensional projections at various angles a two-dimensional image can be constructed, and from the slices of two-dimensional images, a three-dimensional images can be formed'

    4. “single particle” analysis

      KEY POINT=== AVERAGING!!! this gets hid of any heterogenous features!

  5. Mar 2023
    1. can orchestrate the function of anorganelle encoded by two separate genomes, both of which re-quire p53 for their integrity,

      so is p53 encoded in both nDNA and mtDNA? NO, this is all about CHCHD4 regulating the abundance in mito- must be imported into mitochondria

    2. (D)

      Yet again the colours are not explained well

      Images are too small and difficult to compare, blurry upon zooming in too

      perhaps instead of this 'overlapping', to show colocalization use a technique such as FRAP/FRET to show fluorescence upon interaction?

      also why is the condition with siRNA CHCHD4 not shown for these cells? like in B. would expect to see similar results to the cDNA condition? better showing that without the SCO2, CHCH overexpression does not increase p53 nuclear import

    3. (C

      again loading control? also there is still some p53 detected in the column-bound without CHCH present? also;;;; binding to his6 tag? Need another experiment to confirm interaction, this and imaging is not really enough?

    Annotators

    1. What are the column names?

      Var1= gnames Var2= sample grouo value= expression values

    2. day 3

      em_symbols or em_scaled

    Annotators

    1. signal intensity

      units?

    2. GAPDH

      is there a reason for only 3 loading controls?

    3. E

      what is that symbol?

      loading controls?

      why are there so many PINK1 bands?? needs arrows, what am I looking at??

    4. V5

      this is a small peptide tag

    5. C-terminal HOIP

      what's this?

    6. D

      This is soooo confusing! the figure states an anti-V5 antibody, but does this suggest anti-Ub??

      The smear at 80+kDa, no label?? What is this supposed to be?

      all lanes look very similar, arrows should've been used to indicate bands clearly

    7. Input

      this is just totally not explained in the figure legend

    8. C

      No idea what is being shown here, as the legend explains nothing--- the %??, no units

    9. p-S65-Parkin

      more sig

    10. p-S65-ubiquitin

      kinda??

    11. nterestingly, M1-ubiquitination at mito-chondria induced by TNF treatment or OTULIN silencing alsoincreased the processed PINK1 species

      direct proof of link between Ub and PINK processing??

    12. B

      again more unprocessed PINK1 with CCCP treatment, but not really less processed... could this instead be attributed to expression change?

    13. A

      does kinda show more unprocessed PINK1 w/ CCCP, compared to TNF, control, and silenced conditions

      from legend: unprocessed PINK1; processed PINK1. ALSO... P < 0.05 both? is ** attached to any statistical significance?

    14. carbonyl cyanide 3-chlorophenylhydrazon

      chemical inhibitor of oxidative phosphorylation

    15. M1-ubiquitination at mitochondriais a fast and transient response with maximum intensity after15 mi

      not very clear results

    16. G

      The M1-Ub is not very clear...

      'TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies'

    17. F

      molecular weights would be nice... does show bands for 'LUBAC' components HOIP, HOIL, and SHARPIN at the mito only with TNF treatment (or increased co-localisation)

    18. E

      whats yellow? shows colocalisation of hsp60 and M1-Ub... not all HSP60 is mito-localised... couldve done an experiment specifically tracking mito...

      'Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red), and analyzed by SR-SIM. Scale bar, 5 μm'

    19. D

      for mito: Ub only detected in WT with TNF treatment, HOIP KO extinguished Ub

      loading control inconsistency?

    20. indicating that OTULIN regulatesmitochondrial M1-ubiquitination

      there is a clear difference in signal when silenced

    21. B

      shows even less localisation at mito than A... but there is a difference in the amount of mito Ub, as shown by the control vs. tnf bands

    22. A

      the amount of HOIP co-precipitating is much higher than OTULIN. there is almost no diff between the control and TF treatment cond for OTULIN

    23. B to the nuclear.

      ??? to the nucleus

    24. C

      image of actual results, doesn't clearly show whats being highlighted here

    25. A

      Figure error; overlapping numbers on image

    Annotators

  6. Feb 2023
    1. loop

      A loop is a control statement that allows multiple executions of a statement or a set of statements

    2. numbers

      the 3rd number in the 'numbers' vector

    Annotators

    1. $

      $ specifying the column!!!

    2. possible

      the aes() adds the legend, by grouping the data---- by what is specified by colour=

    3. legend

      shows the colours (2) with either TRUE or FALSE.

    4. hat happens to the plot?

      colours the non-sig values red (prev black) and sig values blue (prev red and blue)

    5. blue

      add in colour= "" to aes()

    6. Finally, try changing row.names=1 to row.names=2. What does this do? Why doesn’t it work?

      theses rows are not first, they cannot be shifted to the row names. =1 indicates that the data file has row names, and which column number they are stored in--- column 1

    7. header=TRUE, row.names=1

      use header=TRUE, then the first row is used for the names of the columns

    8. rbind

      joining of 3x tables, that now each have a 'direction' column containing either a, b, or c--- as the data

    9. The tricky part in this method is having a column (or vector) which groups the dots appropriately. Having the sigcolumn is great but it only has two categories, and so only colours the volcano plot sig and not-sig. If we want toauto colour three ways we need to build a new column into master with groups “a”, “b”, “c”

      so need to create 3x cateogories for which colour can be automatically assigned

    10. factor

      'factor returns an object of class "factor" which has a set of integer codes the length of x with a "levels" attribute of mode character and unique '

      changes the data according to the assigned levels

    11. levels

      levels provides access to the levels attribute of a variable. The first form returns the value of the levels of its argument and the second sets the attribute. allows you to assign a factor/level to variables, in this case, we are ordering significant (TRUE) first and unot second (FALSE). Sig is leveled above

    12. What happens if you add a the geom_text layers from your previous volcano plot

      for me, it didn't work, many errors

    13. aes()

      aes() is a quoting function. This means that its inputs are quoted to be evaluated in the context of the data. This makes it easy to work with variables from the data frame because you can name those directly. Quasiquotation

    14. What happens if you change the width / height

      these are the number of pixels wide and long, increasing--- increases the size and file size, because of more pixels

    15. em_symbols_sig = em_symbols[

      em_symbols_sig = em_symbols[sig_genes,] em_scaled_sig = em_scaled[sig_genes,]

    16. ONLY

      sig_genes = master_sig[,17]

    17. p.adj < 0.05 & abs(log2fold) >1)?

      master_sig = subset(master, p.adj < 0.05 & abs(log2fold) >1)

    18. # converts a matrix to a data.frame

      https://www.geeksforgeeks.org/matrix-vs-dataframe-in-r/ v.s. Both represent 'rectangular' data types, meaning that they are used to store tabular data, with rows and columns. The main difference, as you'll see, is that matrices can only contain a single class of data, while data frames can consist of many different classes of data.

    19. transpose

      swap columns and rows around

    20. scaled expression matrix

      a method of normalising the expression values? scale()= generic function which centers and scales the columns of a numeric matrix

      possibly: Centers and scales gene expression matrix so that each row has mean=0 and sd=1

    21. What is abs()?

      abs()= absolute value, takes all positive values and doesn't change them. Takes all negative values, and makes them positive, then extracts them. In this case, this allows us to take values that are significantly upregulated >1 and downregulated <-1

    22. How would you do this using just one line of code?

      names(annotations) = c("chromosome","start","stop","Gname","Gtype")

    Annotators

    1. (B)

      significant difference between NS and CHCH shRNA--- higher ratio (more ox, less glyc) for the NS condition? So depletion of CHCH causes decreased ox. metabolism.

    2. (A)

      significant difference in the p53-/- condition? So even without p53, there is an effect on amount of oxidative metabolism? unexplained

    3. H 2 O

      concentration the same?

    4. CHCHD4-mediated p53 im-portation

      affect trafficking to mito, or the import directly?

    5. CHCHD4 Regulates p53 Function in the Mitochondria.

      !!!!!!!!!!! Suggest doing an in vitro assessment of interaction, to identify whether there are any other molecules involved in this process/needed for interaction--- e.g. CO-IP

    6. basal integrity of nuclear genomic DNA was measured

      how?

    7. CHCHD4 caused exclusion of p53 from the nucleus afterH2O 2 treatment (Fig. 2B)

      I'd say that is clear

    8. Fig. 2A, Right

      useful

    9. (Fig. 2A, lanes 2 and 4

      not a huge change, quantifiable? repeats, imageJ, statistical significance?

    10. Nuclear p53

      How did they extract just the nuclear p53?

    11. At the structural level, the specificity ofthis interaction was further confirmed by substituting the two cys-teine residues in the CPC motif of CHCHD4 with serine, whichabolished p53 binding (Fig. 1F, lane 10).

      Suggest a quantifiable method of measuring interaction difference

    12. (F)

      why is there significantly more p53 here? again, should consider a better measure of interaction

      loading controls!!

    13. (E)

      pull-down assay with TAGGED p53? I still question, is there no CHCH endogenous exp (without transfection??),as would expect less but something in the total lysate?? loading control

    14. as indicated by the absence ofeither yellow (Fig. 1D, merge p53/CHCHD4) or white color(merge all three)

      There is definitely some white/light blue? Again this is so non-conclusive. Need more evidence to back-up the effect of non-respiring on localisation...

    15. Confocal immunofluorescent mi-croscopy showed increased accumulation of p53 in the nucleusof SCO2−/− cells

      WOAH how're you identifying the nucleus here?? it is not indicated in the figure, and not tracked like the mito?

    16. CHCHD4-His6

      evidence of endogenous CHCH expression? an sds-page would be nice

    17. Western blot

      CO-IP? No affinity purification... in vitro

    18. (Fig. 1B, merge all three).

      These images don't really back up this conclusion very well, the white and yellow is drawn on? The images between conditions are so dissimilar that its difficult to compare them

    19. (A) Confocal immunofluorescent localization of CHCHD4 (green) and p53 (red) inmyoblasts transduced with lentivirus containing CHCHD4 cDNA or shRNA and then transfected with WT p53. MitoTracker (blue) indicates mitochondria.

      The colours are not explained in the legend, it can be assumed that p53 + CHCH is yellow. And all 3 (p53, mito, and CHCH) together is indicated by white? Again the images between rows are soo different its hard to conclude anything

    20. (B)

      Images are confusing,1st 5, showing 3 cells? then 2nd row, 1 cell?

      The mitochondria localisation itself changes upon cDNA? NO?

      The images in the different rows need to be more similar to enable comparison...

      Does this really give any information about localisation? The cTRL and shRNA images look the same, p53 (red) and mito (blue) are together?

    21. transfected with p53

      again only showing this when p53 is overexpressed

    22. APDH and VDAC serve

      The GAPDH control shows faint bands in the mito fractions. The p53 bands are not dissimilar- does this suggest crossover? Are the p53 bands in mito actually cytoplasmic, check methods.

    23. A

      Lanes 1-4 show increased CHCH expression w. the cDNA only in the mito fractions (not in nDNA). The amount of p53 increases in the mito, w/ cDNA but stays the same in the cytoplasm. So no evidence of EXP change, but localisation.

      with the shRNA KO: decreased amount of CHCH in mito. Same amount of p53 in cyt, no expression change. But decrease in amount in mito--- marginal?

    24. stable knockdown of CHCHD4 using shRNAdecreased the relative level of p53 in the mitochondrial fraction

      the opposite effect is shown?

    25. markedly increased the levelof p53 in the mitochondrial fraction

      So this suggests that overexpression of CHC CAUSES increased p53 expression?

    26. important implications for cance

      Could be more specific. How does the loss of p53 in cancer link to mitochondria?

    27. A

      Notes: -applications? what are the implications in cancer? Is there any hypothesis of HOW this knowledge could be utilised?

    28. superoxide dismutase

      Superoxide dismutases (SODs) constitute a very important antioxidant defense against oxidative stress in the body. The enzyme acts as a good therapeutic agent against reactive oxygen species-mediated diseases.

    29. Thus, the mitochondrial disulfide relay system allowsp53 to regulate two spatially segregated genomes depending onoxidative metabolic activity.

      is this conclusion backed up? the WHOLE system, or just this specific protein?

    30. (A)

      Loading controls?

    Annotators

    1. H

      H, I trying to show that BAK/BAX are not relevant, instead target BID

    2. G

      BAK not active in the resistant line

    3. D

      tBID necessary for trail to cause cell death

    4. C

      KOs not completely effective, still some BID expression

    5. A

      shows the resistance of the cell line to cancer drugs

    6. E

      shows tBID-mediated release of SMAC, not in TKO only DKO

    7. D

      DKO- compare STS and sf sf--- SMAC released and not in STS tBID-mediated release

    8. B–D

      issues with units!! also how significant? large error bars

    9. CLEM

      light and EM microscopy- correlated

    10. E

      only BID KO- the mutaions on BID dont sig effect the cell death. tBID is not as important if BAX/BAK is present

      the helix mutations arent effecting BAX/BAk activation, by tBID, so this helix is not involved in this interaction

    11. EF

      mouse embroyonic fibroblasts (MEFs)- just another model

    12. K157/K158 at the a-helix 6 in blue

      the helix mutation ONLY shows a decrease in cell death, this helix not the BH3 domain. The helix is important for the BID functioning

    13. K

      supposedly showing significant increase in cell death (by measurement of cytosolic mtDNA) when using the COMBO treatment with either BAX or tBID transfected. No sig diff to cell death without the COMBO treatment, and just with transfection--- need anti-apop proteins neutralised

      BAX and BID both cause mtDNA release. both significant

      but using DKO--- lots and lots of tBID present as its overexpressed and not KOed... can you compare them? less BAX present

    14. I

      TKO- sig less cell death than DKO--- tBID is working

      red release= showing mtDNA outside (away from TOM20) in the tBID condition tBID induces release MOMP

    15. FAM

      mitochondrial DNA--- showing release of mtDNA

    16. G, H

      pretty weak WBs, with varied bands for the loading controls. G) somewhat shows that BID is completely KOed 24h after siRNA silencing, but small tubulin band? H) incomplete KO in DKO cells, good should show BID, but not much?

      TKO- triple BAX/BAK/BID KO

      TKO1 and 2--- different different guide RNA, CRISPR to check both clones work

    17. F

      the silenced phentoype (no cell death) is able to be recovered by overexpression of tBID-GFP--- good control

    18. Percentage of cell death induced by TRAIL (1 μg/ml), COMBO and their combination

      so this is just showing the effect of the treatments on WT cells. the combination treatment only, produces a significant increase in cell death

      so DKO-no BAX/BAK, but still BLCs and tBID. COMBO- inhib BCL TRail activates, cleaves BID

    19. Representative WB

      cleavage shown, but also less expression? and again no representative loading control

      cleaved tBID- induced by trail

    20. B

      TRAIL extrinsic activation of pathway- death receptor activator--- leads to cleavage of BID to tBID (activates)

      COMBO prevents sequestering of tBID

    21. HCT D

      wouldve been nice to have an additional control, of WT cells, showing BAX/BAk having the same effect

    22. ABT737 + S63586)

      Inhibition of anti-apoptotic protein--- leading to activation of apoptosis... Here DKO--- tBID can cause apop without BAX/BAK

      w/o COMBO- tBID is sequestered by anti-apoptotic proteins

    23. DKO cells

      Double BAK BAX KO

    24. ZVAD

      significant reduction in nuclei release? and cell death in the presence of ZVAD?

      Doesn't really explain what is being measured in terms of cyt c and nuclei? From the next figure: it is that cell death is being measured based upon the release of these 2 molecules

      not inhibiting cyt c release, only the downsteam caspases

    25. ell death induce

      using different cell death inhibitors to test which type is occuring. ZVAD=apoptosis (caspase inhib) ferroptosis- ion dependene tmode of cell death NSA= necrosis inhib MOCK= placebo, the vehicle control

    26. G

      ratio of GFP (tBID) and PI (uptaken)

      No information on the time point of this, how long?

    27. F

      Does show increased PI uptake with increasing tBID expression. BUT big standard deviations, reliable method to measure expression?--- RNAseq?

      PI only uptaken in rumptured cells

      comparing amount of tBID and amount of PI uptake, kinetics at the same time. They increase together, correlating cell death takes time, after tBID peaks, for apoptosis to occur

      control, no PI uptaken

    28. Representative western blot analysis of PARP1

      Loading control? Does appear to be the same amount of PARP is the - condition uncleaved and + condition cleaved. However, what is the cleavage product? Should there not be a secondary ~15kDa product?

      caspase cleaved substrate-- example to show activation

    29. Quantification of caspase activity i

      luceriferase reporter

    30. Quantification of the effect

      uptake of PI dye which is only permeable to dead cells, then looking for the presence of the 2 hallmarks. NOT here, just morphological changes

    31. HCT AKO cells

      physiological relevence- will not have AKO occuring naturally. This may not be observable in vivo

    32. pyknotic nuclei

      condensation of nuclei

    33. PARP1

      DNA repair polymerase

    34. alm6 pre-B ALL cells

      Nalm6 is a B cell precursor leukemia cell line initiated from an adolescent male. It is a CD24-positive xenograft model of acute lymphoblastic leukemia

    35. **P < 0.025 and *P < 0.05 with respect to tBID-GFP wt condition

      vague significance here, only 0.025?

    36. B

      significantly less cell death when the lysines are mutated

      slightly more death upon methionine or asparagine mutation?

    37. tBID-GFP

      transfection or...?

    38. BAX/BA

      so are these KO?

    39. Representative confocal immunofluorescence im

      The difference in (cyan) nuclei is obvious, but not so much cyt c--- for which the pattern of (magneta) is similar in the control

    40. D

      Shows: -no sig diff between GFP (control) and BCLXL -low * (12h) and then mid ** (24h) sig diff between tBID and control, but not a high level of significance--- does this conclude that caspase activity is definitely promoted by tBID? -sig dif between BAK and BAX and control

    41. C

      Pellet fractions: consistent amount of SMAC in the membrane fraction. generally more Cyt c in the BCLXL--- to do with level of expression? No control here. There should be cells just expressing GFP for comparison of expression levels. AND no loading control

    Annotators

  7. Nov 2022
    1. (a, b)

      A= RED LIGHT hy5 null mutant= increased hypo elongation= lack of HY5 inhibition of skotomorph

      HY5 (overexp) and S36A show similar patterns of reduced elongation- more so than for WT. This suggests that the inhibitory functioning of HY5 is still occurring with or without phosphorylation. These show elongation less than WT--- because they are both overexpressing the HY5?? And there would not be as much natural expression of HY5 under RL conditions/more degradation?

      S36D shows increased inhibition, suggesting that phosphorylation is decreasing HY5 activity.

      B=far red

      Under FR conditions, all show a high level of inhibition by HY5 of elongation--- suggesting how important HY5 is in this process.

      Compared to the red light condition, the elongation is reduced. This suggests that HY5 is more regulated by far-red signalling than red. This is because red-light does not highly activate skotomorph- like FR.

    2. Whether phosphoryla-tion of HY5 has any influence on interaction with other tran-scription factors awaits further studies

      To find out if S36 phosphorylation on HY5 affects interaction with other TFs, a CO-IP could be performed on the mutated HY5 S36A and S36D to show whether BBX20/21/22 interacts differently. OR a proximity labelling experiment with LS-MS could be used to identify the TFs that interact with non-phospho-HY5 (either in spaQ mutant or S36A) and phosphorylated HY5 (WT/overexpressing SPA1 or S36D) in vivo!

    3. HY5 proteins were signifi-cantly accumulated in both cop1 and spaQ mutants

      Accumulation of HY5 in COP and SPA mutants? The phosphorylation by SPA causes degradation of the HY5? And in these mutants without COP1 or SPA (1-4) there is no degradation--- this is not due to lack of phosp but rather lack of Ub (as phospho-HY5 is more stable in the dark-- later on)

    4. we hardly detected HY5 protein signals in WTseedlings grown in the dark,

      Paper to support lack of HY5 expression in dark conditions?

      If they are never shown light- then HY5 should not accumulate? (if light-to-dark transition, the lack of HY5 would be due to degradation by COP1/SPA1)

    5. (e)

      The multi-bands are again present here- could this suggest degradation or multiple phospho-sites?

      mSPA mutation decreases phosphorylation in the radiogram- but the bands are still strong on the coomassie- Could this mutation cause a conformational cause that reduces its affinity for HY5? Use imaging to understand any structural change?

    6. ime-dependent kinase assays of full-lengthSPA1 on HY5 (autoradiogram in the upper panel, reaction time for 15, 30, or 60 min).

      Radio-labelled ATP- the phosphorylation is producing the bands on the autoradiogram. Only bands when the SPA1 protein is present. The coomassie showed multiple new bands with SPA1 added- multiple phosphorylation sites? Multiple kinases that WOULD interact in vivo?

    7. (HY5-S36A) cannot be phosphorylated by SPA1

      OK, so this may suggest that HY5 is only phosphorylated by SPA1 at s-36, but another phosphorylating protein may interact? A in vivo proximity tagging experiment could be used to track all interacting proteins for HY5.

      BUT--- notice there is still some residual bands in the mutated samples- is this showing some other phosphorylation of HY5 at a different site?

    8. A previous study showed that a deletion of the first40 amino acids of HY5 completely abolished the interaction withCOP1 (Hardtke et al., 2000).

      So this backs up the Ser-36 phosphorylation, but does this study try deletion of residues 80-110 which are not covered in the mass spec data? May not be as essential as the 1st 40, but could still be relevent. after 80 is outside of the COP1 interaction site, but could be involved in supporting the structure/another function?

    9. in vitro phosphorylation assays

      Mapping and analysis of phosphorylation sites: a quick guide for cell biologists. 2017. https://doi.org/10.1091/mbc.e12-09-0677

      Tandem mass spectrometry (MS/MS) analysis of protein phosphorylation. (A) Protein samples are digested with a proteolytic enzyme. The resulting peptides are separated by reverse-phase high-performance liquid chromatography. Peptides enter the mass spectrometer as they elute from the column. Peptide matching is done algorithmically using spectral data and sequence database information. (B) Two basic types of information are generated in the mass spectrometer. The masses of intact peptide ions are determined in a full scan (MS or MS1). Peptides are then isolated one at a time, as depicted for the highlighted peak, and fragmented by colliding them with an inert gas. The resultant fragment ions are detected in a MS/MS (or MS2) scan. With sufficient coverage of fragment ions, the position of the phosphorylated residue, circled in orange, can be identified from the MS/MS (upper path). Fragmentation, however, often liberates the relatively labile phosphate groups at the expense of more informative fragmentation of the peptide backbone (lower path). In extreme cases, MS/MS spectra are dominated by a single ion representing the intact peptide stripped of phosphate. (C) Site localization is dependent on the detection of ions that can distinguish between possible phosphorylatable residues. In the example shown, a threonine and a tyrosine are separated by one amino acid. Fragment ions in the central panel either do not contain a phosphorylatable residue or will have equivalent mass for both possible phosphopeptides. If the threonine is phosphorylated, we would expect to see some or all of the top four site-specific fragment ions shown on the right. If, instead, the phosphate lies on the tyrosine, we would see the bottom four.

    10. trep-tagge

      The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag II is a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin, and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions, it is especially suited for generation of functional proteins.

    11. Pichia pastoris

      methylotrophic yeast.

    12. S. cerevisiae

      The main purpose of recombinant protein expression is often to obtain a high degree of accumulation of soluble product in the bacterial or yeast cells

      However, high level of expression can lead to aggragation of the protein- especially in inclusion bodies- therefore it is important to optimise the expression vecotrs, hosts and conditions

      Yeasts are the simplest eukaryotic organisms and like bacteria are single-celled, genetically well-characterised, easy to grow and mainpulate. since yeast is a eukaryote, it have an intron excision mechanism. Thus, it can be used for producing and expressing recombinant DNA of eukaryotes. while bacteria may produce a larger amount of protein, the protein produced may not have the same types of post-translation modifications as it would if it were produced in eukaryotic cells like yeast. In this case, yeast may therefore be a better host for protein production.

    13. COP1–SPA complex is also reorganized, reducing the E3ligase activity of the COP1–SPA complex, thus stabilizing HY5 under light

      this was important!!! wheres this form?? Measure Ub in light and dark??

    14. skotomorphogenesis.

      Light-grown seedlings exhibit a developmental response termed photomorphogenesis, resulting in short hypocotyls and expanded green cotyledons. In contrast, dark-grown seedlings are characterized by long hypocotyls and unexpanded etiolated cotyledons; this process is called skotomorphogenesis

    Annotators

    1. purified from Sacharomyces cerevisiae and GST-HY5 protein purified from E. coli.

      Were the SPA proteins purified from yeast to improve likelihood of expression of a functioning protein- in a eukaryote rather than prokaryote. Better folding, PTM addition etc.

    Annotators