In B. subtilis, all three proteins are essential for engulfment and deletion mutants demonstrate developmental blockage during sporulation after asymmetric septation and produce bulged septal membranes."

"Interestingly, we found that although both transcriptional and translational machineries are present in the forespore of the mutant strain, there is little fluorescence signal corresponding to the active translation in the spore..."

In the DSP cross-linked samples, we also observed enrichment of translation factors...indicating that we have collected actively translating ribosomes, generally resulting in defective spore formation."

Deletion strains of any component of the complex produce small forespores with membrane invaginations and limited transcriptions during late sporulation."

"Based on the above observations we propose that the ribosomes are packed into the forespore sequentially, after the chromosome translocation, and that this shift is associated with membrane migration or cell wall remodelling during engulfment."

"In vegetative cells of B. subtilis, the ribosomes were shown to be localised at the cell poles, away from centrally located nucleoid and transcriptional machinery.

"Polar modifications of the synthetic agonist that extend toward the phosphoinositol pocket may allow to improve solubility and selectivity while maintaining or even improving potency."

"Relatedly, in MD simulations of the cysteinyl leukotriene receptor 1, it was previously demonstrated that the presence of an orthosteric ligand is essential to prevent the collapse of a similar membrane opening between helices IV and V."

"Admittedly, even single point mutations can affect the receptor’s surface expression, and differential receptor expression may potentially influence the comparison of ligand potency and efficacy or the basal receptor activation. However, none of the investigated GPR55 mutations was completely inactive, and some mutations (e.g., R2536.62A or G1524.56W) even showed opposite effects on the potency of both ligands which cannot be explained solely by altered receptor expression."

"We discovered that intact receptor glycosylation is essential for basal GPR55 activation and ligand potency even though ML184 did not show direct glycan interactions. Therefore, we hypothesize that the removal of the ECL2 glycosylation site may affect the structural integrity or dynamics of the ECL2 which, in consequence, could explain the reduced activation with and without ligands."

Shouldn't the suggestions for future experimentation be located at the end of the paper rather than in the middle? The organization of this paper is throwing me for a loop.

What are these microswitch conformations?

What is the root-mean-square deviation (RMSD)?

"The density map results indicated that CLR remains in the identified CLR binding site in system 1 for all three replicates. For system 2, where ClLR was removed from the CLR binding pocket, we observed a CLR molecule diffusing towards the CLR binding site from the membrane within approximately 600 to 1,300 ns. Within the next few hundred ns, the CLR inserted into the same CLR binding site."

Why did they clarify which species' cell membranes they used for the experimentation so late into the paper, as well as the employed cholesterol molecule? Shouldn't that be something introduced towards the beginning of the paper?

"Surprisingly, mutations of N171^ECL2 to A or Q still significantly reduced the potency of ML184 by 24- and 32-fold."

"Accordingly, the potency of ML184 at the receptor containing the R253^6.62A mutant was not reduced but slightly increased by a factor of three with an efficacy similar to the wt receptor."

"The mutations reduce the potency of LPI by 3- to 5- fold but did not affect efficacy or basal receptor activation, indicating that bulkier amino acids at this position can provide a significant steric hindrance without completely abolishing LPI activity."

"Notably, both mutations (N171^ECL2Q and N171^ECL2A) lead to a significant decreased constitutive receptor activity (19% and 16% basal activation respectively)."

What in the world is the "Ballesteros-Weinstein numbering system"?

What does the "A" with an accent mean when the paper is talking about global resolution?

"The lysophosphatidylinositol findings corroborate that GPR55 can be activated by membrane components in vitro (test tube). However, the receptor currently retains its orphan statue due to the lack of significant in-vivo (organism) evidence for any of the discovered lipids"

We considered two ways in which alpha-syn aggregation could diminish ESCRT-III availability. First, aggregate sequestration may immobilize ESCRT-III subunits, restricting them from reaching sites of action. Second, aggregation may reduce ESCRT-III protein levels, as we observed CHMP2B levels to decline by half in HEK cells and primary neurons upon aggregation." Both hypotheses feel very likely.

"This synergy between CHMP2 knockdown and alpha-syn aggregation is consistent with both perturbations limiting ESCRT-III availability, thereby reducing ESCRT function." So a knockdown CHMP2 variation along with alpha-sun aggregations reduce ESCRT function as well.

"CHMP2B-alpha-2 therefore binds fibrillar but not monomeric alpha-syn." "As for alpha-syn, CHMP2B-alpha-2 specifically bound fibrillar but not monomeric A-beta-42." Interesting how the modified CHMP2B was only able to bind up the fibrillar form of two different amyloid proteins.

"Deleting these helices sequentially, we found that most mutants co-localized with alpha-syn aggregates, but removal of the second helix (alpha-2), spanning residues 55-96, abrogated co-localization." If most mutants are unable to co-localize with the removal of the second alpha helix, then is the other percentage of that group completely unaffected? Or does a different alpha helix need to be removed from the equation for an effect to be seen?

"This result implies that alpha-syn aggregates sequester much of the ESCRT-III system, with CHMP2B-alpha-syn co-localization serving as a proxy for this effect." This aligns with what we discussed in class the other day. About how the Lewy bodies also contain ESCRT proteins. But how many types of ESCRT proteins do Lewy bodies typically contain?

"Proteopathic aggregates thus exert trans- acting loss of function by both sequestering other cellular proteins and targeting these proteins for collateral degradation." So, from what it sounds like so far, ESCRT proteins being aggregated and degraded seems to be more of an unfortunate happenstance rather than an intentional action by these alpha-syn mutations.

"Although CHMP2B has a paralog, CHMP2A, only CHMP2B mutations have been implicated as neurodegenerative risk factors." The paper says CHMP2B is the main antagonist of neurodegenerative diseases, yet our notes from class say its CHMP2A...

"This implies that a sequence of uracils codes for phenylalanine, and our work suggests that it is probably a triplet of uracils." This was only a hypothesis when this paper was written and published, and now it is recognized as a biological fact. It's really crazy to see how quickly science can develop within just a few decades.

"However, exactly how many triplets code amino acids and how many have other functions we are unable to say." This makes more sense in relation to a comment I made at the very beginning of the paper. Still, the fact that the idea was even suggested during this period of time is impressive.

"Normally, an alteration confined to the A cistron (be it a deletion, an acridine mutant, or any other mutant) does not prevent the expression of the B cistron. Conversely, no alteration within the B cistron prevents the function of the A cistron. This implies that there may be a region between the two cistrons which separates them and allows their function to be expressed individually." Are there instances where a mutation on the A cistron would adversely affect the function of the B cistron then? And vice versa? The use of the word "Normally" at the beginning seems to imply that, but I don't know how true that is.

"The fact that we can make these changes and can study so large a region probably come about because this part of the protein is not essential for its function." Which part of the protein are they referring to exactly?

"The simplest postulate to make is that the shift of the reading frame produces some triplets the reading frame of which is 'unacceptable'; for example, they may be 'nonsense' or stand for 'end of the chain', or be unacceptable in some other way due to the complications of protein structure." Nonsense must mean the resulting codon does not produce a viable protein, 'end of the chain' must result in a stop codon, and the last bit seems pretty self explanatory.

"However, when both mutations are present in the same piece of DNA, as in the pseudo-wild double mutant FC (0+1), then although the reading triplets between FC 0 and FC 1 will be altered, the original reading will be restored to the rest of the gene." A mutation that reverses the original mutation as long as everything lines up correct. Once again, a concept that you would think would not be possible to conjure up over 50 years ago.

"Thus in all we have about eighty independent r mutants, all suppressors of FC 0, or suppressors of suppressors, or suppressors of suppressors of suppressors" Although I do understand what they authors are trying to convey, the way that this was communicated was not the most efficient. It could have been worded way better. If someone will no knowledge of the sciences were to read this, it would cause great confusion.

"Another idea is that certain triplets make 'sense' whereas others make 'nonsense', as in the comma-free codes of Crick, Griffith and Orgel." Nonsense could be interpreted within the literal sense, which I am certain is the author's intention, or it could be used in a more humorous sense.

"The code is probably degenerate; that is, in general, one particular amino-acid can coded by one of several triplets of bases." I think it's really interesting that Crick and the other researchers apart of this paper were able to glean such an advanced concept for the 1960s.

The salad bowl or kaleidoscope metaphor could also be something important to include, just like with Brabeck's integration vs. assimilation.

It's ironic how two elements are melted together to form a stronger metal, yet melting together two or more cultures actually weakens the country as a whole because it encourages racial and cultural discrimination.

History of the term. Very important.

This directly relates to what Brabeck was talking about in her own research. How with the melting pot theory, immigrants are expected to become "more American" and completely assimilate into American society.

Once again, layering on different evidence proving the benefits of multiculturalism in children.

This is an interesting idea that I could bring up in my research.

This is quite a bit of information, I probably will not use every piece of it, but what's important is what Brabeck is trying to prove. She is trying to convey to her readers the importance of multicultural identity, what happens when children are allowed to flourish in both their national and acquired identity, and the consequences of trying to silence on or the other.

The fact that a particular president was elected at this particular time does not seem relevant. The young girl may have been having these thoughts before Trump was even elected. Not that him being elected into presidency is the cause of her thoughts.

An eight year old child is already being exposed to this idea of American's culture being an unrelenting homogenous mixture. There is no room for incorporating individual aspects to a melting pot. You either with us, or you get left behind.