This entry intends to be a review of different therapies that use proteins as drugs.
Reversible Pegylation Prolongs the Hypotensive Effect of Atrial Natriuretic Peptide
Nesher, et al. 2008 Bioconjug Chem 19 342-348.
Signficance
I only skimmed this one, but natriuresis (doctors out there) is the excretion of sodium in urine, a physiological response that promotes hypotension and gets rid of excess sodium. This is triggered by endogenous brain and atrial natriuretic peptide.
They wanted to make these peptides stick around longer, to combat things like hypertension, so they reversibly attached a lot of PEG to the end of it.
Criticism
The result? They have a drug that sticks around longer, but it's only 10% as effective as the real thing. They did radiolabelled ligand competition experiments to analyze binding to the receptor, then just looked at the compound sitting in buffer to analyze the rate of hydrolysis (apparently this is similar to looking at it degrade in human serum? I don't really believe that one...). Finally, they administered the prodrug to mice and watched their blood pressure go down.
Thursday, February 05, 2009
Tuesday, February 03, 2009
4: Graduate Science Education
So, I'm curious as to what other people think about this as well: What does a graduate education in the life sciences consist of? This is entirely my own analysis.
Many of my advisors at Muhlenberg said that the highlight of a PhD is learning how to troubleshoot - when you don't know something, or when something's not working, or when you're missing what you need, a PhD gives you the training to figure things out on your own. What should we call this - practicality? Self-sufficiency? Certainly, it's a dimension of critical thinking and problem solving.
Okay, next, one would imagine it's important to learn how to actually carry out science. Perhaps, I can break this down into 3 main areas:
-How to develop strong hypotheses: Both recognizing problems/gaps in knowledge, and coming up with a testable conjecture
-How to develop and carry out strong experiments: Make sure they're logically sound, efficient/optimal, reproducible, and bestow technical knowledge/experience
-How to analyze results
After this, what's next? Well, I imagine we're supposed to learn how to effectively convey our results: This covers giving presentations, writing articles, grants, and a thesis.
The only other thing I can really think of is learning how to interact with other scientists -criticize what we read and see in other people's work, collaborate on projects, and discuss science in social contexts, while considering the interaction of our science with society.
So, how do they go about helping us learn these things?
-We attend classes - read articles, write proposals, take exams
-We write a thesis, on experiments that take us years to finish
I guess, the whole point of this entry is that I have 2 issues with this:
1) Content - What am I supposed to know? One day, I learn the details of the bacterial cell wall, another day, I learn about aging research. There are so many disparate fields of biology. And then, on top of that, there's chemistry (even the occassional upper-level mathematics/statistics and physics). Most of it ends up turning into unnecessary detail, unless that's the specific topic you're interested in pursuing. Another point, is that I thought this would get a lot more complicated than in my upper level undergraduate classes. But it's pretty much the same level of difficulty (except for chemistry classes - they really took things to the next level). So does this mean we just have to try and remember a broader range of material instead of more depth? We don't really get tested (besides the fleeting midterm/final) on these things, and it makes me think that it's only to make us more rounded as scientists. At the same time, all of this coursework is NOT teaching me what I really need to learn about my topic.
You know, you always hear about chemists that switch to bio/biochem after their PhD and really hit it big. But you never hear about people doing the opposite, and moving into chemistry. It seems too difficult or something.
2) Assessment - Everything is highly curved here, so the only real assessments are our thesis proposal (qualifier) and thesis defense. In both scenarios, they can ask you whatever the hell they want - but will you know it?
Hmm, this was kind of a pointless list, not much of a discussion. But I guess it lends insight into why graduate school feels so loosely structured. They can only do so much, really, in providing the resources for us to develop and teach ourselves. They can guide us in our analysis of articles/science (provided they're a good teacher), but really it's all up to us, which is highly unnerving. I don't know much about the qualifier, but I feel like that also won't be so bad. It seems easy to just slip through the cracks, because there's not really much to be held accountable for. As a result, it just leaves this unnerving feeling that I won't have learned enough to function autonomously as a successful scientist once I get out of here.
Will I ever learn to look at molecules like Dan Kahne or Nathan Gray? Only if I teach myself...
Many of my advisors at Muhlenberg said that the highlight of a PhD is learning how to troubleshoot - when you don't know something, or when something's not working, or when you're missing what you need, a PhD gives you the training to figure things out on your own. What should we call this - practicality? Self-sufficiency? Certainly, it's a dimension of critical thinking and problem solving.
Okay, next, one would imagine it's important to learn how to actually carry out science. Perhaps, I can break this down into 3 main areas:
-How to develop strong hypotheses: Both recognizing problems/gaps in knowledge, and coming up with a testable conjecture
-How to develop and carry out strong experiments: Make sure they're logically sound, efficient/optimal, reproducible, and bestow technical knowledge/experience
-How to analyze results
After this, what's next? Well, I imagine we're supposed to learn how to effectively convey our results: This covers giving presentations, writing articles, grants, and a thesis.
The only other thing I can really think of is learning how to interact with other scientists -criticize what we read and see in other people's work, collaborate on projects, and discuss science in social contexts, while considering the interaction of our science with society.
So, how do they go about helping us learn these things?
-We attend classes - read articles, write proposals, take exams
-We write a thesis, on experiments that take us years to finish
I guess, the whole point of this entry is that I have 2 issues with this:
1) Content - What am I supposed to know? One day, I learn the details of the bacterial cell wall, another day, I learn about aging research. There are so many disparate fields of biology. And then, on top of that, there's chemistry (even the occassional upper-level mathematics/statistics and physics). Most of it ends up turning into unnecessary detail, unless that's the specific topic you're interested in pursuing. Another point, is that I thought this would get a lot more complicated than in my upper level undergraduate classes. But it's pretty much the same level of difficulty (except for chemistry classes - they really took things to the next level). So does this mean we just have to try and remember a broader range of material instead of more depth? We don't really get tested (besides the fleeting midterm/final) on these things, and it makes me think that it's only to make us more rounded as scientists. At the same time, all of this coursework is NOT teaching me what I really need to learn about my topic.
You know, you always hear about chemists that switch to bio/biochem after their PhD and really hit it big. But you never hear about people doing the opposite, and moving into chemistry. It seems too difficult or something.
2) Assessment - Everything is highly curved here, so the only real assessments are our thesis proposal (qualifier) and thesis defense. In both scenarios, they can ask you whatever the hell they want - but will you know it?
Hmm, this was kind of a pointless list, not much of a discussion. But I guess it lends insight into why graduate school feels so loosely structured. They can only do so much, really, in providing the resources for us to develop and teach ourselves. They can guide us in our analysis of articles/science (provided they're a good teacher), but really it's all up to us, which is highly unnerving. I don't know much about the qualifier, but I feel like that also won't be so bad. It seems easy to just slip through the cracks, because there's not really much to be held accountable for. As a result, it just leaves this unnerving feeling that I won't have learned enough to function autonomously as a successful scientist once I get out of here.
Will I ever learn to look at molecules like Dan Kahne or Nathan Gray? Only if I teach myself...
Monday, February 02, 2009
3: Lol! Bacterial Lipoprotein and Lipopolysaccharide Processing
A New ABC-Transporter Mediating the Detachment of Lipid-modified Proteins from MebranesYakushi, et al. 2000 Nat Cell Bio
Signficance
The cell membrane has many functions: regulating the passage of solutes in and out of the cell, facilitating cell-cell communication and interaction with the external millieu, defining cell size and shape, and even providing a surface for mediating reactions. Many of these functions are carried out by membrane proteins, but how do they get there? This can get even more complicated, because as you know, gram-negative bacteria (e.g. E. coli) have two plasma membranes...
These researchers identified the protein complex that's responsible for detaching lipoproteins from the inner membrane and bringing them to the outer membrane. This sounds like an arduous task, for these fat-soluble proteins have to cross the polar periplasm before arriving at the nonpolar outer membrane.
The type of transporter that they found is called an ABC transporter. This superfamily is responsible for using ATP energy to transport all different things across membranes, from small molecules and drugs to big proteins, and so have an interesting mix of conserved motifs along with highly variable pore sites. E. coli has around 57 in total (10 subfamilies) - 44 have associated partners and are thought to be involved in solute uptake, and the rest don't have partners, and are thought to be responsible for solute export. But LolCDE don't export - they just detach! And they analyzed sequences to find homologues in other species too, making this a conserved system in gram-negative bacteria.
There are over 80 E. coli lipoproteins, and 90% of them are on the outer membrane. I think many of them may be virulence factors too.
Background
Lipoproteins are transported across the inner membrane via the Sec translocon and a signal sequence. Once there, an N-terminal Cys gets aminoacylated with the lipid component.
LolA is a water-soluble chaperone in the periplasm, that helps to bring lipoproteins across the polar periplasmic space and into the outer membrane. If you don't have LolA, then proteins get stuck in the inner membrane.
LolB is a receptor for the LolA-lipoprotein complex, and basically accepts the lipoprotein for incorporation into the outer membrane.
But! How do proteins get from the inner membrane to LolA?...
Results
I thought they set up a pretty cool assay- they reconstructed proteoliposomes (from solubilized inner-membrane proteins + LolA + outer-membrane lipoprotein Pal + ATP + phospholipids) as basically chunks of membrane, and tested whether lipoproteins were getting transported or not, by analyzing supernatant (soluble) and pellet (insoluble, liposome-bound) fractions with PAGE. Is this an accurate reconstitution of the system? They seem to think so, but I would've liked to see them make it work for a few more candidate lipoproteins besides Pal.
Using classical chromatography, they then figured out what inner-membrane proteins in the mix were responsible for relasing the lipoprotein to LolA. They cloned the gene cluster for these proteins, purified them (LolCDE), and determined their ratios in a complex together.
By comparing the sequence to other ABC transporters, they identified several residues to mutate and test. The mutants inhibited the growth of cells that were Lpp+, but not Lpp- cells (Lpp, when mislocated, binds to peptidoglycan and inhibits cell growth).
They did another cool thing I didn't know about, which was create spheroblasts out of the growth-inhibited cells. You basically inhibit bacterial cell division and form long strings - you can then digest the cell wall, and they turn into big spheres! They found that the mutant LolD prevented proteins from getting released from the spheroplast, even with LolA added.
They also created a mutant lacking LolCDE that couldn't grow, and cells deficient in LolCDE couldn't release lipoproteins. After that, they used proteinase K digestion of the spheroplasts to prove that Pal wasn't translocated, but rather it was detached (completely digested)
Identification of Two Inner-membrane Proteins Required for the Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli
Ruiz, et al. PNAS 2008
Significance
Lipopolysaccharides are, for many bacteria, an essential membrane component, consisting of sugar+fat together in one molecule. The last article talked about Lol's involved in transporting lipoproteins across the periplasm. Lipopolysaccharides, on the other hand, have the Lpt system which also posseses an ABC transporter. They had found LptB, the ATP-binding domain, but couldn't find the transmembrane part until now - LptF and LptG.
Results
How do you find a mystery protein? That's a hard question, and there are different approaches, but usually you screen knockout/deletion mutants, until you find a key phenotype. This group, however, used bioinformatics. They knew the missing transporter must be conserved, because it's essential in gram-negative endosymbionts. What helps is that this family of bacteria lose a lot of their nonessential genes when evolving with their hosts (ah, connection to last entry!).
When probing other species, they found linkage of LptF and LptG to other Lpt genes, and even regions of gene overlap. They also found that deletion mutants were inviable. Mutants that only express low levels are sensitive to hydrophobic antibiotics, signifying a defective outer membrane. These low-level mutants were also found to make defective lipopolysaccharide, as determined by gel electrophoresis. They used an additional waaL knockout to show that LptFG must be acting after lipopolysaccharide export by MsbA (WaaL is involved in assembling lipopolysaccharide O-antigen...lipopolysaccharides are composed of lipid A, core, and O-antigen).
The next step was to demonstrate that LptFG weren't acting on outer membrane proteins. Apparently, when defects in assembly of outer membrane proteins occurs, they get degraded by the periplasmic protease DegP. But LptFG depletion strains all had normal levels of outer membrane proteins (they used OmpA and LptD).
When there are defects in lipopolysaccharide biogenesis, phospholiipids get translocated to the outer leaflet of the outer membrane. This activates PagP, which palmitoylates lipid A (adds a seventh lipid). They detected this increase in the weight of lipid A using MALDI-TOF mass spec for both LptF and LptG depletion mutants.
Finally, they use a pulse-chase experiment to determine whether, when they stop LptFG induction, de novo synthesized lipopolysaccharide was palmitoylated or not. Using TLC (with not good enough resolution, in my opinion), they did not see modified lipopolysaccharide in the outer membrane, suggesting that it never got there (defect in transport from the inner membrane).
2: Microbial Diversity
Ok, this entry's bound to be more entertaining, and reflects some of what we've been talking about in my Microbiology class.
Molecular identification by ‘‘suicide PCR’’ of Yersinia pestis as the agent of Medieval Black Death
Raoult, et al. 2000
This paper confirmed that the Black Death was caused by Yersinia pestis. They did this, by exhuming a few bodies, and removing the pulp from their teeth (apparently, when we die, the pulp gets sealed off from the external environment, and allows pristine preservation of whatever is inside. They used a special technique called "Suicide PCR" that supposedly only uses the primers once (I don't really understand how it works) to prevent contamination.
Detection of a bioluminescent milky sea from space
Miller, et al. 2005
So, apparently, it's a common myth to see the ocean glowing at night. Well, apparently, in January of 1995, this naval ship came across this phenomenon, and it was co-documented by a satellite that took a picture of the area - basically, a spot as big as the state of Connecticut, in the Indian Ocean, glowing. Pretty crazy. They confirmed it was bioluminescent bacteria.
Biomineralization of Gold: Biofilms on Bacterioform Gold
Reith, et al. 2006
Apparently, bacteria can spit out gold. Good job, them.
Coevolved Crypts and Exocrine Glands Support Mutualistic Bacteria in Fungus-Growing Ants Currie, et al. 200
6
So, this type of thing I've seen in several classes, because one of the guys I'm interested in working with here (Jon Clardy) encounters this in his chemical ecology research.
Anyway, in case you haven't figured it out yet from this entry, bacteria are pretty amazing. Without them, we wouldn't be here - They're responsible for making the Earth hospitable for other forms of life (cyanobacteria created all the oxygen from...the nitrogen and carbon dioxide that was there before), they help everyone with nutrition (plants absorb nutrients from the soil, us digest food and absorb nutrients, cows and termites digest cellulose), they help break down detritus and cycle chemicals, and provide us with resources like natural gas.
Anyway, it's pretty common for mutualistic bacteria and their hosts to have coevolved together for millions of years:
The illustrated example is somewhat complicated, with about 5 players: 1) the Southern pine beetle likes to bore into trees and lay eggs, killing our forests. It has special compartments that 2) store a "good" fungus that grows in the tree with the eggs, and then serves as a food source.
But, there's a 3) "bad" fungus that kills off the good fungus and takes over. Fortunately, the beetle also carries a 4) bacterium that kills off the bad fungus with an antifu
ngal compound it produces. Not shown is a 5) "poser" bacterium that hitches along for the ride sometimes, but doesn't actually produce the antifungal.
There's a very similar situation with leaf-cutter ants, which is what the cited article is actually about. People travel to Costa Rica to research this, and I believe Cornell owns a fully functional, big, reconstructed leaf-cutter ant farm.
A Molecular View of Microbial Diversity and the Biosphere
Pace Science 1997
It's hard to categorize bacteria - if you think about it, they all look very similar to us (well, it's pretty hard to see them), and unlike animals, we wouldn't be able to effectively categorize them by morphological differences. Also, because of all the genetic swapping they did over long periods of evolutionary time, it's hard to see lineage. Nevertheless, Woese came up with the brilliant idea of sequencing their 16S rRNA to construct a phylogenetic tree, that after years of harsh disbelief by the scientific community, displaced the five kingdom model, and replaced it with the three domain model.
This has been expanded through various expeditions to the ends of the Earth. In the image, plants, humans, and fungi represent the smallest 3 fingers in Eucarya. Isn't it crazy how much more diverse the monocellular organisms are?
These things can be found everywhere. I won't post the details, but, there are several efforts right now to sequence all the bacteria found in the human intestinal system. The article I have in front of me found 395 different phylotypes of bacteria. I think this works out to something like 100x the number of genes that we actually have. Crazy.
I also learned that bacteria may be responsible for clouds. In the CLAW hypothesis (you can wikipedia this), phytoplankton are responsible for releasing sulfates that nucleate clouds.
Finally, an important idea that has special relevance to me: We still don't understand anything about the majority of bacteria out there because scientists can't figure out how to grow them. The estimate is that we can only grow 1% of the stuff we find in dirt. While eDNA cloning techniques enable us to at least harness and see the DNA of these organisms, and use that to predict what they make and how they're structured, this isn't usually a substitute for the real thing. I've seen a couple of people working on "co-culturing", where it's believed that one bacteria will grow only if it senses the presence of another one nearby (using small molecule signals that probably evolved between the two species over the course of evolution)
Molecular identification by ‘‘suicide PCR’’ of Yersinia pestis as the agent of Medieval Black Death
Raoult, et al. 2000
This paper confirmed that the Black Death was caused by Yersinia pestis. They did this, by exhuming a few bodies, and removing the pulp from their teeth (apparently, when we die, the pulp gets sealed off from the external environment, and allows pristine preservation of whatever is inside. They used a special technique called "Suicide PCR" that supposedly only uses the primers once (I don't really understand how it works) to prevent contamination.
Detection of a bioluminescent milky sea from space
Miller, et al. 2005
So, apparently, it's a common myth to see the ocean glowing at night. Well, apparently, in January of 1995, this naval ship came across this phenomenon, and it was co-documented by a satellite that took a picture of the area - basically, a spot as big as the state of Connecticut, in the Indian Ocean, glowing. Pretty crazy. They confirmed it was bioluminescent bacteria.
Biomineralization of Gold: Biofilms on Bacterioform Gold
Reith, et al. 2006
Apparently, bacteria can spit out gold. Good job, them.
Coevolved Crypts and Exocrine Glands Support Mutualistic Bacteria in Fungus-Growing Ants Currie, et al. 200
6So, this type of thing I've seen in several classes, because one of the guys I'm interested in working with here (Jon Clardy) encounters this in his chemical ecology research.
Anyway, in case you haven't figured it out yet from this entry, bacteria are pretty amazing. Without them, we wouldn't be here - They're responsible for making the Earth hospitable for other forms of life (cyanobacteria created all the oxygen from...the nitrogen and carbon dioxide that was there before), they help everyone with nutrition (plants absorb nutrients from the soil, us digest food and absorb nutrients, cows and termites digest cellulose), they help break down detritus and cycle chemicals, and provide us with resources like natural gas.
Anyway, it's pretty common for mutualistic bacteria and their hosts to have coevolved together for millions of years:
The illustrated example is somewhat complicated, with about 5 players: 1) the Southern pine beetle likes to bore into trees and lay eggs, killing our forests. It has special compartments that 2) store a "good" fungus that grows in the tree with the eggs, and then serves as a food source.
But, there's a 3) "bad" fungus that kills off the good fungus and takes over. Fortunately, the beetle also carries a 4) bacterium that kills off the bad fungus with an antifu
ngal compound it produces. Not shown is a 5) "poser" bacterium that hitches along for the ride sometimes, but doesn't actually produce the antifungal.There's a very similar situation with leaf-cutter ants, which is what the cited article is actually about. People travel to Costa Rica to research this, and I believe Cornell owns a fully functional, big, reconstructed leaf-cutter ant farm.
A Molecular View of Microbial Diversity and the Biosphere
Pace Science 1997
It's hard to categorize bacteria - if you think about it, they all look very similar to us (well, it's pretty hard to see them), and unlike animals, we wouldn't be able to effectively categorize them by morphological differences. Also, because of all the genetic swapping they did over long periods of evolutionary time, it's hard to see lineage. Nevertheless, Woese came up with the brilliant idea of sequencing their 16S rRNA to construct a phylogenetic tree, that after years of harsh disbelief by the scientific community, displaced the five kingdom model, and replaced it with the three domain model.
This has been expanded through various expeditions to the ends of the Earth. In the image, plants, humans, and fungi represent the smallest 3 fingers in Eucarya. Isn't it crazy how much more diverse the monocellular organisms are?
These things can be found everywhere. I won't post the details, but, there are several efforts right now to sequence all the bacteria found in the human intestinal system. The article I have in front of me found 395 different phylotypes of bacteria. I think this works out to something like 100x the number of genes that we actually have. Crazy.
I also learned that bacteria may be responsible for clouds. In the CLAW hypothesis (you can wikipedia this), phytoplankton are responsible for releasing sulfates that nucleate clouds.
Finally, an important idea that has special relevance to me: We still don't understand anything about the majority of bacteria out there because scientists can't figure out how to grow them. The estimate is that we can only grow 1% of the stuff we find in dirt. While eDNA cloning techniques enable us to at least harness and see the DNA of these organisms, and use that to predict what they make and how they're structured, this isn't usually a substitute for the real thing. I've seen a couple of people working on "co-culturing", where it's believed that one bacteria will grow only if it senses the presence of another one nearby (using small molecule signals that probably evolved between the two species over the course of evolution)
1: Protein Evolution
Because this is my first entry, I'd like to just mention: Many times, I will be typing phrases directly from the articles or summarizing viewpoints and conclusions that I don't claim to be my own. Also, I hope to get a lot better at this (in numerous ways) as I practice more and time progresses.Importance
So, this is a technique that, if people can get it to work well, enables several important things:
1) Re-engineering of natural enzymes and pathways to accept novel reactants or produce novel products. Think about creating new antibiotics, at the very least!
2) Creating completely new proteins with previously unheard of functions, catalyzing unprecedented chemical reactions, eliminating the need for hazardous chemicals and improving the yield of synthesis for many important drugs.
3) Exploring the protein folding problem and linking sequences to structure and function.
Summary
We can harness the principles of evolution (selection for a favorable trait or ability, then replication/proliferation) to engineer biomolecules in directed ways, specifically proteins and nucleic acids. These types of polymers are inherently combinatorial, assembled from their monomeric building blocks, and are thus suited to this type of approach.
The basic strategy is to first construct a library of sequences exploring biochemical space, usually through modification of the gene encoding the protein of interest - this can be done using random or targeted mutagenesis, DNA shuffling, and degenerate codons. After creating the library, you can then screen it or use selection, using a variety of techniques lke HPLC, NMR, Mass spec, detection of color/fluorescence, and FACS.
So, there are 3 cool techniques that help this to be feasible: phage, ribosome, and mRNA display
Phage display: Fuse your gene to a bacteriophage surface protein gene. When the virus particles lyse the host, they will display the protein of interest on their surface, and carry the sequence encoding that protein with their DNA inside.
Ribosome display: Attach a spacer sequence right before the stop codon of your protein, so that it gets stuck inside the ribosome as an mRNA:ribosome:fully-folded protein complex
mRNA display: fuse puromycin to the end of the mRNA for your gene of interest. This will get caught in the ribosome's A site and get fused to the protein. So, you get an mRNA-protein hybrid.
Whichever technique you use, you could then use affinity chromatography to pull out the protein that works (for example, if you're looking for a protein that can bind ATP, attach an ATP-analogue to the resin, and it will pull out any proteins fused to their encoding sequences)
The advantage of the latter two is that you can express entirely in vitro without cells, and still retain the sequence if you find a protein that does the job.
You can either: 1) design from scratch or 2) redesign an existing protein
1) Designing from scratch requires little information (if you want to create just something that folds or has a specific secondary structure), but it's difficult to engineer something that binds a small molecule (20 out of 10^13 in an 88aa library could bind streptavidin, and with little affinity). They've also created ATP-binders, which had adopted a fold fiound in native proteins. They can create alpha-helices/beta-sheets by predefining sequence positions of hydrophilic/hydrophobic residues (some even bound heme!)
Computational screening has enormous potential in allowing virtual screening of many more sequence combinations. The program alternates between sequence design and structure prediction to evolve a molecule over computational time.
2) Redesigning can help improve enzymatic stability, tolerance for different conditions or substrates, and introduce the ability to produce new products. In one cool example, they engineered Cre recombinase to excise out HIV provirus from the human genome
The feelings from many scientists that I've spoken to on this are:
-it's only worked for a few examples. And there are a growing number of supporting experimental successes. But could you do this for enzymes that are not so well behaved? Not yet.
-It's all about your ability to come up with a successful screen. You need to wittle your way down from an incredibly large library to something that has the desired function. According to this article, the biggest challenge is that functional sequences are rare compared to the number of theoretical combinations.
-I also have a problem with the "rational" in rational design. With the whole randomized mutation technique, you're kind of waiting to stumble upon a sequence that works. How good are they at determining specifically, "Ok, if I change this one residue, this will happen..."?
This review really didn't help answer that question. :/ But I have another one waiting to be read, and maybe that'll give me better insight. Sorry, this wasn't a good first entry, hopefully things will get better.
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